A class of clostridin-type diterpenoids, biosynthetic method and application thereof

Abstract

The application belongs to the field of genetic engineering and biosynthesis, and particularly relates to biosynthesis of a class of diterpenoids. The application provides genes having a key role in the formation of a 5-8-5 tricyclic mother nucleus and a five-membered heterocyclic ring in biosynthesis of the class of diterpenoids, which are respectively named as PrcA and PrcB and polypeptides encoded by the genes, expression of PrcA and PrcB is carried out through a heterologous expression system of Aspergillus oryzae, and five diterpenoids with novel structures and anti-metabolic related fatty liver disease activity are obtained. The application provides application of the polypeptides in biosynthesis of the class of diterpenoids, and enriches a diterpenoid library, thereby providing lead compound resources for discovering new anti-metabolic related fatty liver disease medicinal raw materials.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and biosynthesis, and specifically relates to a class of clostridium diterpenoid compounds, their biosynthetic methods, and applications. Background Technology

[0002] Fusicoccin-type diterpenes possess a unique 5-8-5 tricyclic skeleton, exhibiting complex and diverse structures and a variety of significant biological activities, making them an important natural resource for screening drug lead compounds. For example, Fusicoccin A not only exhibits strong phytotoxicity but also demonstrates good hypoglycemic and antitumor activities; Periconicin A–B shows significant antibacterial effects against both Gram-positive and Gram-negative bacteria; and Dongtingnoid E can significantly promote seed germination. Therefore, exploring novel fusicoccin-type diterpenes has significant theoretical research and practical application value.

[0003] In recent years, with the rapid development of gene sequencing technology, a large number of fungal genomes have been analyzed. Bioinformatics analysis shows that the number of secondary metabolism gene clusters in fungal genomes is far higher than the number of currently discovered natural products, suggesting that fungal genomes still contain a large amount of untapped natural product resources that need further exploration to obtain compounds with new structures and activities. Therefore, screening for clostridium diterpenoid biosynthetic gene clusters in fungi using gene mining technology and conducting heterologous expression studies is expected to yield more structurally novel clostridium diterpenoid bioactive molecules. Summary of the Invention

[0004] The purpose of this invention is to provide a class of clostridium diterpenoid compounds, their biosynthetic methods, and applications. All of these clostridium diterpenoid compounds contain a 5-8-5 tricyclic skeleton, with some compounds containing a five-membered heterocycle, and their structural formulas are shown in Figures 1-5. .

[0005] Another aspect of the present invention relates to the use of the above-mentioned compounds in the preparation of medicaments for the prevention and / or treatment of metabolic syndrome, metabolic fatty liver disease, liver injury, and liver fibrosis and cirrhosis caused by these liver diseases.

[0006] The PrcA provided in this invention is a diterpenoid cyclase, and the PrcB is a cytochrome P450 enzyme, both derived from fungi of the genus *Nematocystis*. periconia sp. No. 19-4-2-1. The gene encoding terpene cyclase PrcA consists of 1203 nucleotide bases, and the gene encoding cytochrome P450 enzyme PrcB consists of 1719 nucleotide bases. Their gene sequences are shown in SEQ ID NO: 3-4. PrcA The protein encoded by the gene contains 382 amino acid residues. PrcB The protein encoded by the gene contains 515 amino acid residues, and its amino acid sequence is shown in SEQ ID NO.1-2.

[0007] In one specific implementation scheme, PrcA Gene expression in Aspergillus oryzae yields 1, PrcA and PrcB The gene is simultaneously expressed in Aspergillus oryzae, and 1 can be consumed to obtain 2~5.

[0008] A first aspect of the present invention provides an isolated polypeptide comprising: (a) The polypeptide sequence shown in SEQ ID NO: 1, or a polypeptide sequence having at least 70% sequence identity with it, preferably having 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity; or A polypeptide sequence encoded by the nucleic acid sequence shown in SEQ ID NO: 3 or a nucleic acid sequence having at least 70% sequence identity with it, preferably having 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity; and / or (b) The polypeptide sequence shown in SEQ ID NO: 2, or a polypeptide sequence having at least 70% sequence identity with it, preferably having 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity; or A polypeptide sequence encoded by a nucleic acid sequence shown in SEQ ID NO: 4 or a nucleic acid sequence having at least 70% sequence identity with it, preferably having 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity.

