A flavonol-producing Aspergillus pseudoacanthus strain and a method for constructing a gene editing system thereof

Abstract

The present invention discloses an Aspergillus aculeatinus SF1 strain, which has been deposited in the China Center for Type Culture Collection on July 7, 2023, with a deposit number of CCTCCNO.M20231229. The present invention also discloses the preparation of flavonols using the above-mentioned Aspergillus aculeatinus SF1 strain. The present invention also discloses a method for constructing a gene editing system for the above-mentioned Aspergillus aculeatinus SF1 strain. The Aspergillus aculeatinus SF1 provided by the present invention has a short culture cycle, simple culture medium components, and low cost, and can quickly and efficiently ferment and produce flavonoids (flavonols) substances in a short time. The Aspergillus aculeatinus SF1 provided by the present invention can also successfully prepare protoplasts and can be transformed by CRISPR gene editing technology.

Description

Technical Field

[0001] The invention relates to the field of microbial technology, and in particular to a flavonol-producing Aspergillus pseudoacanthus and a method for constructing a gene editing system thereof. Background Art

[0002] Flavonoids represent a highly diverse class of secondary plant metabolites, with approximately 9,000 structures identified to date. These compounds are found in all vascular plants as well as in some mosses. Even within the same species, many different flavonoids may occur. It has been established that flavonoids have a major impact on various aspects of plant biology. They exhibit a wide range of functions in physiology, biochemistry, and ecology, such as UV protection, flower color, interspecific interactions, and plant defense.

[0003] Other very remarkable properties of certain flavonoids are their nutritional value and medicinal value for humans, including antioxidant or anticancer activity. Flavonols, for example, may be the most common and largest flavonoid subgroup in fruits and vegetables, mainly including kaempferol, quercetin, myricetin, rutin, etc. Quercetin is a typical representative of flavonols and is widely found in plants such as fruits, vegetables and grains. Quercetin is relatively abundant in hawthorn, apple, pear, citrus fruits, and in traditional Chinese medicines such as Sophora japonica and Ginkgo biloba. Tea and red wine are also sources of flavonols. Epidemiological and animal studies have shown that a high intake of flavonols in the diet may be associated with a reduced risk of several cancers (such as lung and colon cancer), coronary heart disease, chronic inflammation and osteoporosis.

[0004] Aspergillus fungi are important chassis cells and research model organisms for industrial microorganisms and synthetic biology. Aspergillus can be used to produce cellulase, sugars, small molecules, and high-value alkaloids, flavonoids, paclitaxel precursors and other substances. Although the patent number CN102277304B discloses that Aspergillus aculeatus produces 5,7,8,4'-tetrahydroxyisoflavone, it is an isoflavone, not a flavonol. Isoflavone compounds are a unique subgroup of flavonoid compounds, and the distribution of isoflavones is very limited, almost confined to soybeans and some leguminous plants.

[0005] However, except for a few Aspergillus model fungi, most Aspergillus fungi are still difficult to transform through CRISPR gene editing technology, which greatly limits the potential economic value of this species. The prerequisite for gene editing is to be able to obtain high-quality fungal protoplasts. The cell wall of fungi has complex components and a solid structure, making it difficult to obtain high-quality protoplasts. The L1412 lytic enzyme produced by Sigma, which is commonly used in research, has been discontinued, and a new method for preparing protoplast hydrolysate is urgently needed.

[0006] For example, the article entitled "Two novel species of Aspergillus section Nigri from Thai coffee beans" discloses that Aspergillus pseudoacanthus is a uniseriate species, similar in morphology to Aspergillus aculeatus (a diiseriate species), but produces smaller conidia (2-5 μm). The present invention intends to obtain an Aspergillus pseudoacanthus that can produce flavonols and can be modified by CRISPR gene editing technology. Summary of the invention

[0007] In view of the above-mentioned shortcomings that currently exist, the present invention provides a strain of Aspergillus aculeatus that produces flavonols and a method for constructing a gene editing system thereof. The Aspergillus aculeatus of the present invention can produce flavonols and can be transformed by CRISPR gene editing technology.

