I-type ganoderic acid as well as preparation method and application thereof

Abstract

The invention discloses a Ganoderma lucidum engineering strain delta W2 after gene editing, a preparation method of the Ganoderma lucidum engineering strain delta W2, and a ganoderic acid compound 3alpha, 22alpha-diacetoxylanost-8, 24-dien-26-oic acid generated by fermentation of the Ganoderma lucidum engineering strain delta W2, and the chemical structural formula of the ganoderic acid compound is shown in the specification. The compound can effectively improve NO release of LPS-induced macrophages RAW264.7 and inhibit release of inflammatory factors TNF-alpha and IL-6, and has a wide application prospect in addition to anti-inflammatory drugs, health-care functional foods or cosmetics. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and medicinal chemistry, and specifically relates to type I ganoderic acid, a preparation method thereof, and anti-inflammatory applications. Background Art

[0002] Ganoderma lucidum (also known as Ganoderma lingzhi) is a fungus belonging to the phylum Basidiomycota, class Agaricomycetes, order Polyporales, family Ganodermataceae, and genus Ganoderma. Ganoderic acid is the primary characteristic component of Ganoderma. The search for active ganoderic acids from Ganoderma has been ongoing for over 40 years, resulting in the discovery of numerous structurally diverse ganoderic acid compounds with demonstrated anti-inflammatory, antibacterial, antioxidant, and anti-tumor activities. The ganoderic acid compounds found in fermented Ganoderma mycelium can be divided into Type I (without conjugated double bonds) and Type II (with conjugated double bonds), depending on the presence or absence of conjugated double bonds in the nucleus. Generally, the variety and content of Type II ganoderic acids are far greater than those of Type I.

[0003] The influence of the nucleus and substituents of these two structures on their biological activity has always been a key question in the development of ganoderic acid. However, due to the lack of a corresponding type I ganoderic acid, the structure-activity relationship of ganoderic acid has remained a matter of speculation. Furthermore, type I ganoderic acid is an essential reference for understanding the biosynthetic pathway of ganoderic acid. Therefore, it is necessary to research and develop new type I ganoderic acid compounds. Summary of the Invention

[0004] The present invention first provides a method for preparing an engineered Ganoderma lucidum strain ΔW2, comprising the following steps:

[0005] Using dual-sgRNA-mediated CRISPR / Cas9 technology, we designed dual sgRNA sequences targeting the Ganoderma CYP512W2 gene: sgRNA-1: 5'-gtactatccatattgcgact-3', sgRNA-2: 5'-gaattattgaccgttcgacc-3'. Using PEG-mediated protoplast transformation, the Ganoderma CYP512W2 gene deletion plasmids pUC57-PU6-CYP512W2-1-ophph and pUC57-PU6-CYP512W2-2-ophph were co-transformed into Ganoderma pJW-EXP-intron-opCas9 cells. After selection with 250 mg / L hygromycin B and PCR verification, we successfully obtained the engineered Ganoderma lucidum strain ΔW2, which contained a 952 bp deletion in the CYP512W2 gene.

[0006] The present invention also provides the Ganoderma lucidum engineered strain ΔW2 prepared by the above method.

[0007] The present invention provides a type I ganoderic acid compound, whose chemical name is: 3α,22α-diacetoxylanosta-8,24-dien-26-oic acid, and whose chemical structure is shown in structural formula (1):

[0008]

[0009] The present invention also provides a method for preparing the above-mentioned type I ganoderic acid compound, comprising the following steps:

[0010] (1) Fermentation of Ganoderma lucidum engineered strain ΔW2

[0011] The engineered Ganoderma lucidum strain ΔW2 was inoculated into a primary shake flask and cultured in the dark at 150 rpm and 25°C for 7 days; then transferred to a secondary shake flask at a 10% volume percentage and cultured in the dark at 150 rpm and 25°C for 3 days; then transferred to a static culture for 21 days;

