Antibacterial novel skeleton diterpene compound, synthesis method thereof and application thereof

Abstract

The present application relates to a kind of antibacterial new skeleton diterpene compound and its synthesis method.The present application obtains a terpene cyclase of unknown function and a P450 oxidase of unknown function from the genomic data disclosed in a strain of Aspergillus fumigatiaffinis CNM-CM8980, by heterologous expression and in vitro enzyme catalytic reaction of Aspergillus oryzae, it is found that the terpene cyclase can be catalyzed to generate a diterpene compound with 6 / 5 / 5 / 5 brand new four-ring skeleton, candidate P450 oxidase can act on the diterpene compound to generate the oxidation product with antibacterial activity.The present application first reports the chemical structure of the diterpene compound, biological activity and biosynthesis gene, on the one hand, provides lead compound resources for discovering new antibiotics, on the other hand, lays a foundation for realizing its green and efficient synthesis.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals. Specifically, this invention relates to a diterpenoid compound, its synthesis method, and its application. Technical Background

[0002] Diterpenoids are a diverse class of natural products with important activities, widely distributed in animals, plants, and microorganisms. Their skeletons consist of 20 carbon atoms, exhibiting complex and diverse structures, but all are formed from the linear precursor GGPP (geranylgeranyl diphosphate) under the action of cyclases. Due to their complex chemical structures and broad biological activities, diterpenoids have always been a research hotspot, with some compounds having important clinical applications and significant economic value. For example, the plant-derived taxane-type diterpenoid paclitaxel (Taxol) has outstanding anticancer activity and is widely used clinically to treat various malignant tumors such as ovarian cancer, breast cancer, non-small cell lung cancer, pancreatic cancer, and gastric cancer; the fungal-derived pleuromutilin has significant killing effects on Gram-positive bacteria and has been developed into various antibacterial drugs, such as retaparin and tiamulin. Therefore, diterpenoid compounds are an important source of drug discovery, and discovering more structurally novel and highly active diterpenoid compounds from nature has significant application value.

[0003] In recent years, with the rapid development of gene sequencing technology, an increasing number of fungal genomes have been reported, and databases such as NCBI contain a wealth of genomic information. Bioinformatics analysis shows that the number of gene clusters in fungal genomes is enormous, far exceeding the number of natural products found in fungi. This suggests that many genes in fungal genomes are "silent" genes with unknown functions, urgently awaiting discovery and utilization to find natural products with novel structures and activities. Therefore, obtaining corresponding genes from fungi using gene mining technology, combined with heterologous expression, chemical isolation, structural identification, and activity screening, to discover more compounds with novel structures and good activities is of great significance for the development of fungal-derived drugs. Summary of the Invention

[0004] Based on this, the present invention provides a diterpenoid compound, and provides a method for the biosynthesis of such diterpenoid compound and its application.

[0005] The diterpenoid compounds all contain a 6 / 5 / 5 / 5 tetracyclic skeleton, and their structural formulas are shown in Figures 1 and 2:

[0006]

[0007] The enzymes used to synthesize compounds 1 and 2 are the terpene cyclase AfAS and the P450 oxidase AfP450, respectively. The genes for these two enzymes are derived from the fungus *Aspergillus fumigatiaffinis* CNM-CM8980. The gene encoding terpene cyclase AfAS consists of 1411 nucleotide bases, and its sequence is shown in SEQ ID NO.4. The gene encoding oxidase AfP450 consists of 2005 nucleotide bases, and its sequence is shown in SEQ ID NO.5. The protein encoded by the AfAS gene contains 417 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.1. The protein encoded by AfP450 contains 512 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.2. This invention verifies the functions of cyclase AfAS and oxidase AfP450 through heterologous expression of Aspergillus oryzae and in vitro enzyme catalysis experiments. Cycloase AfAS is responsible for forming a 6 / 5 / 5 / 5 tetracyclic skeleton to obtain compound 1, and oxidase AfP450 is responsible for oxidizing the carboxyl group at C19 in compound 1 to obtain compound 2.

[0008] Furthermore, comparison revealed that the enzyme pocket of AfAS is very similar to that of the known fusicocca-2,10(14)-diene synthase PaFS, with the only difference being the 65th amino acid residue. In AfAS, the 65th residue is isoleucine (I), while in PaFS it is phenylalanine (F), which has a larger side chain. Therefore, the 65th residue may be a key residue catalyzing the formation of either the 6 / 5 / 5 / 5 tetracyclic skeleton compound 1 or the 5 / 8 / 5 tricyclic skeleton compound fusicocca-2,10(14)-diene. We commercially synthesized the cyclase domain (1-350 aa) gene of PaFS, which catalyzes the formation of fusicocca-2,10(14)-diene, with its amino acid sequence shown in SEQ ID NO.3 and its gene sequence shown in SEQ ID NO.6. When PaFS is mutated by F65I, F65V, F65C, F65L, F65M, F65N, F65E, F65T, F65S, and F65A respectively, it can produce a 6 / 5 / 5 / 5 tetracyclic skeleton compound 1. When AfAS was mutated, the results showed that: when I65 of AfAS was mutated to F, the mutant could still produce compound 1, and also produce a small amount of fusicocca-2,10(14)-diene; when I65 of AfAS was mutated to Y, the yield of compound 1 decreased, while fusicocca-2,10(14)-diene became the main product; when I65 of AfAS was mutated to W, the enzyme activity decreased, and the yields of both compound 1 and fusicocca-2,10(14)-diene decreased; when I65 of AfAS was mutated to H, N, M, V, L, D, T, C, or A, it could still produce compound 1, and when mutated to R, Q, P, E, S, K, or G, the enzyme was inactivated. Therefore, in the AfAS and PaFS enzyme pockets, the 65th residue may play a decisive role in shaping the enzyme pocket. When this site is mutated to different amino acids, it leads to the formation of products with different skeletons, such as compound 1 or fusicocca-2,10(14)-diene.

