An anti-inflammatory compound, its biosynthetic gene cluster, synthesis method and application

Abstract

The present invention discloses an anti-inflammatory compound, its biosynthetic gene cluster, synthesis method and application, belonging to the technical field of genetic engineering. An anti-inflammatory compound is provided, and the chemical molecular formula of the anti-inflammatory compound is C<subgt;15< / subgt;H<subgt;22< / subgt;O<subgt;5< / subgt>, and the structural formula is shown in Formula I, named: phaseolinone B. The present invention first discloses the biosynthetic pathway of the anti-inflammatory compound phaseolinone B, laying an important foundation for realizing its green and efficient synthesis; this compound exhibits significant anti-inflammatory activity in the zebrafish Tg 3dpf tail amputation infection model, providing resources for discovering anti-inflammatory drug lead compounds.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and in particular relates to an anti-inflammatory compound and its biosynthetic gene cluster, synthesis method and application. Background Art

[0002] Inflammation is a protective response of the body that clears external stimuli and maintains homeostasis during infection or injury. It is characterized by redness, swelling, heat, pain, and functional impairment. Short-term inflammation helps clear inflammatory substances and promote recovery, making it a key step in the body's self-repair. However, excessive or chronic inflammation can lead to various diseases, such as cardiovascular dysfunction, metabolic disorders, autoimmune diseases, rheumatoid arthritis, cancer, and sepsis, posing a serious threat to human health. Currently, the main drugs used to treat inflammatory diseases include steroidal and nonsteroidal anti-inflammatory drugs (NSAIDs). These drugs have varying degrees of side effects, such as NSAIDs causing alterations in the hematopoietic system, cardiovascular damage, and asthma, posing a significant threat to human health. Therefore, the search for safer and more effective anti-inflammatory drugs from natural products is of great significance for safeguarding human life and health.

[0003] Fungal metabolites are abundant and represent an important natural resource for the development of modern pharmaceutical formulations. Nature is rich in fungal resources, and fungi possess a vast gene pool, potentially allowing for the synthesis of novel and highly active compounds. This makes them an important avenue for developing naturally occurring active pharmaceutical ingredients. Summary of the Invention

[0004] To solve the above technical problems, the present invention proposes an anti-inflammatory compound and its biosynthetic gene cluster, synthesis method and application. The present invention discloses for the first time the biosynthetic pathway of the anti-inflammatory compound farslione B, laying an important foundation for its green and efficient synthesis. The compound exhibits significant anti-inflammatory activity in the zebrafish Tg 3dpf tail-breaking infection model, providing resources for the discovery of lead compounds for anti-inflammatory drugs.

[0005] To achieve the above object, the present invention provides an anti-inflammatory compound, the chemical formula of which is C 15 H 22 O5, the structural formula of which is shown in Formula I, is named: phaseolinone B;

[0006]

[0007] Preferably, the anti-inflammatory compound is synthesized by expressing the biosynthetic gene cluster in Penicillium janthinellum HDN13-494 using Aspergillus nidulans as a host.

[0008] The present invention also provides a biosynthetic gene cluster for synthesizing the anti-inflammatory compound, which is derived from Penicillium purpurogenum HDN13-494 and includes an aristolochene sesquiterpene synthase gene JanA, a short-chain dehydrogenase gene JanC, and three P450 enzyme genes JanB, JanD, and JanE; the nucleotide sequence of the aristolochene sesquiterpene synthase gene JanA is shown in SEQ ID NO.1, the nucleotide sequence of the short-chain dehydrogenase gene JanC is shown in SEQ ID NO.3, the nucleotide sequence of the P450 enzyme gene JanB is shown in SEQ ID NO.2, the nucleotide sequence of the P450 enzyme gene JanD is shown in SEQ ID NO.4, and the nucleotide sequence of the P450 enzyme gene JanE is shown in SEQ ID NO.5.

[0009] The present invention also provides a method for synthesizing the anti-inflammatory compound, comprising the following steps:

[0010] (1) Construction of heterologous gene expression vector;

[0011] (2) constructing an Aspergillus nidulans heterologous expression strain using the gene heterologous expression vector obtained in step (1);

[0012] (3) culturing the Aspergillus nidulans heterologous expression strain obtained in step (2) and fermenting for 5 days to obtain a fermentation product;

[0013] (4) The fermentation product obtained in step (3) was immersed in ethyl acetate overnight, sonicated, and concentrated under reduced pressure to obtain an extract extract, which was separated and enriched by ODS silica gel column chromatography; and then purified by semi-preparative column to obtain farslione B.