[0008] In order to achieve the above object, the present invention provides an Aspergillus aculeatinus SF1 strain, the preservation number of which is CCTCC NO.M 20231229.

[0009] Based on the same inventive concept, the present invention also provides an application of the above-mentioned Aspergillus pseudoaculeatus, wherein the application is to prepare flavonols using the above-mentioned Aspergillus pseudoaculeatus.

[0010] According to one aspect of the present invention, the application comprises the following steps:

[0011] Step 1: adding Aspergillus pseudoacanthus SF1 bacterial liquid into liquid culture medium for cultivation to obtain fermentation liquid;

[0012] Step 2: The fermentation liquid is placed on a cell disruptor to be disrupted to obtain a fermentation liquid with completely disrupted cells;

[0013] Step 3: The fermented liquid after the crushing process is extracted and concentrated using an organic solvent to obtain a flavonol concentrate.

[0014] According to one aspect of the present invention, in step 1, the amount of Aspergillus pseudoacanthus SF1 bacterial solution added is 1-2wt% of the liquid culture medium, the liquid culture medium is PDB liquid culture medium, the culture time is 7-10d, the culture temperature is 28-30°C, and the culture speed is 180-200rpm.

[0015] According to one aspect of the present invention, in step 2, the crushing temperature is 28-30°C, the end diameter of the amplitude change rod used for crushing is 6mm, and the total crushing time is 20min, wherein each ultrasound is 2s and the interval is 3s.

[0016] According to one aspect of the present invention, step 3 specifically comprises:

[0017] Step 31: mixing the fermented liquid after the crushing treatment with ethyl acetate, leaving it to stand, and then separating to obtain an upper organic phase for later use;

[0018] Step 32: the fermentation liquid from which the upper organic phase is separated after the initial extraction is mixed again with ethyl acetate, the upper organic phase is taken after standing, and the organic phases collected from the two extractions are mixed;

[0019] Step 33: Concentrate the organic phase collected by extraction to obtain a flavonol concentrate.

[0020] Based on the same inventive concept, the present invention also provides a method for constructing a gene editing system based on the above-mentioned Aspergillus pseudoacanthus SF1 strain, comprising the following steps:

[0021] Step a: hydrolyzing Aspergillus pseudoacanthus SF1 with a combined enzyme hydrolyzing solution to prepare protoplasts; wherein the combined enzyme hydrolyzing solution is 3% lytic enzyme + 2% yatalase enzyme solution + 1% cellulase hydrolyzing solution;

[0022] Step b: adding plasmid p19ACas9g to the protoplasts, mixing, standing, adding PEG buffer, mixing, and standing; wherein the plasmid p19ACas9g is constructed by pUC19 plasmid and fungal plasmid clone fragment AMA1 plus Cas9 gene and sgRNA fragment;

[0023] Step c: After adding the STC solution, mix the system with the upper isotonic screening MM culture medium, spread it on the lower isotonic screening MM culture medium plate containing hygromycin, and after the upper culture medium solidifies, seal it with a sealing film and culture it at a constant temperature of 37°C for 2 days.

[0024] According to one aspect of the present invention, step a specifically comprises:

[0025] Step a1: inoculate Aspergillus pseudoacanthus SF1 into 100 mL PDB liquid culture medium and culture at 30° C. for 5-7 days;

[0026] Step a2: Use 0.1% Tween 80 solution to wash the surface of the bacteria, collect the spores, then centrifuge and wash twice with sterile water; inoculate the spores into 100 ml PDB liquid culture medium and control the spore concentration to 10 8 , 37°C, 180rpm, 6.5h;

[0027] Step a3: When the spores germinate to 2 to 3 times their length in volume, centrifuge at 8000 rpm, 4°C, 15 min, collect the spores in a sterile conical flask, suspend them in 10 mL of 3% lysozyme + 2% yatalase solution + 1% cellulase enzymatic solution that has been sterilized by filtration, and perform enzymatic hydrolysis;

[0028] Step a4: placing the above solution in a constant temperature shaking incubator, setting the temperature at 30°C and the rotation speed at 90 rpm, and observing it every half an hour after culturing for 2.5-4 hours; when the protoplasts are observed to be hydrolyzed under a microscope, washing with 1.2M sorbitol solution, and filtering the above solution with a falcon cell strainer to remove residual mycelium, and collecting the protoplast fluid;

[0029] Step a5: Place the above protoplast fluid in a centrifuge at 5000 rpm, room temperature, for 10 min; after centrifugation, remove the supernatant to obtain protoplasts.