[0009] In a first aspect, the present invention provides a class of diterpenoid compounds, the structures of which are as follows;

[0010]

[0011] A second aspect of the present invention provides a polypeptide for synthesizing the above-mentioned diterpenoid compound, the polypeptide comprising:

[0012] (a) Terpene cyclase AfAS: The terpene cyclase AfAS is a polypeptide sequence having at least 70% sequence identity with the sequence shown in SEQ ID NO:1; preferably, the terpene cyclase AfAS is a polypeptide sequence having 80%, 85%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:1; and / or

[0013] (b) P450 oxidase AfP450: The P450 oxidase AfP450 is a polypeptide sequence having at least 70% sequence identity with the sequence shown in SEQ ID NO:2; preferably, the P450 oxidase AfP450 is a polypeptide sequence having 80%, 85%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:2.

[0014] A third aspect of the present invention provides a method for synthesizing compound 1, the method comprising using the terpene cyclase AfAS to catalyze geraniol pyrophosphate to generate compound 1.

[0015] In one embodiment, the terpene cyclase AfAS is a polypeptide sequence having at least 70% sequence identity with the sequence shown in SEQ ID NO:1; preferably, the terpene cyclase AfAS is a polypeptide sequence having 80%, 85%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:1.

[0016] In one embodiment, the 55th to 75th amino acids of the terpene cyclase AfAS are mutated to other amino acids.

[0017] In a preferred embodiment, the catalytic site of the terpene cyclase AfAS catalyzing geranylide pyrophosphate is the amino acid residue corresponding to I65 of the amino acid sequence shown in SEQ ID NO:1.

[0018] In a fourth aspect, the present invention provides a method for synthesizing compound 2, the method comprising oxidizing the methyl group at position C19 of the above-mentioned compound 1 to a carboxyl group using the oxidase AfP450, thereby obtaining compound 2.

[0019] In one embodiment, the oxidase AfP450 is a polypeptide sequence having at least 70% sequence identity with the sequence shown in SEQ ID NO:2; preferably, the oxidase AfP450 is a polypeptide sequence having 80%, 85%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:2.

[0020] In a fifth aspect, the present invention provides a cell for synthesizing compound 1 and / or compound 2, said cell comprising the polypeptide described in the second aspect of the present invention.

[0021] In one embodiment, the cells are Aspergillus oryzae cells, Saccharomyces cerevisiae cells, or Escherichia coli cells, preferably Aspergillus oryzae cells.

[0022] In a preferred embodiment, the cells containing synthetic compound 1 and / or compound 2 are constructed by the following method:

[0023] 1) Using Aspergillus fumigatiaffinis CNM-CM8980 genomic DNA as a template, the AfAS and AfP450 encoding genes were amplified by PCR, or the target genes were obtained by chemical synthesis; then, the target genes were ligated into the Aspergillus fumigatiaffinis expression plasmid pTAex3 or pUSA plasmid, respectively, to construct recombinant expression plasmids pTAex3-AfAS and pUSA-AfP450;

[0024] 2) The expression vector pTAex3-AfAS was transformed into the protoplasts of Aspergillus oryzae NSAR1 to obtain the genetically engineered strain AO-AfAS; the expression vectors pTAex3-AfAS and pUSA-AfP450 were co-transformed into the protoplasts of Aspergillus oryzae NSAR1 to obtain the genetically engineered strain AO-AfAS-AfP450.

[0025] In one embodiment, the genetically engineered bacteria generate compound 1 and / or 2 through liquid fermentation or solid fermentation; preferably, the genetically engineered bacteria generate compound 1 and / or 2 through liquid fermentation.

[0026] In a preferred embodiment, the method for generating compound 1 and / or 2 via liquid fermentation is as follows:

[0027] 1) Inoculate the genetically engineered bacteria into liquid culture medium and culture it in a shaker at 28-30℃ and 180-220rpm for 1-3 days to obtain the seed culture of the strain;

[0028] 2) Take the seed culture of the strain obtained in step 1) and inoculate it into the liquid fermentation medium. Culture it in a shaker at 28-30℃ and 180-220rpm for 5-8 days to obtain the fermentation broth of the strain.

[0029] 3) Take the fermentation broth obtained in step 2), filter to obtain mycelium, extract with methanol, sonicate for 30 min, soak overnight, filter to obtain mycelium extract, concentrate under reduced pressure to obtain extract.

[0030] 4) The extract was separated and enriched by silica gel column chromatography, and then monomeric compounds 1 and 2 were prepared by high performance liquid chromatography.

[0031] In a preferred embodiment, the liquid culture medium in step 1) is DPY culture medium, which comprises: 2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.05% MgSO4·7H2O, and 0.5% KH2PO4.

[0032] In a preferred embodiment, the culture medium in step 2) is CD-starch medium, which comprises: 0.3% NaNO3, 0.2% KCl, 0.05% MgSO4·7H2O, 0.1% KH2PO4, 0.002% FeSO4·7H2O, 1% polypeptone, 2% starch, and pH 5.5.

