Application and method of GL10 gene, expression plasmid and genetically engineered bacterium in preparation of geterol

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application and a method of a GL10 gene, an expression plasmid and a genetically engineered bacterium in preparation of geterol. The invention provides an application of a GL10 gene with a nucleotide sequence as shown in SEQ ID NO.1 in preparation of geterol. According to the invention, high-efficiency expression of the GL10 gene can be realized by taking Aspergillus oryzae as a host cell so as to drive mass production of geterol. The method for preparing the geterol is simple to operate, short in culture period, simple and feasible in separation and purification and suitable for large-scale fermentation production, breaks through the resource limitation of traditional separation, can effectively solve the source problem of the geterol, and provides sufficient raw materials for synthesis of natural products containing meroterpenoid skeleton structures and related medical intermediates.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application and method of GL10 gene, expression plasmid and genetically engineered bacteria in preparing gyrophenol. Background Art

[0002] Geroquinol, also known as geranyl-HYQ, is the core skeleton of terpenoids and possesses a unique chemical structure. Its fundamental building blocks are terpenes, linked to heteroatoms (such as oxygen and nitrogen) or non-terpenoid fragments through specific chemical bonds, forming a complex molecular structure. This structure endows geroquinol with a rich chemical reactivity, enabling it to undergo complex chemical reactions such as oxidation and reduction under specific conditions, such as suitable temperature, pressure, and the presence of a catalyst. During oxidation, specific groups within the geroquinol molecule undergo electron transfer, resulting in changes in the molecular structure. During reduction, geroquinol accepts electrons, prompting molecular rearrangements, resulting in products with more complex structures and diverse properties. These modified products occupy a crucial position within the terpenoid natural product family, forming the fundamental building blocks of numerous compounds with unique properties and functions.

[0003] Compounds constructed with girucol as their structural backbone have demonstrated remarkable biological activity. In terms of antibacterial properties, these compounds can effectively interfere with bacterial metabolic processes. By binding to specific targets on the bacterial cell membrane, they disrupt the integrity of the cell membrane, leading to the leakage of intracellular substances and the inability to maintain homeostasis within the cellular environment. This significantly inhibits the growth and reproduction of a wide range of bacteria. In the field of anti-tumor therapy, these compounds have diverse mechanisms of action. On the one hand, they can induce apoptosis in tumor cells, prompting them to follow a specific program of cell death. On the other hand, they can inhibit the migration and invasion of tumor cells, hinder angiogenesis, and deprive them of nutrients, demonstrating potential anti-cancer efficacy. Regarding anti-inflammatory and immunomodulatory properties, girucol compounds can precisely regulate the expression of inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), achieving a balanced immune response and effectively alleviating the symptoms of inflammatory diseases. In the field of neuroprotection, these compounds, through their antioxidant properties, can scavenge free radicals generated by various factors in the body, reducing free radical damage to neurons, offering new possibilities for the treatment of neurological diseases. It can be seen that compounds constructed with girucol as the structural skeleton have potential application value in clinical drug development.

[0004] Although gyrophenol has been commercialized and its CAS registration number is 10457-66-6, its market price is extremely high. The main reason for the high price of gyrophenol is that its source is extremely limited. Currently, extracting gyrophenol from the metabolites of Ganoderma-related fungi is a feasible approach. However, this process faces many difficult problems: in the fermentation stage, the fermentation conditions are extremely sensitive and easily affected by factors such as temperature, pH, and culture medium composition, resulting in an extremely unstable fermentation process and difficulty in achieving stable and continuous production; from the perspective of yield, the content of gyrophenol in the metabolites of Ganoderma fungi is extremely low, and the output of each batch is very limited, which makes large-scale production face many obstacles; in the extraction and purification stage, gyrophenol is mixed with other metabolites, and its chemical structure is relatively special. Conventional extraction and purification methods are difficult to separate it efficiently, and complex and costly technical means are required, which undoubtedly further increases production costs. The above-mentioned problems such as unstable fermentation, low yield and difficulty in extraction and purification have seriously restricted the acquisition of gyrophenol through microbial production, resulting in its scarce market supply and high price.