[0009] A second aspect of the invention provides an expression vector comprising an isolated polynucleotide encoding a polypeptide of the first aspect. In a preferred embodiment, the polynucleotide comprises: (i) The nucleotide sequence shown in SEQ ID NO: 3 or a nucleic acid sequence having at least 70% sequence identity with it, preferably having 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity; and / or (ii) The nucleotide sequence shown in SEQ ID NO: 4 or a nucleic acid sequence having at least 70% sequence identity with it, preferably having 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity; and / or A third aspect of the invention provides an expression cassette comprising a polynucleotide according to a second aspect of the invention.

[0010] A fourth aspect of the invention provides a vector, such as an expression vector, comprising the polynucleotide described in the second aspect of the invention or the expression cassette described in the third aspect of the invention.

[0011] A fifth aspect of the present invention provides a cell comprising the polynucleotide described in the second aspect of the present invention, the expression cassette described in the third aspect of the present invention, or the vector described in the fourth aspect of the present invention.

[0012] In one embodiment, the cell is a fungal cell.

[0013] In a preferred embodiment, the cells are periconia Fungi, for example periconia In another preferred embodiment, sp. No. 19-4-2-1, the cells are Aspergillus oryzae cells.

[0014] The sixth aspect of the present invention provides the use of the polypeptides described in the first aspect, the polynucleotides described in the second aspect, the expression cassettes described in the third aspect, the vectors described in the fourth aspect, or the cells described in the fifth aspect in the synthesis of clostridium compounds.

[0015] A seventh aspect of the present invention provides a kit comprising the polypeptide described in the first aspect of the present invention, the polynucleotide described in the second aspect, the expression cassette described in the third aspect, the vector described in the fourth aspect, or the cell described in the fifth aspect.

[0016] Technical effects of the present invention: The present invention from periconia The genes for synthesizing clostridin-type diterpenoid compounds 1-5 were discovered in sp. No. 19-4-2-1. PrcA and PrcB .Will PrcA Gene expression in Aspergillus oryzae yields 1, PrcA and PrcB The gene is simultaneously expressed in Aspergillus oryzae, consuming 1 unit to obtain 2-5 units, which has advantages such as simple process, high stereoselectivity, and low environmental pollution. This type of clostridium diterpenoid compound has a good protective effect against free fatty acid-induced hepatocellular damage and can be used for the prevention and treatment of metabolic syndrome, metabolic fatty liver disease, liver injury, and liver fibrosis and cirrhosis caused by these liver diseases. Attached Figure Description

[0017] Figure 1 This invention demonstrates the identification of [the source] from [the present invention]. Periconia The biosynthetic gene cluster of clostridium diterpenoids in sp. No. 19-4-2-1 and its heterologous expression products. Among them, Figure 1 A shows from PericoniaBiosynthetic gene cluster of clostridial diterpenoids sp. No. 19-4-2-1; Figure 1 B shows the introduction of Aspergillus oryzae strain (AO) into the culture medium. PrcA Genes and PrcA and PrcB HPLC chromatogram of Aspergillus oryzae extract; Figure 1 C displays the import. PrcA Genes and PrcA and PrcB The structure obtained after gene sequencing from Aspergillus oryzae strain.

[0018] Figure 2 The study demonstrated that clostridin-type diterpenoid compounds 1–5 improved hepatocyte survival and reduced lipid accumulation in a FAA-induced hepatocyte injury model. Detailed Implementation

[0019] The invention can be further understood through embodiments; however, it should 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.