[0033] In a sixth aspect, the present invention provides the use of compound 2 in the preparation of an anti-Staphylococcus aureus preparation.

[0034] Compared with the prior art, the present invention achieves the following beneficial effects:

[0035] This invention uses *Aspergillus oryzae* as the dominant expression host and employs gene introduction to verify the functions of the terpene synthase AfAS and oxidase AfP450 in vivo. Two novel diterpene compounds with a 6 / 5 / 5 / 5 tetracyclic skeleton were isolated and identified, exhibiting certain antibacterial activity and potential applications in the biopharmaceutical field. Furthermore, this invention discovered that the 65th residue plays a decisive role in the formation of products with different skeletons. Saturation mutagenesis at the 65th residue of *PaFS* revealed F65I, F65V, F65C, F65L, F65M, F65N, F65E, F65T, F65S, and F... The 65A mutant produces compound 1; however, saturation mutation verification at position 65 of AfAS revealed that the I65F, I65Y, and I65W mutations not only produce compound 1 but also fusicocca-2,10(14)-diene (a product of the PaFS enzyme). The I65H, I65N, I65M, I65V, I65L, I65D, I65T, I65C, and I65A mutations produce compound 1 and other unidentified diterpenoid compounds, while the I65R, I65Q, I65P, I65E, I65S, I65K, and I65G mutations inactivate the enzyme. Finally, this invention lays a solid foundation for obtaining this type of diterpenoid product and for further in-depth research, providing lead compound resources for enriching the diterpenoid compound library and in order to discover new antibiotics and other pharmaceutical raw materials. Attached Figure Description

[0036] Figure 1 Functional characterization of AfAS in this invention;

[0037] Figure 2 Functional characterization of the AfP450 of this invention;

[0038] Figure 3 Functional characterization of PaFS in this invention;

[0039] Figure 4 Comparative analysis of AfAS and PaFS enzyme pockets, and analysis of key residues;

[0040] Figure 5 GC-MS detection image of the PaFS saturated mutant of this invention;

[0041] Figure 6 GC-MS detection image of the AfAS saturated mutant of this invention;

[0042] Figures 7-12 The NMR spectrum of compound 1 of this invention;

[0043] Figures 13-17 This is the NMR spectrum of compound 2 of the present invention. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. It is to be understood that these embodiments do not limit the invention. Variations of the invention now known or further developed are considered to fall within the scope of the invention described herein and claimed below.

[0045] Example 1: Construction of a heterologous gene expression vector

[0046] Construction of pTAex3-AfAS plasmid: Using commercially synthesized AfAS genomic DNA as a template, the terpene cyclase gene AfAS was amplified by PCR using primers Inf-pT-AfAS-F / Inf-pT-AfAS-R. The PCR reaction system was subjected to agarose gel electrophoresis, and the AfAS target fragment was recovered after purification using a gel extraction kit. Next, the AfAS fragment was integrated into the linear vector pTAex3 digested with SmaI using an in-fusion kit, and then transformed into E. coli DH5α. Positive clones were screened by ampicillin, and selected positive clones were subjected to liquid fermentation. The plasmid was extracted and sequenced for verification, finally obtaining the correct expression vector pTAex3-AfAS.

[0047] Construction of pTAex3-PaFS plasmid: Using the genomic DNA of the PaFS cyclase domain (1-350aa) synthesized commercially available genomic DNA as a template, the terpene cyclase gene PaFS was amplified by PCR using primers Inf-pT-PaFS-F / Inf-pT-PaFS-R; then the correct expression vector pTAex3-PaFS was obtained according to the corresponding method.

[0048] Construction of pUSA-AfP450 plasmid: Using commercially synthesized AfP450 genomic DNA as a template, the oxidase gene AfP450 was amplified by PCR using primers Inf-pU-AfP450-F / Inf-pU-AfP450-R. Using the same method as above, the AfP450 fragment was integrated into the linear vector pUSA digested with SmaI, and finally the expression vector pUSA-AfP450 was obtained.

[0049] Construction of the pYE-AfAS cDNA protein expression plasmid: RNA was extracted from Aspergillus oryzae expressing AfAS, and cDNA was obtained using a reverse transcription kit. The intronless AfAS cDNA gene was then amplified using primers pYE-AfAS-F / pYE-AfAS-R. This cDNA was then inserted into the linearized vector pYE using a seamless cloning kit, resulting in the pYE-AfAS cDNA plasmid.

[0050] Construction of the pYE-PaFS cDNA protein expression plasmid: RNA was extracted from Aspergillus oryzae expressing PaFS, and cDNA was obtained using a reverse transcription kit. The intronless PaFS cDNA gene was then amplified using primers pYE-PaFS-F / pYE-PaFS-R. This cDNA was then inserted into the linearized vector pYE using a seamless cloning kit, resulting in the pYE-PaFS cDNA plasmid.

[0051] Construction of the PaFS 65 residue saturation mutant plasmid:

[0052] Use saturation mutation primers, such as PaFS F65I -F / PaFS F65 -R, the full-length mutant plasmid was amplified from pYE-PaFScDNA, then the template plasmid was degraded with DpnI and transformed into E. coli DH5α. Positive clones were screened by ampicillin or kanamycin, and selected positive clones were subjected to liquid fermentation. The plasmid was extracted and sequenced for verification, finally yielding pYE-PaFScDNA. F65Y pYE-PaFScDNA F65I pYE-PaFScDNA F65V pYE-PaFScDNAF65C pYE-PaFScDNA F65L pYE-PaFScDNA F65M pYE-PaFScDNA F65N pYE-PaFScDNA F65E pYE-PaFScDNA F65T pYE-PaFScDNA F65S pYE-PaFScDNA F65A pYE-PaFScDNA F65W pYE-PaFScDNA F65R pYE-PaFScDNA F65H pYE-PaFScDNA F65D pYE-PaFScDNA F65K pYE-PaFScDNA F65G pYE-PaFScDNA F65P pYE-PaFScDNA F65Q Plasmid.