[0005] UbiA prenyltransferase (UbiA-PT) is capable of prenylation using aromatic compounds of various structures as substrates, transferring geranyl (GPP) to hydroquinone (HYQ), ultimately producing the product geranol. Providing a gene encoding UbiA prenyltransferase, introducing it into a suitable expression system, and applying it to the geranol production process is a feasible and innovative strategy. Summary of the Invention

[0006] To address the above-mentioned deficiencies, the present invention provides the use of a GL10 gene, an expression plasmid, and a genetically engineered bacterium in the preparation of gyrophenol and a method thereof. The present invention provides the use of a GL10 gene having a nucleotide sequence as shown in SEQ ID NO.1 in the preparation of gyrophenol. The present invention uses Aspergillus oryzae as a host cell to achieve efficient expression of the GL10 gene, thereby driving large-scale production of gyrophenol. The method for preparing gyrophenol provided by the present invention is simple to operate, has a short culture cycle, and is simple and easy to separate and purify. It is suitable for large-scale fermentation production, overcomes the resource limitations of traditional separation, can effectively solve the problem of the source of gyrophenol, and provides sufficient raw materials for the synthesis of natural products containing a heteroterpene skeleton structure and related pharmaceutical intermediates.

[0007] The technical solution of the present invention is:

[0008] In a first aspect, the present invention provides an application of the GL10 gene in the preparation of gyrophenol, wherein the nucleotide sequence of the GL10 gene is shown in SEQ ID NO.1.

[0009] SEQ ID NO.1:

[0010] ATGTCGACGAAACCCCTCCTCGAGAACAAACAGGCCACGGCCAAGCCGCCCCAAACTTTTGCGTCCTCGGCATGGGCGTACTTCCGCCTCACCCGTTTGCACAAGTTCCCGGCAGGCTCTGACCTTATTTTCTGGCCTAGTGCCTGGGGCATAATGCTCTGCGCGAGGACCGAAAAACTCGACCCGACCACGATGTGGATCTCGTTGGCTGTCTGCATGATCTGTGGTACCCTGCGCCATAACGCTGGCTGCTGCTGGAACGACATCTGTGATATAGAGCTAGACCGTCAAGTCGAGCGATCCAAGAGCAGACCGCTGGCAAGCGGCGCTGTTTCCTTGGCAggcgccgtgctcgtgctcgctgCACACTATGCGGCTATTACTTGGTTGCTCAACTTCGCCGGGCCCGAAGGAAGACAAGTCGGACTCATCGGGCTATACATCCTCGATCCCATCTACCCCTTCATGAAGAGGTTGACCCATTGGCCCCAGGCGGTGCTCGGAATGGCTATGACTTGGGGGCTACCCACCACTTGGGTTTCCCTCCGCGGGTTCACGGACATGGACGCTCTGACTGTTCTCTATCTCGGTGGGATATGCTGGAGCATTTACTATGACACCATCTATGCGTGCCAGGACCGCCGCGATGACATCAAGGCCGGTGTGAAATCGACGGCGGTTTTGTTTGGAGACTACGTGCGCCCGATTGTATCGGTCTTCGCGACGTTGTTCGTCGCGTCTCTGGTTTACGCTGGGATGGTCACCGAGGCCGGCCCACTCTATTATGTGATTACGGTCGGAGGTGCGGCTGCCTGCTTTGCCTGGCAGATGATCACTATCGACTTCGACAATGGGGAGCAATGCTGGTCTGCGTTCAAGGCAAACGGCACCCTCGTTGGTTTCATTCTGTGGGCTGGGATGTTCGCGGATTACGCGAGTGTCGTTGACCTCGGCTTGTGGTAG。

[0011] Specifically, the GL10 gene exerts its effect by overexpression in a genetically engineered strain.

[0012] Preferably, the method for constructing the genetically engineered strain comprises: introducing an expression plasmid containing the GL10 gene into a host cell.

[0013] Further preferably, the vector backbone of the expression plasmid includes one or more of: PD2, pBR322, pUC18, pUC19, pET-28a, pET-32a, pPIC9K, pPIC9, and pPICZα.

[0014] Still more preferably, the vector backbone of the expression plasmid is a PD2 plasmid.

[0015] Further preferably, the method for constructing the expression plasmid comprises:

[0016] (1) Using Ganoderma lucidum genomic DNA as a template, the GL10 gene was amplified using a primer pair to obtain an amplified fragment;

[0017] (2) Connect the amplified fragment to the vector backbone, transform it into competent cells, screen the positive clones, and obtain the expression plasmid.

[0018] Still more preferably, the primer pair in step (1) is GL10-F and GL10-R, the nucleotide sequence of GL10-F is shown in SEQ ID NO.2; the nucleotide sequence of GL10-R is shown in SEQ ID NO.3.