[0014] Preferably, the construction of the heterologous gene expression vector in step (1) is specifically as follows:

[0015] Using the genomic DNA of Penicillium micropurpurogenum HDN13-494 as a template, PCR amplified the target genes janA, janB, janC, janD and janE; the target genes janA, janB, janC, janD and janE were respectively connected to the Aspergillus nidulans expression plasmids pYTU, pYTR and pYTP to construct recombinant expression plasmids PYTU-janABC, PYTR and PYTP-DE;

[0016] The primers for amplifying the target gene janA are PYTU-janA-F and PYTU-janA-R, the nucleotide sequence of PYTU-janA-F is shown in SEQ ID NO.6, and the nucleotide sequence of PYTU-janA-R is shown in SEQ ID NO.7;

[0017] The primers for amplifying the target gene janB are PYTU-janB-F and PYTU-janB-R. The nucleotide sequence of PYTU-janB-F is as shown in SEQ ID NO.8, and the nucleotide sequence of PYTU-janB-R is as shown in SEQ ID NO.9;

[0018] The primers for amplifying the target gene janC are PYTU-janC-F and PYTU-janC-R. The nucleotide sequence of PYTU-janC-F is as shown in SEQ ID NO.10, and the nucleotide sequence of PYTU-janC-R is as shown in SEQ ID NO.11;

[0019] The primers for amplifying the target gene janD are PYTU-janD-F and PYTU-janD-R. The nucleotide sequence of PYTU-janD-F is as shown in SEQ ID NO.12, and the nucleotide sequence of PYTU-janD-R is as shown in SEQ ID NO.13;

[0020] The primers for amplifying the target gene janE are PYTU-janE-F and PYTU-janE-R. The nucleotide sequence of PYTU-janE-F is as shown in SEQ ID NO.14, and the nucleotide sequence of PYTU-janE-R is as shown in SEQ ID NO.15.

[0021] Preferably, the construction of the Aspergillus nidulans heterologous expression strain using the gene heterologous expression vector obtained in step (2) is specifically as follows: By PEG transformation, the recombinant expression plasmids PYTU-janABC, PYTR, and PYTP-DE are transformed into the protoplasts of Aspergillus nidulans to obtain the transformant AN-janABCDE.

[0022] Preferably, the cultivation of the Aspergillus nidulans heterologous expression strain obtained in step (3) for 5 days to obtain the fermentation product is specifically as follows: The transformant AN-janABCDE is inoculated into a screening medium for cultivation, and then fermented in a fermentation medium for 5 days to obtain the fermentation product.

[0023] More preferably, the screening medium is the CD medium, and the formulation of the CD medium is: 1 g of anhydrous glucose, 5 mL of 20x nitrate, 100 μL of trace elements, and made up to 100 mL with sterile enzyme-free water.

[0024] More preferably, the fermentation medium is the CD-ST medium, and the formulation of the CD-ST medium is: 2 g of soluble starch, 2 g of acid-hydrolyzed casein, 5 mL of 20x nitrate, 100 μL of trace elements, and made up to 100 mL with sterile enzyme-free water.

[0025] The present invention also provides the use of the anti-inflammatory compound in the preparation of anti-inflammatory drugs.

[0026] Compared with the prior art, the present invention has the following advantages and technical effects:

[0027] In the present invention, Aspergillus nidulans is used as the dominant expression host, and the biosynthesis gene cluster of farnesyl ketone B is heterologously reconstructed in its body. The sesquiterpene compound farnesyl ketone B is isolated and identified, and it has good anti-inflammatory biological activity. In addition, the present invention discloses for the first time the biosynthetic pathway of this type of compound, laying an important foundation for realizing its green and efficient synthesis. At the same time, it enriches the sesquiterpene compound library and provides resources for discovering medicinal lead compounds.

[0028] The present invention has established a heterologous biosynthesis system of farnesyl ketone B sesquiterpenes based on the chassis strain of Aspergillus nidulans, providing a reference for the optimization of the yield of this type of compound and the exploration of structural diversity, enriching the sesquiterpene compound library, and providing resources for discovering medicinal lead compounds.