[0053] Construction of the AfAS 65th residue saturation mutant plasmid:

[0054] Use saturation mutation primers, such as AfAS I65F The full-length mutant plasmid was amplified from pYE-AfAScDNA using the -F / R method. The template plasmid was then degraded with DpnI and transformed into *E. coli* DH5α. Positive clones were selected using ampicillin or kanamycin, and selected for liquid fermentation. The plasmid was then extracted and sequenced for verification, ultimately yielding pYE-AfAScDNA. I65F pYE-AfAScDNA I65Y pYE-AfAScDNA I65W pYE-AfAScDNA I65H pYE-AfAScDNA I65N pYE-AfAScDNA I65M pYE-AfAScDNA I65V pYE-AfAScDNA I65L pYE-AfAScDNA I65D pYE-AfAScDNA I65T pYE-AfAScDNA I65C pYE-AfAScDNA I65A pYE-AfAScDNA I65R pYE-AfAScDNA I65Q pYE-AfAScDNAI65P pYE-AfAScDNA I65E pYE-AfAScDNA I65S pYE-AfAScDNA I65K pYE-AfAScDNA I65G Plasmids, etc.

[0055] The primers mentioned are shown in Table 1;

[0056] Table 1: Primer List

[0057]

[0058]

[0059]

[0060] Example 2 Construction of genetically engineered strains

[0061] In the construction of Aspergillus oryzae transfected strains, PEG-mediated protoplast transformation was used to transfect expression plasmids containing different genes into Aspergillus oryzae. The specific operation method is as follows:

[0062] 1) Inoculate E. coli host cells containing the target expression plasmid into 20-30 mL of solution containing Amp + The antibiotic was cultured overnight in LB liquid medium, and then a high concentration (>1 g / L) of recombinant plasmid was extracted for subsequent transfection experiments.

[0063] 2) Pick an appropriate amount of mycelium from A. oryzae NSAR1 plate and place it in 10 mL of DPY medium (2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.05% MgSO4·7H2O, 0.5% KH2PO4, and bring the volume to 1 L). Incubate at 28℃ with shaking at 200 rpm for 1-2 days.

[0064] 3) Add 10 mL of the above culture medium to 100 mL of DPY medium, mix well, and incubate at 28°C and 180 rpm for 1 day.

[0065] 4) Prepare 10 mL TF solution 1: Weigh 0.79 g (NH4)2SO4 and 0.1 g Yatalase lysin into a 15 mL centrifuge tube, add 0-10 mL TF solution, dissolve by inverting, and filter into a 50 mL centrifuge tube using a 0.22 μm microporous membrane.

[0066] 5) Based on the bacterial concentration, take an appropriate amount of bacterial solution into a sterilized syringe, filter the bacterial solution, press dry to collect the bacterial cells, remove the bacterial cells with a sterilized long bamboo stick, and place them into a centrifuge tube containing 10 mL TF solution 1. Incubate at 30°C and 70 rpm in a constant temperature incubator for 3 hours with shaking to break the cell wall. When the supernatant is obviously turbid and light red, filter the protoplastized bacterial solution through a syringe filter into a 50 mL centrifuge tube.

[0067] 6) Add an equal volume of TF solution 2, invert and mix well, centrifuge at 1500 rpm for 10 min at 4°C, discard the supernatant, add 5 mL of TF solution 2 to the precipitate, invert and mix well, take 10 μL, count the number of protoplasts under a microscope using a hemocytometer, centrifuge at 1500 rpm for 10 min at 4°C, discard the supernatant, and then add an appropriate volume of TF solution 2 (dilution or concentration should be appropriate based on the number of protoplasts counted above, so that the protoplast concentration is 1–5 × 10⁻⁶). 7 (each cell / mL), invert and mix thoroughly;

[0068] 7) Take 200 μL of protoplast solution into a 15 mL centrifuge tube, add 10 μL of recombinant plasmid with a concentration of 1 μg / μL, mix well, and let stand on ice for 30 min. Add 250 μL, 250 μL and 850 μL of TFsolution 3 to the suspension in three portions, respectively. After each addition, gently mix with a 1 mL pipette tip and let stand at room temperature for 20 min.

[0069] 8) After standing, add 5 mL of TF solution 2 to a 15 mL centrifuge tube, invert to mix, centrifuge at 1500 rpm for 10 min at 4 °C, discard the supernatant, add 200 μL of TF solution 2, gently suspend and add to the center of the lower culture medium, and quickly add the upper culture medium around the perimeter, and shake quickly to mix.

[0070] 9) After the above culture medium plates are dried, wrap them with parafilm, invert them in an incubator at 28°C and culture for 3-7 days. Pick the transformed strains and inoculate them onto M stable medium for stable passage 1-3 times. After the transformed strains are stable, extract the genome of the transformed strains and perform PCR verification on the target gene. The strains with positive PCR results are used for subsequent fermentation experiments.