[0012] The culture medium in the primary and secondary shake flasks is formulated as follows: 30.0 g / L of anhydrous glucose, 3.0 g / L of yeast powder, 2.0 g / L of KH2PO4, and 2.0 g / L of MgSO4·7H2O, dissolved in 1 L of distilled water;

[0013] (2) Extraction and extraction of mycelium

[0014] The freeze-dried Ganoderma mycelium is soaked and extracted with 80-100% by volume ethanol aqueous solution at room temperature for 1-4 times, the extracts are combined and concentrated to obtain a Ganoderma mycelium extract;

[0015] The Ganoderma mycelium extract is further extracted with an equal volume of petroleum ether for 1-4 times, the petroleum ether extract is discarded, and then extracted with an equal volume of ethyl acetate for 1-4 times, the ethyl acetate extracts are combined, and concentrated under reduced pressure to obtain an ethyl acetate extract;

[0016] (3) Preparation of Ganoderic Acid

[0017] The ethyl acetate extract was separated using YMC ODS C18 (particle size 50 μm, pore size 12 nm) as a filler, with a 0.01% by volume aqueous glacial acetic acid solution as phase A and acetonitrile as phase B by gradient elution: 0-5 min, B: 55%; 5-10 min, B: 55%-60%; 10-30 min, B: 60%-70%; 30-35 min, B: 70%-75%; 35-60 min, B: 75%-80%; 60-80 min, B: 80%-100%; 80-100 min, B: 100%-100% at a flow rate of 200 mL / min; fractions were collected every 400 mL and similar fractions were combined;

[0018] The 40-42 fractions containing the 215nm UV characteristic absorption peak of ganoderic acid were further separated using a Zorbax Eclipse Plus C18 chromatographic column with the following specifications: particle size 5 μm, diameter × column length: 4.6 mm × 250 mm, using a 0.01% volume percent glacial acetic acid aqueous solution as phase A and acetonitrile as phase B for gradient elution: 0-15 min, B: 90%-100%; 15-30 min, B: 100%-100%, detection at a wavelength of 215 nm; collecting the chromatographic peak at 16.95 min to obtain the desired compound.

[0019] The structure of the obtained ganoderic acid compound was analyzed by mass spectrometry, optical rotation, ultraviolet, infrared and nuclear magnetic resonance detection, and its precise chemical structure was determined. The ganoderic acid is named as follows:

[0020] Compound: 3α,22α-diacetoxylanosta-8,24-dien-26-oic acid

[0021] Another object of the present invention is to provide the use of the type I ganoderic acid compound of the above structural formula (1) in the preparation of drugs for preventing or treating inflammation.

[0022] The present invention uses the above-mentioned ganoderic acid compound 3α,22α-diacetoxylanosta-8,24-dien-26-oic acid in the preparation of a drug for preventing or treating inflammation. The component (or active ingredient) of the drug is the above-mentioned type I ganoderic acid compound. One or more excipients acceptable in drug production may also be added to improve the absorption effect of the active ingredient or facilitate use, such as preparing it into capsules or pills, powders, tablets, granules, oral liquids and injections, etc., that is, preparing a suitable dosage form for use.

[0023] The present invention provides for the first time a type I ganoderic acid compound 3α,22α-diacetoxylanosta-8,24-dien-26-oic acid produced by an engineered strain of Ganoderma lucidum, and reveals its anti-inflammatory activity, providing a new approach for the discovery and preparation of anti-inflammatory drugs, and also providing new candidate compounds for the development of anti-inflammatory drugs.

[0024] This invention leverages the latest gene editing technology to guide the large-scale synthesis of downstream ganoderic acids, even allowing for the synthesis of novel ganoderic acids, through gene knockout or overexpression. By constructing an engineered strain, ΔW2, with a CYP512W2 gene deletion in Ganoderma lucidum (CGMCC 5.616), the invention successfully produced a novel type I ganoderic acid compound with anti-inflammatory activity. This provides a new candidate compound for the development of anti-inflammatory drugs or lead compounds, and offers new insights into the use of Ganoderma lucidum as a raw material for the development of anti-inflammatory products.

