Anti-inflammatory compound as well as biosynthetic gene cluster, synthetic method and application thereof

Abstract

The invention discloses an anti-inflammatory compound as well as a biosynthetic gene cluster, a synthetic method and application thereof, belongs to the technical field of gene engineering, and provides the anti-inflammatory compound, the chemical formula of the anti-inflammatory compound is C15H22O5, the structural formula of the anti-inflammatory compound is shown as a formula I, and the anti-inflammatory compound is named as phaseolinone B. The invention discloses a biosynthetic pathway of the anti-inflammatory compound Fardinone B for the first time, and lays an important foundation for realizing green and efficient synthesis of the Fardinone B; the compound shows remarkable anti-inflammatory activity in a zebrafish Tg 3dpf tail-cutting infection mode, and resources are provided for finding lead compounds of anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention belongs to the field of gene 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. During infection or injury, it can remove external stimuli and maintain homeostasis. It is characterized by redness, swelling, heat, pain and dysfunction. Short-term inflammation helps to remove inflammatory substances and promote recovery. It is a key step in the body's self-repair. However, excessive or long-term inflammation can lead to various diseases, such as cardiovascular dysfunction, metabolic disorders, autoimmune diseases, rheumatoid arthritis, cancer, sepsis and other diseases, which seriously threaten human health. At present, drugs for the treatment of inflammatory diseases mainly include steroids and non-steroidal anti-inflammatory drugs. These drugs have more or less side effects, such as non-steroidal anti-inflammatory drugs causing changes in the hematopoietic system, cardiovascular damage, asthma, etc., which have caused serious harm to human health. Therefore, finding safer and more efficient anti-inflammatory drugs from natural products is of great significance to protecting human life and health.

[0003] Fungi are rich in metabolites and are an important natural resource for the development of modern pharmaceutical preparations. Nature is rich in fungal resources, and fungi have a huge gene library and the potential to synthesize novel and highly active compounds, making them an important way to develop natural active ingredients. Summary of the invention

[0004] In order 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 farsligone B, laying an important foundation for achieving 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 is shown in Formula I, 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, the biosynthetic gene cluster 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) is immersed in ethyl acetate overnight, sonicated, and concentrated under reduced pressure to obtain an extract, and the extract is separated and enriched by ODS silica gel column chromatography; and then purified by semi-preparative column to obtain farslenone B.

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

[0015] 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, janC, janD and janE were connected to the Aspergillus nidulans expression plasmids pYTU, pYTR and pYTP, respectively, 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 shown in SEQ ID NO.8, and the nucleotide sequence of PYTU-janB-R is 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 shown in SEQ ID NO.10, and the nucleotide sequence of PYTU-janC-R is 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 shown in SEQ ID NO.12, and the nucleotide sequence of PYTU-janD-R is 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 shown in SEQ ID NO.14, and the nucleotide sequence of PYTU-janE-R is shown in SEQ ID NO.15.

[0021] Preferably, the step (2) of constructing the Aspergillus nidulans heterologous expression strain using the gene heterologous expression vector obtained in step (1) is specifically as follows: transforming the recombinant expression plasmids PYTU-janABC, PYTR and PYTP-DE into the protoplasts of Aspergillus nidulans through PEG transformation to obtain the transformant AN-janABCDE.

[0022] Preferably, the step (3) of 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 culturing, and then fermented in a fermentation medium for 5 days to obtain a fermentation product.

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

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

[0025] The invention also provides application of the anti-inflammatory compound in preparing 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, the farsligone B biosynthetic gene cluster is completely heterologously reconstructed in the organism, and farsligone B sesquiterpene compounds are isolated and identified, which have good anti-inflammatory biological activity; in addition, the present invention discloses for the first time the biosynthetic pathway of such compounds, laying an important foundation for achieving their green and efficient synthesis; at the same time, the sesquiterpene compound library is enriched, providing resources for the discovery of medicinal lead compounds.

[0028] The present invention establishes a heterologous biosynthesis system of farslione B sesquiterpene based on the Aspergillus nidulans chassis strain, provides a reference for optimizing the yield of such compounds and exploring structural diversity, enriches the sesquiterpene compound library, and provides 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 rapidly exploring the diversity of related structures, providing a reference for the biosynthesis and biomimetic synthesis research of such structures.