[0071] To identify the function of the terpene cyclase AfAS, the expression plasmid pTAex3-AfAS was transfected into Aspergillus oryzae according to the transfection method described above, and the transfected strain AO-AfAS containing the AfAS gene was obtained.

[0072] To identify the function of the synthesized terpene cyclase PaFS, the expression plasmid pTAex3-PaFS was transfected into Aspergillus oryzae according to the transfection method described above, resulting in the transfected strain AO-PaFS containing the PaFS gene.

[0073] To identify the function of the P450 enzyme AfP450 in the diterpenoid gene cluster, the present invention transfects expression plasmids pTAex3-AfAS and pUSA-AfP450 into Aspergillus oryzae to obtain transfected strain AO-AfAS-AfP450.

[0074] Example 3: Fermentation and Metabolite Analysis of Genetically Engineered Strain AO-AfAS

[0075] The AO-AfAS Aspergillus oryzae transfected strain obtained in Example 2 was inoculated into CD-Starch liquid medium (0.3% NaNO3, 0.2% KCl, 0.05% MgSO4·7H2O, 0.1% KH2PO4, 0.002% FeSO4·7H2O, 1% polypeptone, 2% Starch, pH 5.5) and cultured at 28℃ and 220 rpm for 5 days. Cells and bacterial culture were collected separately using a Bush funnel. The cells were soaked in an appropriate amount of ethanol for 12 h, sonicated for 30 min, and then concentrated under reduced pressure. The bacterial culture was extracted with ethyl acetate, concentrated under reduced pressure, and the concentrated sample was dissolved in cyclohexane for mass spectrometry. After high-speed centrifugation, the supernatant was collected for GC-MS analysis. The GC-MS results are shown in [Figure number missing]. Figure 1 ;

[0076] The gaseous energy analysis method is as follows:

[0077] The gas chromatography-mass spectrometry (GC-MS) instrument was an Agilent 7890B-7000D triple quadrupole GC-MS system. The chromatographic column was an HP5-MS (30m, 0.32mm id, 0.25μm film). The specific GC settings were: 1) Inlet pressure: 5.8221 psi, nitrogen flow rate 20 mL / min, helium flow rate 2 mL / min; 2) Injection volume: 1 μL; 3) Temperature program: 50°C for 3 minutes, ramped to 70°C at a rate of 20°C / min and held for 1 minute, ramped to 300°C at a rate of 15°C / min and held for 3 minutes; 4) Carrier gas: nitrogen flow rate 20 mL / min. The mass spectrometry settings were: 1) Ion source temperature: 230°C; 2) Transfer line temperature: 250°C; 3) Quadrupole temperature: 150°C; 4) Electron energy: 70 eV.

[0078] Depend on Figure 1GC-MS results showed that, compared with the blank control group (Aspergillus oryzae wild-type strain AO-Wild type), the AO-AfAS strain produced a significant difference peak (compound 1) at 16.8 min, and the molecular ion peak was 272.

[0079] Example 4: Isolation and structural identification of metabolites from genetically engineered bacteria AO-AfAS

[0080] Aspergillus oryzae expressing AfAS was cultured in a 20L CD-starch fermenter for 5 days. The culture medium was filtered off using a nylon mesh, and the bacterial cells were pressed to remove excess liquid. The cells were placed in a 5L glass conical flask, and an appropriate amount of ethanol was added. After soaking for 1 day, the ethanol extract was obtained by filtration through a nylon mesh. The cells were then soaked in ethanol again, and the extract was concentrated using a rotary evaporator for a total of 3 extractions. After the 3 concentrations, an appropriate amount of ethyl acetate was added directly to the rotary flask, dissolved by sonication, and transferred to a separatory funnel. An appropriate amount of deionized water was added to the rotary flask, dissolved by sonication, and transferred to a separatory funnel. Ethyl acetate and water were then added to the rotary flask to ensure all the material was transferred to the separatory funnel. After repeated extractions three times, the ethyl acetate layer was concentrated using a rotary evaporator to obtain a crude extract. The crude extract was purified by silica gel column chromatography, and the fraction containing the target compound was detected by thin-layer chromatography. Compound 1 was enriched by concentration using a rotary evaporator. Finally, compound 1 was purified using high performance liquid chromatography. The prepared chromatographic column was YMC-Pack ODS-A, the flow rate was 3 mL / min, and the mobile phase was 95% acetonitrile-water.

[0081] This invention determined the planar structure of compound 1 by nuclear magnetic resonance spectroscopy, determined its relative configuration by chemical derivatization, and finally determined its absolute configuration by isotope labeling.

[0082] Compound 1: Colorless oil; specific rotation [α] 28D = -27.0 (c 0.50, CH2Cl2); Infrared IR (diamond ATR): 2955(s), 2860(s), 1455(m), 1376(m), 1368(m), 1288(m), 1000(w), 803(w); GC-MS (positive) m / z 272.2, NMR data are shown in Table 3, Appendix Figure 7-12 .