[0025] The present invention constructs a CYP512W2 gene-deficient engineered strain ΔW2 in Ganoderma lucidum (Ganoderma lucidum CGMCC 5.616), realizes the conversion of type II ganoderic acid to type I ganoderic acid, and successfully prepares a new ganoderic acid compound with anti-inflammatory activity. This provides a new candidate compound for the development of anti-inflammatory drugs or lead compounds, and also offers new ideas for using Ganoderma lucidum as a raw material for the development of anti-inflammatory products. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the Ganoderma lucidum CYP512W2 gene deletion vector structure, where

[0027] The CYP512W2-target sequence of pUC57-PU6-CYP512W2-1-ophph is 5′-gtactatccatattgcgact-3′, and the CYP512W2-target sequence of pUC57-PU6-CYP512W2-2-ophph is 5′-gaattattgaccgttcgacc-3′;

[0028] Figure 2 Schematic diagram of the construction of the Ganoderma lucidum CYP512W2 gene deletion engineered strain, where A is the primary screening plate image on CYM plates containing 250 mg / L hygromycin B; B is the secondary screening plate image on 250 mg / L PDA medium; C is the electrophoresis image of the genomic PCR amplification of the CYP512W2 gene, where 5000M is the DL5000 marker, N is the negative control, WT is CK, and 1-7 are transformants; D is the CYP512W2 gene sequencing results of transformant No. 2;

[0029] Figure 3 Semi-preparative liquid chromatogram of fractions 40-42;

[0030] Figure 4 Liquid chromatogram (215 nm) of the purified ganoderic acid compound 3α,22α-diacetoxylanosta-8,24-dien-26-oic acid

[0031] Figure 5 Effects of ganoderic acid compound 3α,22α-diacetoxylanosta-8,24-dien-26-oic acid on LPS-induced TNF-α release in RAW264.7 cells

[0032] Figure 6 Effects of ganoderic acid compound 3α,22α-diacetoxylanosta-8,24-dien-26-oic acid on LPS-induced IL-6 release in RAW264.7 cells DETAILED DESCRIPTION

[0033] The present invention is further described in detail below by way of examples, but the scope of protection of the present invention is not limited to the contents described above. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0034] The CYM medium (g / L) in the following examples includes the following: 20g of glucose, 10g of maltose, 2g of yeast powder, 2g of peptone, 0.5g of MgSO, 4.6g of KH2PO4, and 10g of agar.

[0035] The culture medium formulations for the primary and secondary shake flasks in the following examples were as follows: 30.0 g / L of anhydrous glucose, 3.0 g / L of yeast powder, 2.0 g / L of KH2PO4, and 2.0 g / L of MgSO4·7H2O, dissolved in 1 L of distilled water.

[0036] The engineered Ganoderma lucidum strain pJW-EXP-intron-opCas9 was prepared using the method described in the following literature:

[0037] Liu K, Sun B, You H, et al. Dual sgRNA-directed gene deletion inbasidiomycete Ganoderma lucidum using the CRISPR / Cas9 system[J]. MicrobialBiotechnology, 2020, 13(2):386-396.

[0038] Plasmid pUC57-PU6-26016-1-ophph was prepared using the following method:

[0039] Zhou Dingqian, Xu Junwei. Effects of Cys2His2 transcription factor GL26016 on mycelial growth and ganoderic acid synthesis in Ganoderma lucidum[J]. Journal of Edible Fungi, 2023, 30(2): 1-8.

[0040] All primer synthesis and sequencing were completed by Shanghai Sangon Biotechnology Co., Ltd.

[0041] The complete CYP512W2 gene sequence is shown in SEQ ID NO.1:

[0042]

[0043] The CYP512W2 gene sequence with a partial gene deletion is shown in SEQ ID NO.2:

[0044]

[0045] Example 1: Construction of Ganoderma lucidum engineered strains

[0046] 1. Construction of Ganoderma lucidum CYP512W2 gene deletion vector

[0047] The CRISPOR online tool (http: / / crispor.gi.ucsc.edu / ) was used to design dual sgRNA sequences targeting the Ganoderma lucidum CYP512W2 gene (SEQ ID NO. 1), sgRNA-1: 5'-gtactatccatattgcgact-3', sgRNA-2: 5'-gaattattgaccgttcgacc-3'.