[0019] SEQ ID NO.2:

[0020] ccggaattcgagctcggtaccATGTCGACGAAACCCCTCCTCGAGAAC.

[0021] SEQ ID NO.3:

[0022] ATCGGGTACGAcccgggtaccCTACCACAAGCCGAGGTCAACGACAC.

[0023] Still more preferably, the molar ratio of the amplified fragment to the vector backbone in step (2) is 1:1.

[0024] Still further preferably, the competent cells in step (2) include one or more of Escherichia coli cells, yeast competent cells, plant competent cells, and animal competent cells.

[0025] More preferably, the competent cells in step (2) are Escherichia coli DH5α.

[0026] More preferably, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0027] Still more preferably, the host cell is Aspergillus oryzae.

[0028] In a second aspect, the present invention provides use of an expression plasmid comprising a GL10 gene in the preparation of gyrophenol, wherein the nucleotide sequence of the GL10 gene is shown in SEQ ID NO.1.

[0029] Specifically, the vector backbone of the expression plasmid includes one or more of: PD2, pBR322, pUC18, pUC19, pET-28a, pET-32a, pPIC9K, pPIC9, and pPICZα.

[0030] Preferably, the vector backbone of the expression plasmid is a PD2 plasmid.

[0031] Preferably, the method for constructing the expression plasmid comprises:

[0032] (1) Using Ganoderma lucidum genomic DNA as a template, the GL10 gene was amplified using a primer pair to obtain an amplified fragment;

[0033] (2) Connect the amplified fragment to the vector backbone, transform it into competent cells, screen the positive clones, and obtain the expression plasmid.

[0034] Preferably, the primer pair in step (1) is GL10-F and GL10-R, the nucleotide sequence of GL10-F is shown in SEQ ID NO.2; the nucleotide sequence of GL10-R is shown in SEQ ID NO.3.

[0035] Preferably, the molar ratio of the amplified fragment to the vector backbone in step (2) is 1:1.

[0036] Preferably, the competent cells described in step (2) include one or more of Escherichia coli cells, yeast competent cells, plant competent cells, and animal competent cells.

[0037] Further preferably, the competent cells in step (2) are Escherichia coli DH5α.

[0038] In a third aspect, the present invention provides the use of a genetically engineered bacterium overexpressing the GL10 gene in the preparation of gyrophenol, wherein the nucleotide sequence of the GL10 gene is shown in SEQ ID NO.1.

[0039] Specifically, the GL10 gene exerts its effect by overexpression in a genetically engineered strain.

[0040] Preferably, the method for constructing the genetically engineered strain comprises: introducing an expression plasmid containing the GL10 gene into a host cell.

[0041] More preferably, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0042] Still more preferably, the host cell is Aspergillus oryzae.

[0043] Preferably, the method for constructing the genetically engineered strain comprises:

[0044] 1) Add buffer II and buffer III to Aspergillus oryzae protoplasts, then add the expression plasmid containing the GL10 gene, and mix and place in an ice bath;

[0045] 2) Add buffer III, incubate at room temperature, add buffer II, centrifuge, remove the supernatant, and then add buffer II again;

[0046] 3) Transfer the culture to a nutrient-deficient medium plate, cover with the medium, and culture inverted for 3-7 days. Screen for positive transformants and obtain genetically engineered bacteria after subculturing.

[0047] Further preferably, the method for preparing the Aspergillus oryzae protoplasts described in step 1) comprises inoculating Aspergillus oryzae spores into a culture medium, and culturing the culture medium with shaking to obtain Aspergillus oryzae cells; adding a cell wall lysate to the Aspergillus oryzae cells, and culturing the culture medium with shaking to obtain protoplasts;

[0048] Still further preferably, the culture medium is CD+Dextrin culture medium; the CD+Dextrin culture medium comprises: dextrin, NaNO3, KCl, KH2PO4, MgSO4·7H2O, 2FeSO4·7H2O, ammonium sulfate, methionine, Uracil and Uridine.

[0049] Still more preferably, the cell wall dissolving solution is prepared by dissolving saccharidase in solution I; and the solution I consists of sodium chloride solution and sodium dihydrogen phosphate solution.

[0050] More preferably, the ice bath time in step 1) is 18-20 minutes.

[0051] Further preferably, the buffer II in step 1) or step 2) consists of sorbitol, calcium chloride, sodium chloride and tromethamine (Tris).

[0052] Further preferably, the buffer III in step 1) or step 2) consists of polyethylene glycol-4000, calcium chloride and tromethamine.