[0083] Example 5: Fermentation and Metabolite Analysis of Genetically Engineered Bacteria AO-AfAS-AfP450

[0084] The transfected strain AO-AfAS-AfP450 of this invention was inoculated into CD-Starch liquid medium (same as in Example 4) and cultured at 28°C and 220 rpm for 5 days. Cells and bacterial culture were collected using a Bush funnel. The cells were soaked in an appropriate amount of ethanol overnight, sonicated for 30 min, and then concentrated under reduced pressure. The bacterial culture was extracted with ethyl acetate, concentrated under reduced pressure, and the concentrated sample was dissolved in chromatographic methanol, centrifuged at high speed, and the supernatant was analyzed by HPLC-MS. The results are shown in the appendix. Figure 2 ;

[0085] Liquid chromatography-mass spectrometry (LC-MS) analysis was performed on the above samples:

[0086] The liquid chromatography-mass analysis method used was HPLC-DAD-ELSD-MS;

[0087] Analytical instruments: Dionex Ultimate 3000, equipped with UltiMate3000 Diode Array Detector and Amazon SL ion trap electrospray mass spectrometer;

[0088] Chromatographic columns: YMC C18 (5μm, 4.6×250mm) liquid chromatography column, Phenomenex Gemini C18 (5μm, 4.6×250mm);

[0089] Mobile phase: Acetonitrile-water was used as the mobile phase, and gradient elution was performed at a flow rate of 1 mL / min;

[0090] Gradient elution program: 50%-100% acetonitrile (0-30 min), 100%-100% acetonitrile (30-50 min).

[0091] Depend on Figure 2 The results showed that, compared with Aspergillus oryzae strains containing only AfAS, the fermentation products of Aspergillus oryzae strains with the AfP450 gene were largely consumed by cyclized product 1 and produced a new product peak 2.

[0092] Example 6: Isolation and Structural Identification of Metabolites from Genetically Engineered Bacteria AO-AfAS-AfP450

[0093] Aspergillus oryzae expressing AfAS and AfP450 was fermented in a 20L CD-starch container for 5 days. Extraction was performed using the same method as in Example 6. Silica gel column chromatography was used with a gradient elution from 100% cyclohexane to ethyl acetate (1:1) to obtain the enriched fraction of compound 2. This fraction was further purified using high-performance liquid chromatography (HPLC). The chromatographic column was a YMC-Pack ODS-A, the flow rate was 3 mL / min, and the mobile phase was 85% acetonitrile-water. The purified product was a colorless oily compound 2.

[0094] Compared to compound 1, compound 2 has one less methyl signal and one more carboxyl carbon signal (δ). C 185.5), by two-dimensional NMR analysis, the planar structure of compound 2 was determined, which is a compound in which the methyl group at C19 of compound 1 is oxidized to a carboxyl group. Furthermore, since compound 2 does not add a new chiral center, the absolute configuration of compound 2 was determined by the stereoconfiguration of compound 1.

[0095] Compound 2 is a colorless oil with a specific rotation [α]. 28D =-30.0(c 0.50,CH2Cl2); ESIMS(positive)[M+H] + m / z 303.2. NMR data are shown in Table 4. Figure 13-17 .

[0096] Example 7: Analysis, isolation, and structural identification of metabolites from Aspergillus oryzae-transfected strain AO-PaFS

[0097] The AO-PaFS Aspergillus oryzae transfected strain obtained in Example 2 was inoculated into CD-Starch liquid medium (0.3% NaNO3, 0.2% KCl, 0.05% MgSO4·7H2O, 0.1% KH2PO4, 0.002% FeSO4·7H2O, 1% polypeptone, 2% Starch, pH 5.5) and cultured at 28℃ and 220 rpm for 5 days. Cells and bacterial suspension were collected separately using a Bush funnel. The cells were soaked in an appropriate amount of ethanol for 12 h, sonicated for 30 min, and then concentrated under reduced pressure. The bacterial suspension was extracted with ethyl acetate, concentrated under reduced pressure, and the concentrated sample was dissolved in cyclohexane for mass spectrometry. After high-speed centrifugation, the supernatant was collected for GC-MS detection. The results showed that it could produce compound 3, and the mass spectrometric fragments of compound 3 were highly similar to fusicocca-2,10(14)-diene. GC-MS results are shown below. Figure 3 ;

[0098] The GC-MS analysis method is as described in Example 3;

[0099] To further determine the structure of compound 3, *Aspergillus oryzae* expressing PaFS was cultured in a 10L CD-starch fermenter for 5 days. The culture medium was filtered off using a nylon mesh, and the bacterial cells were pressed to remove excess liquid. The cells were then placed in a 5L glass conical flask, and an appropriate amount of ethanol was added. After soaking for one day, the ethanol extract was filtered through a nylon mesh. The cells were then soaked in ethanol again, and the extract was concentrated using a rotary evaporator for a total of three extractions. After each concentration, an appropriate amount of ethyl acetate was added directly to the rotary flask, dissolved by sonication, and transferred to a separatory funnel. Deionized water was added to the rotary flask, dissolved by sonication, and transferred to the separatory funnel. Ethyl acetate and water were then added to the rotary flask to ensure complete transfer of the contents to the separatory funnel. After three extractions, the ethyl acetate layer was concentrated using a rotary evaporator to obtain a crude extract. The crude extract was purified by silica gel column chromatography, and thin-layer chromatography was used to detect fractions containing the target compound. The target compound-enriched fraction was then concentrated using a rotary evaporator. Finally, high-performance liquid chromatography (HPLC) was used for purification. The prepared chromatographic column was YMC-Pack ODS-A, the flow rate was 3 mL / min, and the mobile phase was 95% acetonitrile-water. After NMR analysis and optical rotation comparison, we determined that compound 3 was fusicocca-2,10(14)-diene.

[0100] Example 8: Comparison of enzyme pockets between AfAS and PaFS, and analysis of key amino acid residues.