[0020] Example 1: Obtaining candidate genes Periconia sp. was activated on potato agar (PDA) medium. After two days of culture, a small amount of mycelium was inoculated into potato liquid (PDB) medium and cultured at 28 °C with shaking at 220 rpm for 3 days. The mycelium was collected by filtration, ground in liquid nitrogen, and total DNA was extracted using the phenol-chloroform method. Sequencing was performed using the Illumina HiSeq 2500 sequencing platform. Sequence analysis was performed using the SOAPdenovo software (version 2.04, http: / / soap.genomics.org.cn / soapdenovo.html). A total of 12,573 contigs were obtained, with a total length of approximately 36.97 Mb. Gene prediction was performed using the AUGUSTUS software (http: / / bioinf.uni-greifswald.de / augustus / ).

[0021] Genes related to the biosynthesis of clostridium diterpenoids were identified using AUGUSTUS software gene function prediction. Figure 1 A). PrcA (The polypeptide sequence is shown in SEQ ID NO: 1, and the nucleic acid sequence is shown in SEQ ID NO: 3.) PrcB (The polypeptide sequence is shown in SEQ ID NO: 2, and the nucleic acid sequence is shown in SEQ ID NO: 4) is used as the target gene for the study.

[0022] SEQ ID NO: 1 >PrcA MEYNYSTVVDPSSYDLEGLCDGIPLRIHKYADLEDIGAIRAQEDWRKYVSPFEEYRGGMGPRYSFMSVSVPECIPERLEIISYANEFAFLHDDITDISSQDIIDKENLEMKDAFTKVARSGRAKLEKFGKKRIQGQILLEMMVLDRERALTTAKSWAKFVEIASGKEHHKNFATLDEYLPYRSHDVGQMFWHGMVTFGMGLTIPEHEMPLCEKLMLPAWQAASLQNDLFSYDKEYRDAVKHGQPDVVNAVWVIMKEHNMNVDQAKQLCRTKIKEVVAQYLEVVERVRKDESISLDLRKYVEAMQYSLSGNVAWSLECPRYHAKADYNDLQLKRMRHGVKKYPSPIQFTSRRSSILARAATIAFLFSLLIVAYTFRGYFLRGY SEQ ID NO: 2 >PrcB MALGGSHVLEVVSEHYLAVPAALFAAYLFSLVTYRLFFSPLAKFPGPKIAAATGYYEFYHDYFRKGQYVKVIRSLHDKYGPIIRINPNELSINDAFYNSVYVAGSVRKTNYAHFARGIEFDGSHFLSTDHDLHRRRRKPLEPFFSRLGVTKLEPMVFNIAKRLSERLESYRGTDQIIRLDQAYVALAGDVIGGICCDNSSDLVSQEDFGADWYTILHDFIHSIPLVMAFPQLISIARMIPDSVMRWLDPRLKTFD KFQRLALQHILDAKREKESNSKPSLQNQNSIFRHILSSDLPESDLSPQRLSREAQVLLGAGTVSSARTMDFLTFYVLNNPEYLKKVQEELAPVMQGYPEKIPSWAELEKLPFIQALLKETLRLSYGVMH RLPRVSPDLPIQYGKWTIPPGVPVGMSAYMMHSDPQVYPEPFKFKPERWMNNVTPAMNRSLVPFSKGSRNCLGMNAYLELNMTLAVIFRPGGPKIQLFETDESDIVQEHDYLIPLPKLSSKGRIKVV SEQ ID NO: 3 > PrcA SEQ ID NO: 4 > PrcB Example 2: Construction of a heterologous gene expression vector pTAex3- PrcA Plasmid construction: Periconia Using sp. genomic DNA as a template, primers Inf - pT - PrcA -F(TCGAGCTCGGTACCCCATAATGGAGTACAATTATTCTACCG) / Inf - pT -PrcA -R (CTACTACAGATCCCCCTAGTAGCCCCTGAGAAAATAAC) for terpene cyclase gene PrcA PCR amplification was performed, and the PCR reaction system was subjected to agarose gel electrophoresis. After purification using a gel extraction kit, the PCR reaction was recovered. PrcA The target fragment will then be processed using an In-fusion kit. PrcA Fragment integration Small The enzyme-digested linear vector pTAex3 was then transformed into E. coli. E.coli In DH5α, positive clones were screened using ampicillin, and the positive clones were selected for liquid fermentation. Plasmids were extracted and sequenced for verification, and finally the correct expression vector pTAex3-PrcA and its Escherichia coli host were obtained.