[0048] Synthesize two pairs of primers:

[0049] CYP512W2-sg-1-F:5'-gtactatccatattgcgactgttttagagctagaaatagcaagtt-3',

[0050] CYP512W2-sg-1-R:5'-agtcgcaatatggatagtacaacggttgagggggacttagg-3';

[0051] and

[0052] CYP512W2-sg-2-F:5'-gaattattgaccgttcgaccgttttagagctagaaatagcaagtt-3'

[0053] CYP512W2-sg-2-R:5'-ggtcgaacggtcaataattcaacggttgagggggacttagg-3',

[0054] And paired with flanking primers pUC57-F:5'-aagcataaagtgtaaagcctggg-3' and pUC57-R:5'-ccaggctttacactttatgct-3' respectively.

[0055] Plasmid pUC57-PU6-26016-1-ophph was used as a template for PCR amplification. The PCR reaction system was: Max DNA Polymerase (Takara, China, R045Q) (25 μL), 1 μL each of forward and reverse primers (10 μmol L⁻¹), 10 ng of template, and 50 μL of ddH₂O. The reaction procedure was as follows: 98°C initial denaturation for 30 s; 30 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 5 s, and extension at 72°C for 1 min; then extension at 72°C for 5 min, followed by storage at 4°C. The amplified product was purified by agarose gel electrophoresis and subjected to homologous recombination using the ClonExpress MultiS One-Step Cloning Kit (Vazyme, China, C112).

[0056] The recombinant product was transformed into Escherichia coli DH5α competent cells (Tsingke, China, DH5α), and the transformants were verified by colony PCR using primers PUC57-PU6-F: 5'-gcacagatgcgtaaggagaaaat-3' and PU6-sgRNA-R: 5'-actcggtgccactttttcaagttg-3'. Plasmids were extracted and sequenced to obtain the CYP512W2 gene deleted plasmids pUC57-PU6-CYP512W2-1-ophph and pUC57-PU6-CYP512W2-2-ophph ( Figure 1 ).

[0057] 2. Construction of Ganoderma lucidum CYP512W2 gene deletion strain

[0058] 30 μg of each of the knockout plasmids pUC57-PU6-CYP512W2-1-ophph and pUC57-PU6-CYP512W2-2-ophph were co-transformed into the Ganoderma lucidum engineered strain pJW-EXP-intron-opCas9 (WT strain) expressing the Csa9 protein using the PEG-mediated protoplast transformation method (refer to Zhang Dehuai. High expression of oxidized squalene synthase gene alone and co-expression of 3-hydroxy-3-methylglutaryl-CoA reductase gene in Ganoderma lucidum to increase ganoderic acid production [D]. Kunming University of Science and Technology, 2018). Then, positive transformants were selected from CYM selective medium containing 250 mg / L hygromycin B and rescreened on PDA plates containing 250 mg / L hygromycin B. The transformants were verified using primers CYP512W2-F: 5'-atggcgacgttggaggaccc-3' and CYP512W2-R: 5'-tcaagaagcctgcgcatgcc-3'. The results are as follows: Figure 2 The PCR reaction system is: Max DNA Polymerase (Takara, China, R045Q) 25 μL, 1 μL each of forward and reverse primers (10 μmol L⁻¹), 100 ng of template, and up to 50 μL of ddH₂O. The reaction procedure was as follows: 98°C initial denaturation for 30 s; 30 cycles of 98°C denaturation for 10 s, 61.7°C annealing for 5 s, and 72°C extension for 1 min; 72°C extension for 5 min, and storage at 4°C.