[0101] The amino acid sequence of the cyclase AfAS was uploaded to the AlphaFold2 server. AlphaFold automatically searched for templates and performed structure prediction, resulting in a successfully modeled AfAS PDB file. Then, the cyclization intermediate was docked into the AfAS enzyme pocket using AutoDock Vina software. Simultaneously, PaFS crystals (PDB: 5er8) were downloaded from a crystallography website and molecular docking was performed using the same method. Post-docking analysis using PyMOL software revealed that the enzyme pocket contained molecules less than [a certain distance] from the substrate. The amino acids within the range are mostly similar, differing only at residue position 65. Figure 4 In AfAS, the 65th amino acid is isoleucine, while in PaFS it is phenylalanine. Figure 4 Therefore, a saturation mutation was performed on the amino acid at position 65 to investigate the effect of this site on the products in both enzymes.

[0102] Example 9: Protein Expression, Purification, and Functional Verification of Wild-Type and Mutant Plasmids

[0103] The protein expression plasmid of this invention was introduced into the protein expression strain for protein expression and purification. The relevant operations are as follows:

[0104] 1) E. coli protein expression strains containing the target gene were revived on LB plates containing the corresponding resistance.

[0105] 2) Inoculate the revived single clones into 10 mL of LB liquid medium containing the corresponding antibiotic, and incubate at 37°C and 220 rpm for 12 hours. Then transfer the bacterial culture to 1 L of LB liquid medium containing the corresponding antibiotic and incubate at 37°C and 220 rpm for about 6 hours until the OD value is 0.4-0.6. Add 1 mL of 0.4 M IPTG and incubate at 18°C ​​and 180 rpm for 16 hours.

[0106] 3) Centrifuge at 4 degrees Celsius and 10,000 rpm for 20 minutes to obtain bacterial precipitate.

[0107] 4) Add buffer A (50mM Tris-HCl, 250mM NaCl, 5mM MgCl2, 5mM Mimidazole, 10% glycerol, pH 8.4) to the bacterial cells to fully suspend the cells, and then use a high-pressure ultrasonic disruptor to break up the cells.

[0108] 5) After the cells are lysed, centrifuge at 4°C, 5800g, for 20 minutes to obtain the protein supernatant.

[0109] 6) After rinsing the nickel column with ultrapure water, equilibrate the column with buffer A. Add the protein supernatant to the nickel column and load the target protein onto the column at a low flow rate.

[0110] 7) Rinse with a washing buffer prepared in a 97:3 ratio (buffer A:buffer B:buffer B (50mM Tris-HCl, 250mM NaCl, 5mM MgCl2, 300mM Mimidazole, 10% glycerol, pH 8.4) to remove impurities. Then, rinse with buffer B to remove the target protein.

[0111] 9) Transfer the flow-through buffer containing the target protein into an ion-dip column, centrifuge at 4°C and 4000g for 30 minutes to concentrate the flow-through buffer, add 10 mL of buffer A to dilute the flow-through buffer, and centrifuge again under the same conditions for 30 minutes to remove imidazole from the flow-through buffer. The recombinant protein obtained after imidazole removal is analyzed for protein concentration using a UV spectrophotometer and its purity is checked using SDS-PAGE.

[0112] In vitro enzyme catalysis experiment:

[0113] Take 30 μL of in vitro reaction buffer (1M Tris-HCl, 100 mM MgCl2, 100 mM DTT) and add the target enzyme protein to make the final enzyme concentration 1 μg / μL; then add 3 μL of GGPP at a concentration of 2 mg / mL; finally, add dH2O to make the reaction volume 300 μL. The in vitro reaction was carried out overnight at 30℃. After the reaction, the product was extracted with ethyl acetate in three fractions of 300 μL each. After drying, the product was dissolved in 300 μL of chromatographic hexane and filtered through a membrane before being analyzed by GC-MS. The reaction results are shown in the appendix. Figure 5 and Figure 6 .

[0114] The GC-MS analysis conditions are as described in Example 3.

[0115] PaFS mutation results show that... Figure 5 Compared to the wild type, the F65I, F65V, F65C, F65L, F65M, F65N, F65E, F65T, F65S, and F65A mutants can produce compound 1. The F65Y mutation does not affect enzyme activity, while other mutations lead to enzyme inactivation or the production of unknown compounds. This indicates that when the F (phenylalanine) at position 65 in PaFS is mutated to I, V, C, L, M, N, E, T, S, or A, the enzyme function of PaFS is transformed into AfAS function.

[0116] The AfAS mutation results showed that... Figure 6 Compared to the wild type, the I65F, I65Y, and I65W mutations not only produce compound 1 but also compound 3 (fusicocca-2,10(14)-diene). The I65H, I65N, I65M, I65V, I65L, I65D, I65T, I65C, and I65A mutations produce compound 1 and other unidentified diterpenoid compounds. The I65R, I65Q, I65P, I65E, I65S, I65K, and I65G mutations inactivate the enzyme. This indicates that when the I (isoleucine) at position 65 in AfAS is mutated to phenylalanine, tryptophan, or tyrosine, the enzyme function of AfAS is transformed into PaFS function.