[0030] The present invention finds that the compound Faslione B exhibits significant anti-inflammatory activity in a zebrafish Tg (Lyz: ds red) 3dpf tail-breaking infection model, and reduces the number of myeloid cells near the infected site of the zebrafish caudal fin. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 A flow chart of the farslione B compound, biosynthetic gene cluster and synthesis method provided in the embodiments;

[0033] Figure 2 A schematic diagram of the composition of the gene sequence of Penicillium purpurogenum HDN13-494 provided in the embodiment;

[0034] Figure 3 The effect of the effective compounds on the number of myeloid cells near the infected site of the zebrafish caudal fin;

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

[0036] Figure 5 The bar scatter plot shows the decrease in the number of myeloid cells;

[0037] Figure 6 is the HRESIMS positive mode spectrum of farslione B;

[0038] Figure 7 is the concentration of Faslidinone B in DMSO-d6 1 H NMR spectrum (500 MHz);

[0039] Figure 8 Faslidinone B in DMSO-d 6 In 13 C-NMR spectrum (126MHz);

[0040] Fig. 9 Faslidinone B in DMSO-d 6 HSQC spectrum in;

[0041] Fig.10 Faslidinone B in DMSO-d 6 HMBC spectrum in;

[0042] Fig.11 Faslidinone B in DMSO-d 6 In 1 H- 1 H COSY spectrum;

[0043] Fig.12 Faslidinone B in DMSO-d 6 NOESY spectrum in . DETAILED DESCRIPTION

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0046] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0047] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0048] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0049] Example

[0050] 1. Synthesis of the anti-inflammatory compound Faslidon B

[0051] The anti-inflammatory compound Faslidinone B (eg Figure 4 The synthesis of Figure 1 As shown, the details are as follows:

[0052] S1. Construction of heterologous gene expression vector: Using PCR technology, the genomic DNA of Penicillium purpurogenum HDN13-494 was used as a template to innovatively amplify 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) (such as Figure 2 Then, the target gene was connected to the Aspergillus nidulans expression plasmids pYTU, pYTR and pYTP plasmids to construct recombinant expression plasmids PYTU-janABC, PYTR and PYTP-janDE.

[0053] The primer sequences are shown in Table 1, including: primers for amplifying the target gene janA are PYTU-janA-F (SEQ ID NO.6) and PYTU-janA-R (SEQ ID NO.7), primers for amplifying the target gene janB are PYTU-janB-F (SEQ ID NO.8) and PYTU-janB-R (SEQ ID NO.9), primers for amplifying the target gene janC are PYTU-janC-F (SEQ ID NO.10) and PYTU-janC-R (SEQ ID NO.11), primers for amplifying the target gene janD are PYTU-janD-F (SEQ ID NO.12) and PYTU-janD-R (SEQ ID NO.13), and 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: The expression vectors PYTU-janABC, PYTR and PYTP-janDE were transformed into the protoplasts of Aspergillus nidulans A1145 by PEG transformation to obtain transformant AN-janABCDE.

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

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

[0065] The fermentation medium is CD-ST medium, which is: soluble starch 2g, acid hydrolyzed casein 2g, 20x nitrate 5mL, trace elements 100μL, dilute to 100mL with sterile enzyme-free water, sterilize at 121℃ for 20min. Before use, add U mother solution (1%), R mother solution (1‰) and P mother solution (1‰) as needed.

[0066] U mother solution: Weigh 0.056 g uracil and 2.442 g uridine, add sterile enzyme-free water to make up to 10 mL, filter through a 0.22 μm filter membrane for sterilization, and store at 4°C.

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

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

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

[0070] Trace elements: weigh 2.20g ZnSO4·7H2O, 1.10g H3BO3, 0.50g MnCl2·4H2O, 0.16g FeSO4·7H2O, 0.16g CoCl2·5H2O, 0.16g CuSO4·5H2O, 0.11g (NH4)6Mo7O 24 4H2O, 5.00gNa4EDTA. Dissolve with sterile enzyme-free water and make up to 1L.

[0071] S4, after 5 days of large-scale fermentation, the fermentation product was immersed in an appropriate amount of ethyl acetate overnight, sonicated, and concentrated under reduced pressure to obtain an extract. The obtained extract was separated and enriched by ODS silica gel column chromatography; then it was further purified by semi-preparative column to obtain a monomer compound.

[0072] Experimental example

[0073] 1. Bioinformatics analysis of the gene cluster for the biosynthesis of sesquiterpenoids of farslione B

[0074] Based on Local Blast homology search, the farslione B sesquiterpene compound biosynthesis gene cluster was found from the Penicillium janthinellum HDN13-494 genome. The functions of the genes in this gene cluster were predicted using the NCBI database. The gene cluster contained the aristolochene sesquiterpene synthase gene JanA, one short-chain dehydrogenase gene JanC, and three P450 enzyme genes JanB, JanD and JanE.