[0059] Experimental results: The complete CYP512W2 gene band (2118 bp) was amplified in the WT strain, and bands of the same size as the WT strain were amplified in transformants 1, 3, and 6. No band was amplified in transformant 7, while bands of approximately 1000 bp were observed in transformants 2, 4, and 5. Transformant No. 2 was randomly selected for sequencing, confirming that the 952 bp sequence of the sgRNA-1 and sgRNA-2 target sites in transformant No. 2 was knocked out, successfully obtaining the Ganoderma lucidum CYP512W2 gene deletion engineered strain ΔW2 ( Figure 2 ).

[0060] Example 2: Preparation of Type I Ganoderic Acid Compound of Structural Formula (1)

[0061] (1) Fermentation of Ganoderma lucidum engineered strain ΔW2

[0062] The engineered Ganoderma lucidum strain ΔW2 prepared in Example 1 was inoculated into a primary shake flask and cultured in the dark at 150 rpm and 25°C for 7 days. The strain was then transferred to a secondary shake flask at a 10% volume fraction and cultured in the dark at 150 rpm and 25°C for 3 days. The strain was then transferred to a static culture for 21 days. The upper mycelium was collected and freeze-dried.

[0063] (2) Extraction and extraction of mycelium

[0064] The freeze-dried mycelia of Ganoderma lucidum was soaked and extracted three times with 95% by volume ethanol aqueous solution at room temperature, and the extracts were combined and concentrated to obtain the mycelia extract of Ganoderma lucidum;

[0065] The Ganoderma mycelium extract was further extracted three times with an equal volume of petroleum ether, the petroleum ether extract was discarded, and then extracted three times with an equal volume of ethyl acetate, the ethyl acetate extracts were combined, concentrated under reduced pressure, and dried to obtain an ethyl acetate extract;

[0066] (3) Preparation of Ganoderic Acid

[0067] The ethyl acetate extract was separated using YMC ODS C18 (particle size 50 μm, pore size 12 nm) as a filler, 0.01% by volume aqueous glacial acetic acid as phase A, and acetonitrile as phase B. Gradient elution was performed: 0-5 min, B: 55%; 5-10 min, B: 55%-60%; 10-30 min, B: 60%-70%; 30-35 min, B: 70%-75%; 35-60 min, B: 75%-80%; 60-80 min, B: 80%-100%; 80-100 min, B: 100%-100% at a flow rate of 200 mL / min. Fractions were collected every 400 mL and similar fractions were combined.

[0068] The fractions 40-42 containing the characteristic UV absorption peak of 215 nm containing ganoderic acid were further separated using a Zorbax Eclipse Plus C18 column with the following specifications: particle size 5 μm, diameter × column length: 4.6 mm × 250 mm. A 0.01% volume percent aqueous glacial acetic acid solution was used as phase A and acetonitrile was used as phase B. Gradient elution was performed: 0-15 min, B: 90%-100%; 15-30 min, B: 100%-100%. Detection was performed at a wavelength of 215 nm. Figure 3 ); collecting the chromatographic peak at 16.95 min to obtain compound 3α, 22α-diacetoxylanosta-8, 24-dien-26-oic acid ( Figure 4 ).