[0053] More preferably, the incubation time at room temperature in step 2) is 18-20 minutes.

[0054] Further preferably, the nutrient-deficient culture medium in step 3) comprises: glucose, NaNO3, KCl, KH2PO4, MgSO4·7H2O, FeSO4·7H2O, ammonium sulfate, methionine, sorbitol and agar.

[0055] Further preferably, the covering medium in step 3) consists of sorbitol and agar powder.

[0056] More preferably, the inverted culture time in step 3) is 3-7 days.

[0057] In a fourth aspect, the present invention provides a method for preparing gyrophenol, which comprises using the GL10 gene, an expression plasmid comprising the GL10 gene, or a genetically engineered bacterium overexpressing the GL10 gene; the nucleotide sequence of the GL10 gene is shown in SEQ ID NO.1.

[0058] Specifically, the method includes:

[0059] S1. Introducing the expression plasmid containing the GL10 gene into the host cell to obtain a genetically engineered strain;

[0060] S2, inoculating the genetically engineered strain into a culture medium for fermentation, adding hydroquinone and continuing the culture, extracting, and concentrating to obtain a fermentation broth extract;

[0061] S3. The fermentation broth extract is segmented by high performance liquid chromatography, and the eluate corresponding to the chromatographic peak where giluophenol is located is collected, which is giluophenol.

[0062] Specifically, the culture medium in step S2 is a culture medium containing maltose.

[0063] Preferably, the culture medium in step S2 comprises: one or more of MPY medium, malt extract agar medium, PMA medium, MYA medium, DMYA medium, wort medium, and MEAYM medium.

[0064] Further preferably, the culture medium in step S2 is MPY culture medium.

[0065] Specifically, the fermentation culture conditions in step S2 are 25-28° C. for 12-48 hours.

[0066] Preferably, the fermentation culture condition in step S2 is 25-28° C. for 24 hours.

[0067] Specifically, the conditions for continuing the culture in step S2 are to continue the culture at 28-32° C. for 2-4 days.

[0068] Preferably, the continued culturing condition in step S2 is culturing at 30° C. for 3 days.

[0069] Specifically, the extraction in step S2 is performed using ethyl acetate for 3-5 times.

[0070] Preferably, the extraction in step S2 is performed by extracting with ethyl acetate three times.

[0071] Preferably, the concentration in step S2 is low-pressure concentration.

[0072] Specifically, the segmentation by high performance liquid chromatography in step S3 includes: using forward chromatography to roughly segment the fermentation broth extract, and then using reverse chromatography to finely segment it.

[0073] Preferably, the normal phase chromatography is normal phase chromatography with silica gel filler.

[0074] Preferably, the reverse phase chromatography is reverse phase chromatography with C18 filler.

[0075] Preferably, the wavelength detection of the high performance liquid chromatograph is 290 nm.

[0076] The beneficial effects of the present invention are:

[0077] The present invention provides the use of the GL10 gene, whose nucleotide sequence is shown in SEQ ID NO. 1, in the production of gyrophenol. The expression plasmid pD2-GL10 provided by the present invention carries the pAmyB amylase promoter, which can drive efficient expression of the GL10 gene when using maltose as a culture medium. The present invention uses Aspergillus oryzae as a host cell, which has strong amino acid synthesis capabilities and can provide abundant precursors for the MVA pathway. The GL10 gene can be efficiently expressed to drive large-scale production of gyrophenol.

[0078] The method for preparing gyrophenol provided by the present invention is simple to operate, has a short culture cycle, and is easy to perform separation and purification. It is suitable for large-scale fermentation production, overcomes the resource limitations of traditional separation methods, and can effectively solve the problem of gyrophenol's source, providing sufficient raw materials for the synthesis of natural products containing terpene skeleton structures and related pharmaceutical intermediates. This method can produce over 50 mg of gyrophenol per 1 L of liquid culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1HPLC analysis of gyrophenol; "AO-WT+HYQ" in the figure indicates that wild-type Aspergillus oryzae is fed with HYQ; "AO-GL10+HYQ" indicates that engineered Aspergillus oryzae (Aspergillus oryzae / pD2-GL10) is fed with HYQ.

[0080] Figure 2 This is the LC-MS detection spectrum of gyrophenol.

[0081] Figure 3 Girophenol 1 H NMR spectroscopy.