[0117] Example 10: Antibacterial activity test of compounds 1 and 2

[0118] This invention uses a two-fold dilution method to test the antifungal and antibacterial activities of compounds 1 and 2; the determination method is as follows:

[0119] Terpenoids possess a variety of biological activities, including antibacterial, anti-inflammatory, and antitumor effects. This study will use a two-fold dilution method to test the antibacterial activity of compounds 1-2 in 96-well plates for initial screening. Staphylococcus aureus (209P) and Escherichia coli (ATCC0111) were cultured in beef extract medium, while Candida albicans (FIM709) and Aspergillus niger (R330) were cultured in Sabouraud dextrose agar. After the strains grew, the bacterial cells were suspended in physiological saline and diluted to 10⁻¹⁰ using a cell counting chamber. 7 -10 9 The bacterial culture was tested at concentrations of 1 / mL. Tobramycin was used as the positive control for antibacterial activity, and itraconazole was used as the positive control for antifungal activity. Compounds 1-2 and the positive control were each prepared into 50 mg / mL solutions using DMSO. 100 μL of the bacterial culture and 200 mL of the corresponding culture medium were mixed, and 200 μL of this mixture was added to the first well of a 96-well plate along with 0.5 μL of either compound or positive control. The solutions were then sequentially diluted halfway, ranging from 128 to 0.25 mg / mL. The 96-well plates for the antibacterial test were incubated at 37°C for 1 day, and the 96-well plates for the antifungal test were incubated at 30°C for 2-3 days.

[0120] The results (Table 2) showed that compound 2 exhibited good anti-Staphylococcus aureus activity, with a minimum inhibitory concentration (MIC) of 1 μg / mL.

[0121] Table 2 Antibacterial activity of compounds 1-2

[0122]

[0123]

[0124] The structure, name, number, and NMR confirmation data of the compounds involved in the above embodiments are as follows:

[0125] Table 3. NMR data assignment for compound 1 (solvent: deuterated benzene, 150MHz CMR, 600MHz HMR).

[0126]

[0127]

[0128]

[0129] a Indistinguishable signals due to overlap or complex multiplicity were not specified in the report.

[0130] Table 4. NMR data for compound 2 (solvent: deuterated chloroform, 100MHz CMR, 400MHz HMR)

[0131]

[0132]

[0133]

[0134] a Indistinguishable signals due to overlap or complex multiplicity are not reported with multiplicity specified.

Claims

1. A diterpenoid compound, characterized in that, The compound contains a 6 / 5 / 5 / 5 tetracyclic skeleton, and its structural formula is shown in compound 2: 。 2. The method for preparing the diterpenoid compound according to claim 1, characterized in that, The method includes the following steps: a) The AfAS polypeptide with the amino acid sequence shown in SEQ ID NO:1 is responsible for generating a 6 / 5 / 5 / 5 tetracyclic skeleton to obtain compound 1, which is shown below: ； b) The AfP450 polypeptide with the amino acid sequence shown in SEQ ID NO:2 catalyzes intermediate compound 1 to generate compound 2; Compound 2 and its intermediate compound 1 are synthesized using the AfAS polypeptide and AfP450 polypeptide in chassis strain Aspergillus oryzae cells, Saccharomyces cerevisiae cells or Escherichia coli cells.

3. The preparation method according to claim 2, characterized in that, The key catalytic site for the AfAS polypeptide to catalyze the formation of compound 1 is the 65th amino acid of AfAS, which is selected from isoleucine, leucine, valine, histidine, threonine, cysteine, methionine, asparagine, aspartic acid, or phenylalanine.

4. The preparation method according to claim 2, characterized in that, The method is as follows: 1) with Aspergillus fumigatiaffinis Using CNM-CM8980 genomic DNA as a template, the encoding genes of AfAS and AfP450 were amplified by PCR; then, the target genes were ligated into the Aspergillus oryzae expression plasmid pTAex3 or pUSA, respectively, to construct the recombinant expression plasmid pTAex3- AfAS and pUSA- AfP450 ; 2) The expression vector pTAex3- AfAS Transformation to Aspergillus oryzae A.oryzae From the protoplasts of NSAR1, genetically engineered bacteria AO- were obtained. AfAS The expression vector pTAex3- AfAS With pUSA- AfP450 Co-transformation to Aspergillus oryzae A.oryzae From the protoplasts of NSAR1, genetically engineered bacteria AO- were obtained. AfAS-AfP450 ; The genetically engineered bacteria produce compounds 1 and 2 through liquid fermentation or solid fermentation.

5. The preparation method according to claim 4, characterized in that, The method is as follows: 1) Inoculate the genetically engineered bacteria into liquid culture medium and culture it in a shaker at 28-30 ℃ and 180-220 rpm for 1-3 days to obtain the seed culture of the strain; 2) Take the seed culture of the strain obtained in step 1) and inoculate it into the liquid fermentation medium. Culture it in a shaker at 28~30 ℃ and 180~220 rpm for 5-8 days to obtain the fermentation broth of the strain. 3) Take the fermentation broth obtained in step 2), filter to obtain mycelium, extract with methanol, sonicate for 30 min, soak overnight, filter to obtain mycelium extract, concentrate under reduced pressure to obtain extract. 4) The extract was separated and enriched by silica gel column chromatography, and then monomeric compounds 1 and 2 were prepared by high performance liquid chromatography.

6. The method as described in claim 5, characterized in that, In step 1), the liquid culture medium is DPY medium, which consists of: 2% dextrin, 1% polypeptone, 0.5% yeast extract, 0.05% MgSO4·7H2O, and 0.5% KH2PO4; in step 2), the culture medium is CD-starch medium, which consists of: 0.3% NaNO3, 0.2% KCl, 0.05% MgSO4·7H2O, 0.1% KH2PO4, 0.002% FeSO4·7H2O, 1% polypeptone, 2% starch, and a pH of 5.

5.

7. The use of the compound according to claim 1 in the preparation of antibacterial drugs, wherein the bacterium is Staphylococcus aureus. 。

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