[0069] The separated compounds were subjected to optical rotation, ultraviolet, infrared, mass spectrometry and nuclear magnetic resonance detection. (c 0.250, MeOH); UV(MeOH)λ max (logε)213(0.635);IR(ATR)v max 2941,1732,1685,1374,1242cm -1 ;HRFIMSm / z:555.3697[MH] - (C 34 H 52 O6, calculated value 555.3691). 1H NMR(CDCl3,500MHz)δ:6.82(m,H-24),5.10(m,H-22),4.66(t,J=2.9Hz,H-3),2.56(m,H-23),2.38(m,H-23),2.07(s,COOHCH3-3), 2.05(s,COOHCH3-22),2.04(m,H-12),2.02(m,H-11),1.99(m,H-7),1.87(m,H-2),1.87(s,H-27),1.76(m,H-16),1.67(m,H-16),1. 64(m,H-2),1.61(m,H-15),1.61(m,H-17),1.60(m,H-6),1.54(m,H-20),1.49(m,H-5),1.49(m,H-6),1.46(m,H-1),1.43(m,H-15), 1.31(m,H-7),1.20(m,H-1),0.99(s,H-19),0.99(d,J=4.8Hz,H-21),0.92(s,H-29),0.90(s,H-30),0.86(s,H-28),0.69(s,H-18); 13 C-NMR(CDCl3,125MHz)δ:172.6(C-26),170.9(COOHCH3-3),170.7(COOHCH3-22),139.7(C-24),134.6(C-9),134.1(C-8) ,129.1(C-25),78.1(C-3),74.8(C-22),49.9(C-14),46.9(C-17),45.3(C-5),44.4(C-13),39.7(C-20),36.9(C-4,10),3 1.9(C-23),31.0(C-16),30.8(C-15),30.7(C-1),27.9(C-7),27.6(C-28),26.0(C-12),24.3(C-30),23.3(C-2),21.8(C- 29), 21.4(COOHCH3-3), 21.1(COOHCH3-22), 20.9(C-11), 19.0(C-19), 18.0(C-6), 15.6(C-18), 12.9(C-21), 12.3(C-27).

[0070] Example 2 Anti-inflammatory Activity Detection of Compounds

[0071] Take RAW264.7 cells in the logarithmic phase and dilute them to 2×10 5A single cell suspension of 100 cells / mL was inoculated into a 96-well plate, 194 μL of the cell suspension was added to each well, and after culturing at 37°C in a CO2 incubator for 24 h, 1 μL of the ganoderic acid compound 3α,22α-diacetoxylanosta-8,24-dien-26-oicacid prepared in Example 1 and the positive control dexamethasone were added respectively. The compound and the positive control were set at three initial concentration gradients of 5, 2, and 1 μmol / mL, and three replicates were set for each concentration.

[0072] After incubating the cells with the compound or positive control for another 1 hour, 5 μL of 100 μg / mL LPS was added to induce damage. An LPS group (5 μL of LPS and 1 μL of PBS) and a blank control group (6 μL of PBS) were also maintained. After 48 hours of incubation, the cell culture supernatant was aspirated and subjected to the following two tests:

[0073] (1) Effects of compounds on NO release from LPS-stimulated RAW264.7 cells:

[0074] Pipette 100 μL of cell culture supernatant into a microplate. Add 50 μL of Griess reagent to each well. Incubate at room temperature in the dark for 10 minutes. Measure the OD value at 543 nm using a microplate reader. Calculate the NO concentration in the cell culture supernatant of each group and the inhibition rate of NO release using a NaNO2 standard curve. Calculate the inhibition rate of the sample according to Publication 1.

[0075]

[0076] The half inhibition rate was calculated based on the inhibition rate under different concentration gradients. The experimental results showed that the ganoderic acid compound 3α,22α-diacetoxylanosta-8,24-dien-26-oic acid has a strong activity in inhibiting NO production, and its half inhibition rate IC 50 The value was 9.86nmol / mL, which was better than the IC of the positive control dexamethasone. 50 The value was 14.56 nmol / mL.

[0077] (2) Effects of ganoderic acid compounds on the expression of inflammatory factors in RAW264.7 cells induced by LPS:

[0078] The cell culture supernatant was aspirated and the TNF-α and IL-6 levels in the cell supernatant were detected according to the ELISA method instructions.

[0079] Experimental results ( Figure 5 、 Figure 6) showed that the ganoderic acid compound 3α,22α-diacetoxylanosta-8,24-dien-26-oic acid significantly inhibited LPS-induced TNF-α and IL-6 release in RAW264.7 cells. At a final concentration of 25 nmol / mL, TNF-α levels decreased by 62.56% and IL-6 levels decreased by 14.87%.

[0080] The above results indicate that ganoderic acid 3α,22α-diacetoxylanosta-8,24-dien-26-oic acid has a significant inhibitory effect on the release of NO and inflammatory factors by LPS-induced macrophages RAW264.7, showing its potential for development as an anti-inflammatory drug, health functional food or cosmetic additive.