[0029] The present invention can analyze the enzymatic reaction steps in the biosynthesis process of such novel sesquiterpenes and establish a heterologous expression system for quickly exploring relevant structural diversity, providing a reference for the biosynthesis and biomimetic synthesis research of this type of structure.

[0030] The present invention discovers that the compound farnesyl ketone B shows significant anti-inflammatory activity in the zebrafish Tg(Lyz:ds red) 3dpf tail amputation infection model, reducing the number of myeloid cells near the infected area of the zebrafish tail fin. Brief Description of the Drawings

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0032] Figure 1 Flow chart of the farnesyl ketone B compound, biosynthesis gene cluster and synthesis method provided for the embodiment;

[0033] Figure 2 Schematic diagram of the gene sequence composition of Penicillium janthinellum HDN13-494 provided for the embodiment;

[0034] Figure 3 Effect of the active compound on the number of myeloid cells near the infected area of the zebrafish tail fin;

[0035] Figure 4It is the chemical structure diagram of Fasilitone B compound;

[0036] Figure 5 It is the column scatter statistical chart of myeloid cell number reduction;

[0037] Figure 6 It is the HRESIMS positive mode spectrogram of Fasilitone B;

[0038] Figure 7 It is for Fasilitone B in DMSO-d6 1 1H nuclear magnetic resonance spectrogram (500 MHz);

[0039] Figure 8 It is for Fasilitone B in DMSO-d 6 in 13 13C-NMR spectrogram (126 MHz);

[0040] Figure 9 It is for Fasilitone B in DMSO-d 6 in HSQC spectrogram;

[0041] Figure 10 It is for Fasilitone B in DMSO-d 6 in HMBC spectrogram;

[0042] Figure 11 It is for Fasilitone B in DMSO-d 6 in 1 1H- 1 1H COSY spectrogram;

[0043] Figure 12 It is for Fasilitone B in DMSO-d 6 in NOESY spectrogram. Detailed implementation manners

[0044] Now, the various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0045] It should be understood that the terms described in the present invention are only for describing special implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0047] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the description of the present invention, which will be obvious to those skilled in the art. Other embodiments obtained from the description of the present invention will be obvious to those skilled in the art. The description and examples of the present invention are merely illustrative.

[0048] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0049] Examples

[0050] I. Synthesis of anti-inflammatory compound fathli ketone B

[0051] The synthesis of the anti-inflammatory compound fathli ketone B (as shown in Figure 4 ), is as shown in Figure 1 , and is specifically as follows:

[0052] S1. Construction of gene heterologous expression vector: Using PCR technology, the target genes janA (SEQ ID NO.1), janB (SEQ ID NO.2), janC (SEQ ID NO.3), janD (SEQ ID NO.4) and janE (SEQ ID NO.5) (as shown in Figure 2 ) were innovatively amplified with the genomic DNA of Penicillium janthinellum HDN13-494 as the template; then, the target genes were respectively ligated into the Aspergillus nidulans expression plasmids pYTU, pYTR and pYTP plasmids to construct the recombinant expression plasmids PYTU-janABC, PYTR and PYTP-janDE.

[0053] The primer sequences are shown in Table 1 and include: the primers for amplifying the target gene janA are PYTU-janA-F (SEQ ID NO.6) and PYTU-janA-R (SEQ ID NO.7); the primers for amplifying the target gene janB are PYTU-janB-F (SEQ ID NO.8) and PYTU-janB-R (SEQ ID NO.9); the primers for amplifying the target gene janC are PYTU-janC-F (SEQ ID NO.10) and PYTU-janC-R (SEQ ID NO.11); the primers for amplifying the target gene janD are PYTU-janD-F (SEQ ID NO.12) and PYTU-janD-R (SEQ ID NO.13); the primers for amplifying the target gene janE are PYTU-janE-F (SEQ ID NO.14) and PYTU-janE-R (SEQ ID NO.15).

[0054]

[0055]

[0056]

[0057]

[0058]

[0059] Table 1 Primer Sequences

[0060]

[0061]

[0062] S2, Construction of heterologous expression strain of Aspergillus nidulans: By the method of PEG transformation, the expression vectors PYTU-janABC, PYTR and PYTP-janDE were transformed into the protoplasts of Aspergillus nidulans A1145 to obtain the transformant AN-janABCDE.