[0082] Figure 4 Girophenol 13 C NMR spectroscopy. DETAILED DESCRIPTION

[0083] The present invention will be further clarified and fully described below by way of examples. The following examples are only a portion of the present invention and are not intended to limit the present invention, but are merely for illustration. The experimental methods used in the following examples are all routine experiments unless otherwise specified, and the materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0084] Preparation of basic example 1 culture medium and reagents

[0085] 1. CD+Dextrin medium: 2 g dextrin, 0.3 g NaNO3, 0.2 g KCl, 0.1 g KH2PO4, 50 mg MgSO4·7H2O, 2 mg FeSO4·7H2O, 925 mg ammonium sulfate, 150 mg methionine, 0.2 g Uracil, 0.5 g Uridine, add water to 100 mL.

[0086] 2. Nutrient-deficient medium: 2 g glucose, 0.3 g NaNO3, 0.2 g KCl, 0.1 g KH2PO4, 50 mg MgSO4·7H2O, 2 mg FeSO4·7H2O, 925 mg ammonium sulfate, 150 mg methionine, 1.2 M sorbitol, 1.5 g agar, add water to 100 mL.

[0087] 3. Covering medium: composed of 1.2 M sorbitol and 0.5% agar powder.

[0088] 4. MPY medium: 3g maltose, 1g polypeptone, 0.5g yeast extract, 0.925g (NH4)2SO4, 0.15g methionine, 0.5g uridine, 0.2g uracil, add ddH2O to 100mL, and sterilize in an autoclave (121°C, 15min). When the corresponding amino acid is removed, the corresponding nutrient-deficient medium is obtained.

[0089] 5. Solution I: consists of 0.8 M sodium chloride solution and 10 mM sodium dihydrogen phosphate solution.

[0090] 6. Buffer II: composed of 1.2 M sorbitol, 50 mM calcium chloride, 50 mM sodium chloride and 10 mM tromethamine (Tris), with pH adjusted to 7.5.

[0091] 7. Buffer III: composed of 40% polyethylene glycol-4000, 50 mM calcium chloride and 50 mM tromethamine.

[0092] 8. Cell wall lysis solution: Prepared by dissolving glycosidase in solution I, the final concentration of glycosidase is 0.5% w / v.

[0093] Example 1 A method for preparing gyrophenol using engineered Aspergillus oryzae

[0094] A method for preparing gyrophenol using an engineered bacterium Aspergillus oryzae is carried out according to the following steps:

[0095] Step S1: Screening of GL10 gene:

[0096] First, the Ganoderma lucidum genome sequence was downloaded from the JGI database. The sequence was then compared with previously reported UbiA prenyltransferase genes from Basidiomycetes. Possible UbiA genes were identified using BioEdit. Using tools such as 2ndFind, antiSMASH, and Snapgene, three genes encoding potential UbiA prenyltransferases were identified. Among them, the GL10 gene was found to transfer geranyl (GPP) to p-phenol (HYQ), ultimately enabling the efficient production of the product geranyl-HYQ.

[0097] Step S2: Construction of expression plasmid pD2-GL10:

[0098] Ganoderma lucidum is a basidiomycete that synthesizes terpenoid compounds with a geranol skeleton. Its ITS NCBI accession number is KX358403.1. Using Ganoderma lucidum genomic DNA as a template, the GL10 gene (SEQ ID NO. 1) was amplified using primers GL10-F (SEQ ID NO. 2) and GL10-R (SEQ ID NO. 3). The GL10 gene was then mixed with the KpnI-digested pD2 plasmid at a 1:1 molar ratio and ligated using the ClonExpress MultiS One Step Cloning Kit. The ligation product was then transformed into Escherichia coli DH5α, cultured overnight, and positive clones were screened. Finally, the expression plasmid pD2-GL10 was obtained through PCR and enzyme digestion verification.

[0099] Step S3: Obtaining an engineered Aspergillus oryzae bacterium carrying the GL10 gene:

[0100] Aspergillus oryzae spore is accessed CD+Dextrin substratum, shaking culture 2-3 days, obtains aspergillus oryzae thalline.Add cell wall lysate, vibrate 2-3 hour, obtain protoplast.Add buffer II and buffer III, then add pD2-GL10 plasmid, ice bath 18-20 minute after mixing.Add buffer III subsequently, incubate at room temperature 18-20 minute, then add buffer II, add buffer II after centrifugation and remove supernatant, be transferred to nutrient deficiency type culture flat board, cover substratum and invert culture 3-7 days.PCR detects positive transformant, and subculture 2 times obtains aspergillus oryzae engineering bacteria (Aspergillus oryzae / pD2-GL10).