[0023] pUSA- PrcB Plasmid construction: Periconia Using sp. genomic DNA as a template, primers Inf - pU - PrcB -F(TCGAGCTCGGTACCCATGGCTTTAGGAGGCTTCCA) / Inf - pU -PrcB -R (CTACTACAGATCCCCTCATACCACCTTGATACGAA) pair PrcB Perform PCR amplification using the same method described above. PrcB Fragment integration Small The expression vector pUSA-PrcB and its Escherichia coli host were finally obtained from the linear vector pUSA after enzyme digestion.

[0024] Example 3: Construction of Aspergillus oryzae expression strain (1) In the construction of Aspergillus oryzae transfected strains, the expression plasmids containing different genes were transfected into Aspergillus oryzae using the PEG-mediated protoplast transformation method. The specific operation method is as follows: 1) The above-mentioned Example 2 contains pTAex3- PrcAor pUSA- PrcB E. coli host cells were inoculated 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. 2) From A. oryzae Pick an appropriate amount of mycelium from an 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, bring the volume to 1L). Incubate at 28℃ with shaking at 200 rpm for 1-2 days. 3) Add 10 mL of the above culture medium to 100 mL of DPY medium, mix well, and incubate at 28 ℃ with shaking at 180 rpm for 1 day; 4) Prepare 10 mL TF solution 1: Weigh 0.79 g (NH4)2SO4 and 0.1 g Yatalase lyase into a 15 mL centrifuge tube, add 0~10 mL of TF solution, dissolve by inverting, and filter into a 50 mL centrifuge tube using a 0.22 μm microporous membrane. 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 ℃ and 70 rpm in a constant temperature incubator for 3 h 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. 6) Add an equal volume of TF solution 2, invert and mix well, centrifuge at 4 ℃, 1500 rpm for 10 min, 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 4 ℃, 1500 rpm for 10 min, 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; 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. 8) After standing, add 5 mL of TF solution 2 to a 15 mL centrifuge tube, invert to mix, centrifuge at 4 ℃ and 1500 rpm for 10 min, 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. 9) After the above culture medium plates are dried, wrap them with parafilm, invert them in a 28 ℃ incubator and culture for 3-7 days. Pick out the transformed plants and inoculate them onto M stable medium for stable passage 1-3 times.

[0025] 10) To identify the function of the terpene cyclase PrcA in the diterpene gene cluster, the expression plasmid pTAex3- was transfected according to the transfection method described above. PrcA Transfection into Aspergillus oryzae yields a product containing... PrcA Gene-transfected strain AO- PrcA ; 11) To identify the function of the P450 enzyme PrcB in the diterpenoid gene cluster, this invention uses the expression plasmid pTAex3- PrcA and pUSA- PrcB Transfected into Aspergillus oryzae, resulting in transfected strain AO- PrcA-PrcB ; (2) The AO- strain PrcA or AO- PrcA-PrcB The seed culture was inoculated into 5 mL of DPY medium and cultured at 28 °C with shaking at 220 rpm for 2 days. Then, the seed culture was inoculated into a 500 mL culture flask containing 100 mL of CD-Starch medium and cultured at 28 °C with shaking at 200 rpm for 3 days. After fermentation, the culture was filtered to separate the mycelia and bacterial culture. The bacterial cells were soaked in 100 mL of ethanol for 1 day, concentrated under reduced pressure, dissolved in 2 mL of methanol, centrifuged at high speed, and the supernatant was used for HPLC analysis. The HPLC results are shown in the figure. Figure 1 B, the products generated are shown in [the diagram]. Figure 1 C. Compared to AO strains, AO- PrcA Strain generation 1; AO- PrcA - PrcBIn this strain, the peak area representing compound 1 decreased, and new peaks representing compounds 2-5 were generated. That is, compared to the AO-PrcA strain, the AO- PrcA-PrcB 1 of the strain is consumed, and 2~5 are produced.