[0063] S3, Culture and fermentation of heterologous expression strain of Aspergillus nidulans: The transformant AN-janABCDE was inoculated into the screening medium for culture, and further cultured in the fermentation medium to obtain metabolites.

[0064] The screening medium was CD medium, and the formula of CD medium was: 1 g of anhydrous glucose, 5 mL of 20x nitrate, 100 μL of trace elements, made up to 100 mL with sterile and enzyme-free water, sterilized at 121 °C for 20 min. Before use, add U stock solution (1%), R stock solution (2‰) and P stock solution (2‰) as needed.

[0065] The fermentation medium was CD-ST medium, and the CD-ST medium was: 2 g of soluble starch, 2 g of acid-hydrolyzed casein, 5 mL of 20x nitrate, 100 μL of trace elements, made up to 100 mL with sterile and enzyme-free water, sterilized at 121 °C for 20 min. Before use, add U stock solution (1%), R stock solution (1‰) and P stock solution (1‰) as needed.

[0066] U stock solution: Weigh 0.056 g of uracil and 2.442 g of uridine, make up to 10 mL with sterile and enzyme-free water, filter and sterilize with a 0.22 μm filter membrane, and store at 4 °C.

[0067] R stock solution: Weigh 0.00125 g of riboflavin and dissolve it in 10 mL of sterile and enzyme-free water, filter and sterilize with a 0.22 μm filter membrane, and store at 4 °C.

[0068] P stock solution: Weigh 0.005 g of pyridoxal hydrochloride and dissolve it in 10 mL of sterile and enzyme-free water, filter and sterilize with a 0.22 μm filter membrane, and store at 4 °C.

[0069] 20x nitrate: Weigh 120 g of NaNO3, 10.4 g of KCl, 10.4 g of MgSO4·7H2O, 30.4 g of KH2PO4 respectively, dissolve with ddH2O and make up to 1 L.

[0070] Trace elements: Weigh 2.20 g of ZnSO4·7H2O, 1.10 g of H3BO3, 0.50 g of MnCl2·4H2O, 0.16 g of FeSO4·7H2O, 0.16 g of CoCl2·5H2O, 0.16 g of CuSO4·5H2O, 0.11 g of (NH4)6Mo7O 24 ·4H2O, and 5.00 g of Na4EDTA respectively. Dissolve them with sterile and enzyme-free water and make up the volume to 1 L.

[0071] S4. After large-scale fermentation culture for 5 days, the fermentation product was immersed in an appropriate amount of ethyl acetate overnight, ultrasonicated, and concentrated under reduced pressure to obtain an extract. The obtained extract was separated and enriched with the target component by ODS silica gel column chromatography; then further purified by a semi-preparative column to obtain monomeric compounds.

[0072] Experimental Example

[0073] I. Bioinformatics analysis of the biosynthetic gene cluster of farnesylone B sesquiterpenoid compounds

[0074] Based on Local Blast homology search, the biosynthetic gene cluster of farnesylone B sesquiterpenoid compounds was found in the genome of Penicillium janthinellum HDN13-494. The functions of the genes in this gene cluster were predicted through the NCBI database. The gene cluster contains the aristolene sesquiterpene synthase gene JanA, one short-chain dehydrogenase gene JanC, and three P450 enzyme genes JanB, JanD, and JanE.

[0075] Table 2 Functions of predicted genes in the jan gene cluster

[0076]

[0077] II. Construction of gene heterologous expression vectors

[0078] Construction of PYTU-janABC plasmid: Using the genome of Penicillium janthinellum HDN13-494 as a template, the genes janABC were amplified by PCR with primers PYTU-janA-F (SEQ ID NO.6) / PYTU-janA-R (SEQ ID NO.7), PYTU-janB-F (SEQ ID NO.8) / PYTU-janB-R (SEQ ID NO.9), and PYTU-janC-F (SEQ ID NO.10) / PYTU-janC-R (SEQ ID NO.11). The PCR reaction system was subjected to agarose gel electrophoresis, and the target fragment of janABC was recovered after purification with a gel extraction kit (Omega). Using the method of yeast homologous recombination, the fragments of janA, janB, and janC were integrated into the linear vector pYTU digested with NotI. After screening and verification by auxotrophy, the correct yeast plasmid was transformed into Escherichia coli DH10B, and positive clones were selected by ampicillin resistance screening. Positive clones were selected for verification, and the plasmids of the verified transformants were extracted and sequenced to obtain the correct expression vector PYTU-janABC;

[0079] Among them, Aspergillus nidulans A1145 and Saccharomyces cerevisiae BJ5464 are common model strains, and Escherichia coli DH10B is a common Escherichia coli strain, which can be purchased commercially.