Claims

1. A method for preparing an engineered Ganoderma lucidum strain ΔW2, characterized in that The steps include: Using dual-sgRNA-mediated CRISPR / Cas9 technology, we designed dual sgRNA sequences targeting the Ganoderma CYP512W2 gene: sgRNA-1: 5'-gtactatccatattgcgact-3', sgRNA-2: 5'-gaattattgaccgttcgacc-3'. Using PEG-mediated protoplast transformation, the Ganoderma CYP512W2 gene deletion plasmids pUC57-PU6-CYP512W2-1-ophph and pUC57-PU6-CYP512W2-2-ophph were co-transformed into Ganoderma pJW-EXP-intron-opCas9 cells. After selection with 250 mg / L hygromycin B and PCR verification, we successfully obtained the engineered Ganoderma lucidum strain ΔW2, which contained a 952 bp deletion in the CYP512W2 gene. The sequence of the Ganoderma lucidum CYP512W2 gene is shown in SEQ ID NO.

1.

2. The engineered Ganoderma lucidum strain ΔW2 prepared by the method of claim 1.

3. A type I ganoderic acid compound, the chemical structure of which is shown in structural formula (1):

4. The method for preparing the type I ganoderic acid compound according to claim 3, comprising the following steps: (1) Fermentation of Ganoderma lucidum engineered strain ΔW2 The engineered Ganoderma lucidum strain ΔW2 according to claim 2 was inoculated into a first-level shake flask and cultured under dark conditions at 150 rpm and 25°C for 7 days; the strain was transferred to a second-level shake flask at a volume percentage of 10%, and cultured under dark conditions at 150 rpm and 25°C for 3 days; and then cultured statically for 21 days; The culture medium in the primary and secondary shake flasks is formulated as follows: 30.0 g / L of anhydrous glucose, 3.0 g / L of yeast powder, 2.0 g / L of KH2PO4, and 2.0 g / L of MgSO4·7H2O, dissolved in 1 L of distilled water; (2) Extraction and extraction of mycelium The freeze-dried Ganoderma mycelium is soaked and extracted with 80-100% by volume ethanol aqueous solution at room temperature for 1-4 times, the extracts are combined and concentrated to obtain a Ganoderma mycelium extract; The Ganoderma mycelium extract is further extracted with an equal volume of petroleum ether for 1-4 times, the petroleum ether extract is discarded, and then extracted with an equal volume of ethyl acetate for 1-4 times, the ethyl acetate extracts are combined, and concentrated under reduced pressure to obtain an ethyl acetate extract; (3) Preparation of Ganoderic Acid The ethyl acetate extract was packed with YMC ODS C18, particle size 50 μm, pore size 12 nm, 0.01% by volume aqueous glacial acetic acid as phase A and acetonitrile as phase B, with gradient elution (0-5 min, B: 55%). 5-10min, B: 55%-60%; 10-30min, B: 60%-70%; 30-35min, B: 70%-75%; 35-60min, B: 75%-80%; 60-80 min, B: 80%-100%; 80-100 min, B: 100%-100%, separation, flow rate is 200 mL / min; collect one fraction every 400 mL, and combine similar fractions; The 40-42 fractions containing the 215nm ultraviolet characteristic absorption peak of ganoderic acid were further separated, and a Zorbax Eclipse Plus C18 chromatographic column was selected with the following specifications: particle size 5 μm, diameter × column length: 4.6 mm × 250 mm, and a 0.01% volume percentage glacial acetic acid aqueous solution as phase A and acetonitrile as phase B. Gradient elution was performed: 0-15 min, B: 90%-100%; 15-30 min, B: 100%-100%, and detection was performed at a wavelength of 215 nm. The chromatographic peak at 16.95 min was collected to obtain the desired compound.

5. Use of the type I ganoderic acid compound of structural formula (1) according to claim 3 in the preparation of drugs for preventing or treating inflammation.

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