[0026] The HPLC conditions are as follows: Instruments: Dionex UltiMate 3000 equipped with UltiMate 3000 Diode Array Detector (DIONEX, USA) and Amazon SL ion trap electrospray mass spectrometer (BRUKER, Germany), Alltech (Grace) 2000ES evaporative light scattering detector (ELSD) (Alltech Co., Ltd., Portland, USA). Liquid chromatography column: COSMOSIL-Pack C18 column (5 μm, 4.6 × 250 mm) Mobile phase A: Water (0.1% formic acid); Mobile phase B: Acetonitrile (0.1% formic acid) Gradient setting: 0 30 min 50%-100% B, 30 50 min 100% B, 50 60 min 50% B Flow rate: 1 mL / min Example 4: Isolation, purification, and structural identification of compounds The methods and results of compound analysis and purification involved in Example 3 above are summarized as follows: Example 4.1 Compound Isolation and Purification Isolation of Compound 1: AO- was cultured using 10 L of CD-starch fermentation. PrcAAspergillus oryzae was extracted 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 5 L 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 extracted twice more with ethanol, and the extracts were combined. The extract was concentrated using a rotary evaporator. Appropriate amounts of ethyl acetate and deionized water were added to the rotary flasks to dissolve the concentrated sample, which was then transferred to a separatory funnel. After three extractions, the ethyl acetate layer was concentrated using a rotary evaporator to obtain 1.3 g of crude extract. The extract was subjected to silica gel column chromatography with cyclohexane and ethyl acetate as the mobile phase, using a gradient separation (1:0, 50:1, 10:1, 5:1, 1:1, 0:1). The 1:0 fraction weighed 121.5 mg and was purified by HPLC [YMC-pack ODS-A (5 μm, 10 nm, 250 mm), 95% acetonitrile-water, 3 mL / min] to obtain compound 1 (20.0 mg).

[0027] Isolation of compound 2: AO- fermentation culture in 20 L CD-starch. PrcA - PrcB Aspergillus oryzae was extracted 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 5 L 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 extracted twice more with ethanol, and the extracts were combined. The extract was concentrated using a rotary evaporator. Appropriate amounts of ethyl acetate and deionized water were added to the rotary flasks to dissolve the concentrated sample, which was then transferred to a separatory funnel. After repeated extraction three times, the ethyl acetate layer was concentrated using a rotary evaporator to obtain 3.15 g of crude extract. The extract was subjected to silica gel column chromatography with cyclohexane and ethyl acetate as the mobile phase, using a gradient separation (1:0, 50:1, 10:1, 5:1, 1:1, 0:1). The 50:1 fraction weighed 851.3 mg and was purified by HPLC [YMC-pack ODS-A (5 μm, 10 nm, 250 mm), 87% acetonitrile-water, 3 mL / min] to obtain compound 2 (7.1 mg).

[0028] Compound 3 separation: the above AO- PrcA - PrcB The extract fraction 5:1 weighed 247.2 mg and was purified by HPLC [YMC-packODS-A (5 μm, 10 nm, 250 mm), 83% acetonitrile-water, 3 mL / min] to give compound 3 (5.3 mg).

[0029] Compound 4 separation: The above AO- PrcA - PrcBThe extract fraction, 1:1 by weight 228.4 mg, was purified by HPLC [YMC-packODS-A (5 μm, 10 nm, 250 mm), 80% acetonitrile-water, 3 mL / min] to give compound 4 (5.6 mg).

[0030] Compound 5 separation: the above AO- PrcA - PrcB The extract fraction, 1:1 by weight 228.4 mg, was purified by HPLC [YMC-packODS-A (5 μm, 10 nm, 250 mm), 80% acetonitrile-water, 3 mL / min] to give compound 5 (1.3 mg).