[0080] Construction of PYTP-janDE plasmid: Using the genome of Penicillium janthinellum HDN13-494 as a template, the genes janD and janE were amplified by PCR with primers PYTP-janD-F (SEQ ID NO.12) / PYTP-janD-R (SEQ ID NO.13) and PYTP-janE-F (SEQ ID NO.14) / PYTP-janE-R (SEQ ID NO.15). Using the same method as above, the fragments of janD and janE were integrated into the linear vector pYTP digested with BamHI to obtain the correct expression vector PYTP-janDE.

[0081] III. Construction of Aspergillus nidulans mutant strains

[0082] Take out the frozen Aspergillus nidulans spore suspension from -80 °C, add an appropriate amount to 40 mL of CD (supplemented with elements U, R, and P) liquid medium, and culture at 37 °C and 220 rpm for 9 h until most spores complete germination.

[0083] Transfer the culture medium to a 50 mL centrifuge tube, centrifuge at 4000 rpm for 10 min, and collect the bacterial cells. Add 15 mL of OM buffer (weigh 147.88 g of MgSO4·7H2O, 25 mL of 0.2 M sodium phosphate buffer, dissolve in appropriate ddH2O, adjust the pH to 5.8 with 1 M Na2HPO4, and make up to 500 mL. Filter and sterilize with a 0.22 μm filter membrane, and store at 4°C) to the bacterial cells and resuspend the cells. Centrifuge at 4000 rpm for 10 min and collect the bacterial cells.

[0084] Add 10 mL of protoplast enzyme solution to the germinated spores (weigh 0.02 g of Yatalase TM and 0.03 g of lytic enzyme of Trichoderma harzianum and dissolve in 10 mL of OM buffer, filter with a 0.22 μM sterile filter membrane, and use immediately). Resuspend the cells. Incubate at 28°C and 80 rpm overnight for enzymatic hydrolysis. Observe the enzymatic hydrolysis status under a microscope, and perform subsequent operations after the germinated spores are basically enzymatically hydrolyzed into protoplasts.

[0085] Slowly transfer the enzymatic hydrolysis system to a pre-cooled 50 mL centrifuge tube in advance, and slowly add an equal volume of capture buffer (weigh 109.3 g of D-sorbitol, 100 mL of 1 M Tris-HCl buffer (pH 7.0), add ddH2O to make up to 1 L. Filter and sterilize with a 0.22 μm filter membrane, and store at 4°C). Centrifuge at 4°C and 4000 rpm for 15 min (the centrifuge needs to be pre-cooled in advance). An obvious layered state can be observed. The middle layer is the protoplast. Aspirate the protoplasts in the middle layer with a sterile Pasteur pipette into a new centrifuge tube, add 2 volumes of pre-cooled STC buffer (weigh 218.6 g of D-sorbitol, 1.47 g of CaC12·2H2O, 10 mL of 1 M Tris-HCl buffer (pH 7.5), make up to 1 L. Filter and sterilize with a 0.22 μm filter membrane, and store at 4°C), and gently mix. Centrifuge at 4°C and 4000 rpm for 10 min. Discard the supernatant, and the precipitate is the protoplast.

[0086] Resuspend the protoplasts with an appropriate amount of pre-cooled STC buffer. Aliquot 100 μL into each 1.5 mL EP tube. Add the correctly constructed recombinant plasmid to the protoplasts, gently pipette and mix well, and incubate in an ice bath for 45 min. Add 600 μL of PEG solution (weigh 60 g of PEG4000, 0.735 g of CaCl2·2H2O, 5 mL of 1 M Tris-HCl buffer (pH 7.5), add ddH2O to make up to 100 mL. Sterilize and store at room temperature, gently pipette and mix well. Let stand at room temperature for 25 min.

[0087] Coat the above transformation system on the corresponding defective CDS regeneration medium (supplemented with corresponding nutrient elements). Incubate in a 37°C incubator for 3 days. Transfer the grown transformants to the corresponding defective CD medium for scale-up culture, which is used for strain preservation and subsequent fermentation.