[0075] Table 2 Predicted functions of genes in the jan gene cluster

[0076]

[0077] 2. Construction of heterologous gene expression vector

[0078] Construction of PYTU-janABC plasmid: The genome of Penicillium purpurogenum HDN13-494 was used as a template, and the janABC gene was amplified by PCR using 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 janABC target fragment was recovered after purification using a gel recovery kit (Omega). Using the yeast homologous recombination method, the janA, janB and janC fragments were integrated into the linear vector pYTU after NotI digestion. The correct yeast plasmid was transformed into Escherichia coli DH10B through nutritional deficiency screening and verification. The positive clones were screened by ampicillin resistance and selected for verification. 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 commonly used model strains, and Escherichia coli DH10B is a commonly used Escherichia coli strain and is commercially available.

[0080] Construction of PYTP-janDE plasmid: Using the genome of Penicillium purpurogenum HDN13-494 as a template, PCR amplification of genes janD and janE was performed using 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 janD and janE fragments were integrated into the linear vector pYTP after BamHI digestion to obtain the correct expression vector PYTP-janDE.

[0081] 3. 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 CD (supplemented with U, R, and P elements) liquid culture medium, and culture at 37°C and 220 rpm for 9 hours until most of the spores have germinated.

[0083] Transfer the culture solution to a 50mL centrifuge tube, centrifuge at 4000rpm for 10min, and collect the bacteria. Add 15mL OM buffer (147.88g MgSO4·7H2O, 25mL 0.2M sodium phosphate buffer, dissolve in appropriate amount of ddH2O, adjust pH to 5.8 with 1MNa2HPO4, and make up to 500mL. Filter with 0.22μm filter membrane for sterilization, and store at 4℃), resuspend the bacteria. Centrifuge at 4000rpm for 10min, and collect the bacteria.

[0084] Add 10 mL of protoplast hydrolysate (0.02 g Yatalase TM 0.03g of Trichoderma harzianum lytic enzyme was dissolved in 10mL OM buffer, filtered with a 0.22μM sterile filter membrane, and the cells were resuspended. The cells were enzymolyzed overnight at 28℃ and 80rpm. The enzymolysis state was observed under a microscope, and subsequent operations were performed after the spores were basically enzymolyzed into protoplasts.

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

[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 blow and mix with a pipette tip, and place on ice for 45 minutes. Add 600 μL of PEG solution (take 60g PEG4000, 0.735g CaCl2·2H2O, 5mL 1M Tris-HCl buffer (pH 7.5) to the transformation system, add ddH2O to make up to 100mL. After sterilization, store at room temperature and gently blow and mix. Let stand at room temperature for 25 minutes.

[0087] The above transformation system was applied to the corresponding defective CDS regeneration medium (supplemented with corresponding nutrient elements) and cultured in a 37°C incubator for 3 days. The grown transformants were transferred to the corresponding defective CD medium for expansion culture for seed preservation and subsequent fermentation.

[0088] IV. Cultivation, fermentation and metabolite analysis of Aspergillus nidulans transfected strains

[0089] The Aspergillus nidulans mutant strain obtained in "III. Construction of Aspergillus nidulans mutant strain" was inoculated into CD solid culture medium and cultured at 37°C for 3 days. The obtained mutant strain was inoculated into CD-ST solid culture medium and fermented at 28°C for 5 days. The extract was extracted with ethyl acetate and concentrated under reduced pressure. The concentrated sample was dissolved in chromatographic methanol and centrifuged at high speed. The supernatant was taken 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, and 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, mobile phase ratio is phase A / phase B (volume ratio): 50:50-0:100, 5-30 min, detection wavelength is 190-600 nm, and flow rate is 1 mL / min.

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

[0092] 5. Large-scale fermentation of Aspergillus nidulans mutants and structure determination of the resulting sesquiterpenoid compounds

[0093] (1) Fermentation production

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

[0095] (2) Obtaining the extract

[0096] The fermentation medium was extracted three times with an equal amount of ethyl acetate, and all ethyl acetate phases were combined and concentrated under reduced pressure to obtain a crude extract, a total of 21 g.