[0031] Example 4.2 Structure, name, number, and NMR confirmation data of the compounds involved in the examples. NMR data assignment for compound 1 (solvent: deuterated chloroform, 100 MHz carbon spectrum, 400 MHz proton spectrum) a Indistinguishable signals due to overlap or complex multiplicity were not specified in the report. NMR data assignment for compound 2 (solvent: deuterated chloroform, 100 MHz carbon spectrum, 400 MHz proton spectrum) a Indistinguishable signals due to overlap or complex multiplicity were not specified in the report. NMR data assignment for compound 3 (solvent: deuterated dimethyl sulfoxide, 100 MHz carbon spectrum, 400 MHz proton spectrum) a Indistinguishable signals due to overlap or complex multiplicity were not specified in the report. NMR data assignment for compound 4 (solvent: dimethyl sulfoxide, 100 MHz carbon spectrum, 400 MHz proton spectrum) a Indistinguishable signals due to overlap or complex multiplicity were not specified in the report. NMR data assignment for compound 5 (solvent: dimethyl sulfoxide, 100 MHz carbon spectrum, 400 MHz proton spectrum) a Indistinguishable signals due to overlap or complex multiplicity were not specified in the report. Example 5: Free fatty acid (FFA)-induced hepatocyte injury model and activity test The models are as follows: Preparation of 20 mM palmitic acid (PA) stock solution: Weigh 0.02 g PA and dissolve in 3.9 mL of anhydrous ethanol. After dissolving by heating in a metal bath at 65 ℃, immediately add 150 μL to 1.35 mL of 40% BSA. Heat at 65 ℃ for a short time until clear and transparent. Filter through a 0.22 μM sterile filter membrane. Prepare and use immediately. Preparation of 40% bovine serum albumin (BSA): Weigh 4 g BSA and dissolve in 10 mL of phosphate buffered saline (PBS). Preparation of 20 mM oleic acid (OA) stock solution: Dissolve 127 μL OA in 10 mL of 0.2 M sodium hydroxide. After dissolving by heating in a metal bath at 75 ℃ for 30 minutes, quickly add 40% BSA and shake to mix. Then place in a water bath at 55 ℃ for 30 minutes until the solution is clear. Preparation of 20 mM FFA stock solution: Mix 20 mM PA stock solution and 20 mM OA stock solution in a 2:1 ratio, filter through a 0.22 μM sterile filter membrane, and use immediately after preparation.

[0032] AML-12 cells were seeded into 96-well plates at densities of 8 × 10³ cells per well and 24-well plates at densities of 12 × 10⁴ cells per well. After 24 h of adherent growth, the densities reached 30% in the 96-well plates and 60% in the 24-well plates. The original culture medium was discarded, and the cells were starved in serum-free medium for 12 h to synchronize cell growth. Then, the original culture medium was discarded, and both the model group and the model + drug group were treated with 300 μM FFA-BSA. The drug treatment group was treated with the corresponding concentration of the test drug, while the control group was treated with an equal volume of BSA-PBS. After 24 h of culture, CCK-8 cell viability assays and Oil Red O staining assays were performed.

[0033] Cell viability assay: After culturing cells in 96-well plates for the required time, the original culture medium was discarded, and 100 μL of 10% CCK-8 reagent was added. The plates were then incubated at 37 ℃ in the dark for 2.5 hours. The absorbance was then measured at 450 nm using a microplate reader. Cell viability was calculated as: (absorbance of treatment group / absorbance of control group) × 100%.