[0088] IV. Culture, Fermentation, and Metabolite Analysis of Transfected Strains of Aspergillus nidulans

[0089] Inoculate the Aspergillus nidulans mutant strain obtained in "III. Construction of Aspergillus nidulans Mutant Strains" on a CD solid medium and incubate at 37°C for 3 days. Inoculate the obtained mutant strain into a CD-ST solid medium for fermentation, incubate at 28°C for 5 days for fermentation, extract with ethyl acetate, concentrate under reduced pressure, dissolve the concentrated sample in chromatographic methanol, centrifuge at high speed, and take the supernatant for HPLC detection.

[0090] The HPLC analysis conditions are as follows: The chromatographic column is a Capcell Park C18 column: 5 μL, 20 mm × 250 mm. The mobile phase includes phase A and phase B. Mobile phase A: Chromatographic methanol + 1‰ (volume fraction) trifluoroacetic acid; Mobile phase B: Water + 1‰ (volume fraction) trifluoroacetic acid; Injection program: 0 - 45 min, the mobile phase ratio is A phase / B phase (volume ratio): 50:50 - 0:100, 5 - 30 min, the detection wavelength is 190 - 600 nm, and the flow rate is 1 mL / min.

[0091] The Aspergillus nidulans mutant strain produced obvious differential peaks compared with the wild type. Through large-scale fermentation, the structure of the compound was determined by separation and purification through NMR.

[0092] V. Large-Scale Fermentation of Aspergillus nidulans Mutant Strains and Determination of the Structure of Sesquiterpene Compounds in the Products

[0093] (1) Fermentation production

[0094] Perform large-scale fermentation on the heterologous expression mutant strain AN-janABCDE of Aspergillus nidulans according to the conditions of small-scale fermentation. Use a CD-ST medium, ferment 10 L, and incubate statically on a 28°C plate for 5 days to obtain the fermentation product.

[0095] (2) Obtaining the extract

[0096] Extract the fermented medium with an equal volume of ethyl acetate three times, combine all the ethyl acetate phases, and concentrate under reduced pressure to obtain a crude extract, totaling 21 g.

[0097] (3) Separation and purification of compounds

[0098] The crude extract was separated by an ODS (octadecylsilyl) column, and reverse-phase column chromatography was performed with a methanol-water elution system, resulting in 7 fractions. Component 6 was subjected to reverse-phase semi-preparative high-performance liquid chromatography (methanol:water = 80:20) to obtain compound fasciculitone B (54.3 mg). The structure of the compound was analyzed by NMR and HRMS. As Figure 6 shown, it is the positive mode HRESIMS spectrum of fasciculitone B; as Figure 7 shown, it is the 1 1H nuclear magnetic resonance spectrum (500 MHz) of fasciculitone B in DMSO-d6; as Figure 8 shown, it is the 6 13C-NMR spectrum (126 MHz) of fasciculitone B in DMSO-d 13 ; as Figure 9 shown, it is the HSQC spectrum of fasciculitone B in DMSO-d 6 ; as Figure 10 shown, it is the HMBC spectrum of fasciculitone B in DMSO-d 6 ; as Figure 11 shown, it is the 6 1H- 1 1H COSY spectrum of fasciculitone B in DMSO-d 1 ; as Figure 12 shown, it is the NOESY spectrum of fasciculitone B in DMSO-d 6 .

[0099] Compound fasciculitone B is a yellow solid with the molecular formula C 15 H 22 O5, ESI-MS m / z: 283 [M+H]+, and the 1H and 13C NMR data assignments are shown in Table 3.

[0100] Table 3 1H and 13C NMR data of compound fasciculitone B

[0101]

[0102]

[0103] VI. Anti-inflammatory activity test

[0104] Inflammation, as the most common and complex physiological response in humans, is usually a natural reaction of the body to injury, infection, or other stimuli. It is usually manifested as redness, swelling, heat, pain, and dysfunction of local tissues. Although the inflammatory response is beneficial to maintaining physical health and resisting infection to a certain extent, continuous or excessive inflammatory responses may also lead to tissue damage, disease development, and the occurrence of inflammatory diseases.

[0105] The zebrafish Tg(Lyz:ds red) was used to establish a tail-trimming injury infection model at 3 days post-fertilization (dpf), and 71 isolated marine compounds were used for screening of inflammatory phenotypes. Sudan Black (SB) is a medium-lipid-soluble dye that can stain mature myeloid cells and can be used for the preliminary judgment of inflammatory infection injury.