[0097] (3) Separation and purification of compounds

[0098] The crude extract was separated by ODS (octadecylsilyl) column, and then chromatographed by reverse phase column chromatography with methanol-water as the elution system, and was divided into 7 fractions. Component 6 was subjected to reverse phase semi-preparative HPLC (methanol: water = 80:20) to obtain compound Faslione B (54.3 mg). The structure of the compound was analyzed by NMR and HRMS, as shown in FIG. Figure 6 As shown, this is the HRESIMS positive mode spectrum of farslione B; Figure 7 The following is the result of the reaction of Faslidinone B in DMSO-d6. 1 H NMR spectrum (500MHz); Figure 8 The following is the result of the reaction of Faslidinone B in DMSO-d 6 In 13 C-NMR spectrum (126MHz); Fig. 9 The following is the result of the reaction of Faslidinone B in DMSO-d 6 HSQC spectrum in; Fig.10 The following is the result of the reaction of Faslidinone B in DMSO-d 6 HMBC spectrum in Fig.11 The following is the result of the reaction of Faslidinone B in DMSO-d 6 In 1 H- 1 H COSY spectrum; Fig.12 The following is the result of the reaction of Faslidinone B in DMSO-d 6 NOESY spectrum in .

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

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

[0101]

[0102]

[0103] 6. Anti-inflammatory activity test

[0104] Inflammation is the most common and complex physiological response of humans. It is usually a natural response of the body to injury, infection or other stimulation. It usually manifests as redness, swelling, heat, pain and dysfunction of local tissues. Although the inflammatory response is beneficial to maintain health and resist infection to a certain extent, persistent or excessive inflammatory response may also lead to tissue damage, disease development and the occurrence of inflammatory diseases.

[0105] Zebrafish Tg (Lyz: ds red) were used as a tail-cutting injury infection model at 3 dpf, and 71 isolated marine compounds were used for inflammatory phenotype screening. SB (Sudan Black) is a medium-lipid-soluble dye that can stain mature myeloid cells and can be used for preliminary inflammatory infection injury judgment.

[0106] This experiment hopes to screen out marine active compounds through the tail injury infection model.

[0107] The compounds were preliminarily tested for their anti-inflammatory activity in vitro. The transgenic zebrafish Tg (Lyz: ds red) were self-pollinated to obtain fish eggs, which were immersed in 3% methylene blue solution. An infection model was established after 3 dpf, and dexamethasone (DEX) was used as a positive control drug. After 6 hours of drug immersion treatment, paraformaldehyde solution was added for fixation for 2 hours. SB staining was used to observe the SB positive signal after tail fin injury, and statistical data was collected.

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

[0109] Table 4: The effective group in the initial screening and the group with all samples dying

[0110]

[0111]

[0112] like Figure 3 As shown, 3254 in the figure represents Faslidinone B, DEX represents dexamethasone, and Faslidinone B effectively reduces the number of myeloid cells near the infected tail fin of zebrafish; Figure 5 As shown, it can be seen that the number of myeloid cells in the group treated with farsligone B represented by 3254 was reduced. The compound farsligone B showed certain antibacterial activity in the zebrafish Tg (Lyz: ds red) 3dpf tail-breaking infection model at a concentration of 10 μM.

[0113] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all 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 is shown in Formula I, 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.

3. A biosynthetic gene cluster for synthesizing the anti-inflammatory compound of claim 1 or 2, characterized in that: The biosynthetic gene cluster 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.

4. The method for synthesizing the anti-inflammatory compound according to claim 1 or 2, characterized in that: 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) is immersed in ethyl acetate overnight, sonicated, and concentrated under reduced pressure to obtain an extract, and the extract is separated and enriched by ODS silica gel column chromatography; and then purified by semi-preparative column to obtain farslenone B.

5. The synthesis method according to claim 4, characterized in that: The construction of the gene heterologous 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, janC, janD and janE were connected to the Aspergillus nidulans expression plasmids pYTU, pYTR and pYTP, respectively, to construct recombinant expression plasmids PYTU-janABC, PYTR and 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 step (2) of using the gene heterologous expression vector obtained in step (1) to construct the Aspergillus nidulans heterologous expression strain is specifically 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 4 or 6, characterized in that: Step (3) culturing the Aspergillus nidulans heterologous expression strain obtained in step (2), fermenting for 5 days, and obtaining a fermentation product is specifically as follows: inoculating the transformant AN-janABCDE in a screening medium for culturing, and then fermenting 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 culture medium is a CD culture medium, and the formula of the CD culture medium is: 1 g of anhydrous glucose, 5 mL of 20x nitrate, 100 μL of trace elements, and sterile enzyme-free water to make up to 100 mL.

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 make up to 100 mL.

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

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