[0101] Step S4: Preparation of gyrophenol using engineered Aspergillus oryzae

[0102] (1) The mycelia of the engineered Aspergillus oryzae bacteria (Aspergillus oryzae / pD2-GL10) carrying the GL10 gene were inoculated into a sterilized MPY medium at an inoculum size of 1%, and the culture was statically fermented at 25-28°C for 1 day. After the fermentation was cultured for 1 day, 20 mg / L HYQ substrate was added for feeding. After culturing at 30°C for 3 days, a fermentation broth was obtained, which was a crude extract of the metabolites of the engineered Aspergillus oryzae bacteria.

[0103] The crude extract of the metabolites of wild-type Aspergillus oryzae was obtained by the same operation steps as above as a control. The crude extract of the metabolites of engineered Aspergillus oryzae and the crude extract of the metabolites of wild-type Aspergillus oryzae were analyzed by HPLC. Figure 1 shown.

[0104] (2) The crude extract of the metabolites of the engineered Aspergillus bacteria was extracted three times with equal amounts of ethyl acetate, and the combined extracts were concentrated under low pressure to obtain a fermentation broth extract. The fermentation broth extract was roughly fractionated using normal phase chromatography with silica gel packing, and the segments where gyrophenol was distributed were preliminarily determined using TLC. The segments containing gyrophenol were combined and then further subdivided using reverse phase chromatography with C18 packing. The location of gyrophenol was determined using TLC, and finally, the extract was prepared using an Agilent HPLC1260 Infinity II liquid chromatograph at a wavelength of 290 nm. According to the characteristic retention time of gyrophenol, the eluent corresponding to the chromatographic peak where gyrophenol was located was collected to obtain pure gyrophenol (purity>98%) with a yield of 80%.

[0105] Its structure was confirmed by 400M nuclear magnetic spectrometry, and its structural data (solvent is deuterated chloroform) are shown in Table 1:

[0106] Table 1

[0107]

[0108]

[0109] The normal phase chromatographic filler used in the above separation and preparation process is 200-300 mesh silica gel, the reverse phase chromatographic filler is C18 chromatographic filler, the high performance liquid chromatograph used in the preparation is Agilent 1260 Infinity II, the chromatographic column is Agilent ZORBAXRX-C18, 9.4×250 mm, and the detection wavelength is 290 nm.

[0110] The above detailed description is a specific description of one feasible embodiment of the present invention and is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the patent of this invention should be based on the attached requirements.

Claims

1. The application of GL10 gene in the preparation of gyrophenol is characterized in that: The nucleotide sequence of the GL10 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The GL10 gene exerts its effect by overexpression in a genetically engineered strain.

3. The use according to claim 2, characterized in that The method for constructing the genetically engineered strain comprises: introducing an expression plasmid containing the GL10 gene into a host cell.

4. The use according to claim 3, characterized in that The vector skeleton of the expression plasmid includes one or more of PD2, pBR322, pUC18, pUC19, pET-28a, pET-32a, pPIC9K, pPIC9, and pPICZα.

5. The use according to claim 3, characterized in that The host cells include prokaryotic cells or eukaryotic cells.

6. The use according to claim 5, characterized in that The host cell is Aspergillus oryzae.

7. Use of an expression plasmid comprising the GL10 gene in the preparation of gyrophenol, characterized in that: The nucleotide sequence of the GL10 gene is shown in SEQ ID NO.

1.

8. Use of a genetically engineered bacterium overexpressing the GL10 gene in the preparation of gyrophenol, characterized in that: The nucleotide sequence of the GL10 gene is shown in SEQ ID NO.

1.

9. A method for preparing gyrophenol, characterized in that: The method comprises using the GL10 gene, an expression plasmid containing the GL10 gene, or a genetically engineered bacterium that overexpresses the GL10 gene; the nucleotide sequence of the GL10 gene is shown in SEQ ID NO.

1.

10. The method according to claim 9, characterized in that The method includes: S1. Introducing the expression plasmid containing the GL10 gene into the host cell to obtain a genetically engineered strain; S2, inoculating the genetically engineered strain into a culture medium for fermentation, adding hydroquinone and continuing the culture, extracting, and concentrating to obtain a fermentation broth extract; S3. The fermentation broth extract is segmented by high performance liquid chromatography, and the eluate corresponding to the chromatographic peak where giluophenol is located is collected, which is giluophenol.

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