[0106] This experiment aimed to screen out marine bioactive compounds through the tail-trimming injury infection model.

[0107] For the preliminary in vitro anti-inflammatory activity test of the compounds, transgenic zebrafish Tg(Lyz:ds red) were self-crossed to obtain fish eggs, which were soaked in 3% methylene blue solution. After 3 dpf, an infection model was established. Dexamethasone (DEX) was used as a positive control drug. After 6 h of drug administration and soaking treatment, paraformaldehyde solution was added for fixation for 2 h. Sudan Black staining was used to observe the SB-positive signal after tail fin injury, and the data were statistically analyzed.

[0108] Through the primary screening, 11 effective compounds were obtained from 71 compounds, and the rest were ineffective, as shown in Table 4 below.

[0109] Table 4 Primary screening effective group and all-sample death group

[0110]

[0111]

[0112] As Figure 3 shown, 3254 in the figure represents fathiazole B, and DEX represents dexamethasone. Fathiazole B effectively reduced the number of myeloid cells near the infected tail fin of zebrafish; as Figure 5 shown, it can be seen the reduction of the number of myeloid cells in the fathiazole B treatment group represented by 3254. The compound fathiazole B showed certain antibacterial activity against the 3 dpf tail-trimming infection model of zebrafish Tg(Lyz:ds red) at a concentration of 10 μM.

[0113] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An anti-inflammatory compound, characterized in that The chemical formula of the anti-inflammatory compound is C 15 H 22 O5, the structural formula of which is shown in Formula I, is named: phaseolinone B; 2. The anti-inflammatory compound according to claim 1, characterized in that The anti-inflammatory compound is synthesized by expressing the biosynthetic gene cluster in Penicillium janthinellum HDN13-494 using Aspergillus nidulans as a host; The biosynthetic gene cluster includes an aristolochene sesquiterpene synthase gene JanA, a short-chain dehydrogenase gene JanC, and three P450 enzyme genes JanB, JanD, and JanE; the nucleotide sequence of the aristolochene sesquiterpene synthase gene JanA is shown in SEQ ID NO.1, the nucleotide sequence of the short-chain dehydrogenase gene JanC is shown in SEQ ID NO.3, the nucleotide sequence of the P450 enzyme gene JanB is shown in SEQ ID NO.2, the nucleotide sequence of the P450 enzyme gene JanD is shown in SEQ ID NO.4, and the nucleotide sequence of the P450 enzyme gene JanE is shown in SEQ ID NO.

5.

3. A biosynthetic gene cluster for synthesizing the anti-inflammatory compound according to claim 1 or 2, characterized in that: The biosynthetic gene cluster is derived from Penicillium purpurogenum HDN13-494, and includes the aristolochene sesquiterpene synthase gene JanA, one short-chain dehydrogenase gene JanC, and three P450 enzyme genes JanB, JanD and JanE; the nucleotide sequence of the aristolochene sesquiterpene synthase gene JanA is shown in SEQ ID NO.1, the nucleotide sequence of the short-chain dehydrogenase gene JanC is shown in SEQ ID NO.3, the nucleotide sequence of the P450 enzyme gene JanB is shown in SEQ ID NO.2, the nucleotide sequence of the P450 enzyme gene JanD is shown in SEQ ID NO.4, and the nucleotide sequence of the P450 enzyme gene JanE is shown in SEQ ID NO.

5.

4. The method for synthesizing the anti-inflammatory compound according to claim 1 or 2, wherein: The following steps are involved: (1) Construction of heterologous gene expression vector; (2) constructing an Aspergillus nidulans heterologous expression strain using the gene heterologous expression vector obtained in step (1); (3) culturing the Aspergillus nidulans heterologous expression strain obtained in step (2) and fermenting for 5 days to obtain a fermentation product; (4) The fermentation product obtained in step (3) was immersed in ethyl acetate overnight, sonicated, and concentrated under reduced pressure to obtain an extract, which was separated and enriched by ODS silica gel column chromatography; and then purified by semi-preparative column to obtain farslione B; The construction of the heterologous gene expression vector specifically comprises the following steps: using the genomic DNA of Penicillium micropurpurogenum HDN13-494 as a template, PCR amplifying the target genes janA, janB, janC, janD and janE; connecting the target genes janA, janB and janC into the Aspergillus nidulans expression plasmid pYTU to construct a recombinant expression plasmid PYTU-janABC; constructing the Aspergillus nidulans expression plasmid PYTR; and connecting the target genes janD and janE into the Aspergillus nidulans expression plasmid pYTP to construct a recombinant expression plasmid PYTP-DE.