[0034] Cellular lipid accumulation detection: Weigh 0.5 g of Oil Red O solid in the dark, sonicate to dissolve in 100 mL of isopropanol, and then filter twice through a 0.22 μm microporous membrane to obtain the Oil Red O stock solution, which was stored at 4 ℃. The Oil Red O working solution was obtained by mixing the Oil Red O stock solution and ultrapure water in a 3:2 ratio, and filtering twice through a 0.22 μm microporous membrane. After culturing cells in 24-well plates for the appropriate time, discard the original culture medium and wash twice with PBS. Then, fix the cells with 4% paraformaldehyde in the dark for 30 minutes, rinse twice with 60% isopropanol (5 minutes each time), and then stain with Oil Red O working solution in the dark for 30 minutes. After staining, wash the 24-well plates with PBS until no excess red is left, and then take pictures using a 20× inverted microscope. After taking pictures, discard the PBS in the 24-well plates, add 250 μL of isopropanol to each well, and gently pipette. 200 μL was taken from each well and placed in a 96-well plate. The absorbance was measured at 560 nm using a microplate reader. The lipid accumulation rate was calculated as: (absorbance of the treatment group / absorbance of the control group) × 100%.

[0035] The results of cell viability and lipid accumulation assays showed that, at a concentration of 1 μM, compounds 1–5 significantly improved the decrease in hepatocyte viability and increase in lipid accumulation induced by FFA, demonstrating varying degrees of hepatoprotective activity. Figure 2 It is worth mentioning that compounds 3-5, with a 5-5-8-5 ring structure containing a five-membered oxygen heterocycle, were more effective than compounds 1-2 in improving cell viability; compounds 3-5 were also more effective than compounds 1-2 in improving lipid accumulation. Using Oil Red staining as a ratio to cell viability, compounds 3-5 were more effective than compounds 1-2 and comparable to resmetirom. This indicates that clostridial diterpenoid compounds 1-5, especially compounds 3-5, have a good protective effect against FFA-induced hepatocellular damage and can be used for the prevention and treatment of metabolic syndrome, metabolic fatty liver disease, liver injury, and liver fibrosis and cirrhosis caused by these liver diseases.

Claims

1. A diterpenoid compound of the clostridial type, characterized in that, All of the compounds contain a 5-8-5 tricyclic skeleton, and some of the compounds contain a five-membered heterocycle, the structural formulas of which are shown in Figures 1-5: 。 2. A polypeptide used to synthesize the compound of claim 1, characterized in that, The polypeptide is selected from: a) the PrcA polypeptide containing the amino acid sequence shown in SEQ ID NO: 1, and b) the PrcB polypeptide containing the amino acid sequence shown in SEQ ID NO:

2.

3. An isolated polynucleotide, characterized in that, The polynucleotide encodes the polypeptide of claim 2.

4. The isolated polynucleotide according to claim 3, characterized in that, The polynucleotide is: (i) The nucleotide sequence shown in SEQ ID NO: 3; or (ii) The nucleotide sequence shown in SEQ ID NO:

4.

5. A carrier, characterized in that, The vector comprises the polynucleotide as described in claim 3 or 4.

6. A host cell, characterized in that, The cell comprises the carrier of claim 5; the cell is a fungal cell or a bacterial cell; preferably, the fungal cell is selected from... Periconia It belongs to the fungal and Aspergillus oryzae cell family; the bacterial cell is Escherichia coli cell.

7. A method for synthesizing a class of diterpenes, comprising contacting the polypeptide PrcA of claim 2 with the compound geranylgeranyl pyrophosphate, or the polypeptide PrcA and PrcB in combination with the compound geranylgeranyl pyrophosphate; wherein the structure of the compound geranylgeranyl pyrophosphate is shown in the following formula: 。 8. Use of the polypeptide of claim 2, the polynucleotide of claim 3 or 4, the carrier of claim 5, and / or the host cell of claim 6 in the synthesis of this type of diterpene.

9. A reagent kit, characterized in that, It comprises the polypeptide of claim 2, or the polynucleotide of claim 3 or 4, or the vector of claim 5, or the host cell of claim 6.

10. The use of the compound of claim 1 in the preparation of a medicament for the prevention and / or treatment of metabolic syndrome, metabolic fatty liver disease, liver injury, and liver fibrosis and cirrhosis caused by these liver diseases.

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