5. The synthesis method according to claim 4, characterized in that The construction of the heterologous gene expression vector in step (1) is specifically as follows: Using the genomic DNA of Penicillium micropurpurogenum HDN13-494 as a template, PCR amplification was performed to obtain the target genes janA, janB, janC, janD and janE; the target genes janA, janB and janC were ligated into the Aspergillus nidulans expression plasmid pYTU to construct a recombinant expression plasmid PYTU-janABC; an Aspergillus nidulans expression plasmid PYTR was constructed; and the target genes janD and janE were ligated into the Aspergillus nidulans expression plasmid pYTP to construct a recombinant expression plasmid PYTP-DE. The primers for amplifying the target gene janA are PYTU-janA-F and PYTU-janA-R, the nucleotide sequence of PYTU-janA-F is shown in SEQ ID NO.6, and the nucleotide sequence of PYTU-janA-R is shown in SEQ ID NO.7; The primers for amplifying the target gene janB are PYTU-janB-F and PYTU-janB-R, the nucleotide sequence of PYTU-janB-F is shown in SEQ ID NO.8, and the nucleotide sequence of PYTU-janB-R is shown in SEQ ID NO.9; The primers for amplifying the target gene janC are PYTU-janC-F and PYTU-janC-R, the nucleotide sequence of PYTU-janC-F is shown in SEQ ID NO.10, and the nucleotide sequence of PYTU-janC-R is shown in SEQ ID NO.11; The primers for amplifying the target gene janD are PYTU-janD-F and PYTU-janD-R, the nucleotide sequence of PYTU-janD-F is shown in SEQ ID NO.12, and the nucleotide sequence of PYTU-janD-R is shown in SEQ ID NO.13; The primers for amplifying the target gene janE are PYTU-janE-F and PYTU-janE-R. The nucleotide sequence of PYTU-janE-F is shown in SEQ ID NO.14, and the nucleotide sequence of PYTU-janE-R is shown in SEQ ID NO.

15.

6. The synthesis method according to claim 4 or 5, characterized in that The method of step (2) of constructing the Aspergillus nidulans heterologous expression strain using the gene heterologous expression vector obtained in step (1) is as follows: The recombinant expression plasmids PYTU-janABC, PYTR and PYTP-DE were transformed into the protoplasts of Aspergillus nidulans by PEG transformation to obtain transformant AN-janABCDE.

7. The synthesis method according to claim 6, characterized in that Step (3) culturing the Aspergillus nidulans heterologous expression strain obtained in step (2) and fermenting it for 5 days to obtain a fermentation product is specifically as follows: the transformant AN-janABCDE is inoculated into a screening medium for cultivation, and then fermented in a fermentation medium for 5 days to obtain a fermentation product.

8. The synthesis method according to claim 7, characterized in that The screening medium is CD medium, and the formula of the CD medium is: 1 g of anhydrous glucose, 5 mL of 20x nitrate, 100 μL of trace elements, and sterile enzyme-free water to 100 mL; The preparation method of the 20x nitrate is as follows: 120g NaNO3, 10.4g KCl, 10.4g MgSO4·7H2O, and 30.4g KH2PO4 are weighed respectively, dissolved in ddH2O, and the volume is adjusted to 1L to obtain 20x nitrate.

9. The synthesis method according to claim 7, characterized in that: The fermentation medium is CD-ST medium, and the formula of the CD-ST medium is: 2 g of soluble starch, 2 g of acid hydrolyzed casein, 5 mL of 20x nitrate, 100 μL of trace elements, and sterile enzyme-free water to 100 mL; The preparation method of the 20x nitrate is as follows: 120g NaNO3, 10.4g KCl, 10.4g MgSO4·7H2O, and 30.4g KH2PO4 are weighed respectively, dissolved in ddH2O, and the volume is adjusted to 1L to obtain 20x nitrate.

10. Use of the anti-inflammatory compound according to claim 1 or 2 in the preparation of anti-inflammatory drugs.

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