[0030] According to one aspect of the present invention, the STC solution comprises: 1.2M sorbitol, 50mM calcium chloride, and 50mM Tris-HCl; the upper isotonic screening MM culture medium comprises: 1.2M sorbitol, 20×Salts solution 2%, 1000×trace elements solution 0.1%, glucose 1%, agar 1%, supplemented with 100ug / ml hygromycin, natural pH, and sterilized at 115°C for 20min; the lower isotonic screening MM culture medium comprises: 1.2M sorbitol, 20×Salts solution 2%, 1000×trace elements solution 0.1%, glucose 1%, agar 1.5%, supplemented with 100ug / ml hygromycin, natural pH, and sterilized at 115°C for 20min.

[0031] According to one aspect of the present invention, the PDB liquid culture medium comprises: 20% potato extract, 2% glucose, natural pH value, and sterilized at 115° C. for 20 min.

[0032] Beneficial effects of the present invention:

[0033] (1) The Aspergillus aculeatinus SF1 provided by the present invention has a short culture cycle, simple culture medium components, and low cost, and can quickly and efficiently ferment and produce flavonoids (flavonols) in a short time.

[0034] (2) The Aspergillus aculeatinus SF1 strain provided by the present invention can successfully prepare protoplasts and can be transformed by CRISPR gene editing technology, so it has potential economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 (a) shows the colonies formed by the Aspergillus pseudoacanthus SF1 strain described in Example 1 of the present invention growing on a PDA plate medium; Figure 1 (b) The colonies formed by the Aspergillus pseudoacanthus SF1 strain described in Example 2 of the present invention growing on PDB liquid culture medium;

[0036] Figure 2 This is the phylogenetic tree of the Aspergillus pseudoacanthus SF1 strain described in Example 1 of the present invention;

[0037] Figure 3 The chromatogram of the methanol washing liquid after the Aspergillus pseudoacanthus SF1 fermentation liquid described in Example 2 of the present invention is extracted;

[0038] Figure 4 This is an electron microscopic image of the protoplasts of the Aspergillus pseudoacanthus SF1 strain described in Example 3 of the present invention;

[0039] Figure 5 This is a gel imaging image of the colony PCR product described in Example 3 of the present invention. DETAILED DESCRIPTION

[0040] To make the present invention easier to understand, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by professional and technical personnel in the field; unless otherwise specified, the raw materials and reagents involved in this article can be purchased from the market or prepared by known methods.

[0041] the term:

[0042] Colony PCR does not require the extraction of target gene DNA or enzyme digestion identification. Instead, it directly uses the DNA exposed after pyrolysis of the bacteria as a template for PCR amplification and uses primers to screen positive clones. This PCR method is usually used to screen the inserted target gene or DNA sequencing analysis. The final PCR product size is the size of the fragment between the primers.

[0043] Example 1

[0044] Isolation, purification and identification of Aspergillus aculeatinus SF1

[0045] The Aspergillus aculeatinus SF1 is isolated from the ginkgo root of Yuelu Mountain in Changsha City, Hunan Province, and the specific steps are as follows:

[0046] Take an appropriate amount of ginkgo root samples from Yuelu Mountain, Changsha City, Hunan Province, and rinse the surface soil and impurities with running water. Place the rinsed ginkgo roots in an ultrasonic cleaner for ultrasonic cleaning (160W, 15min) to further remove the residual sand and other organic matter on the surface. In the clean bench, the ginkgo roots rinsed with sterile water were disinfected with 75% alcohol for 30s, and repeatedly rinsed with sterile water 5 times; then soaked in 4% sodium hypochlorite for 3min, and immediately rinsed with sterile water 5 times. Spread it flat on a plate containing sterile filter paper, and the sterile filter paper absorbs the surface moisture. Take 10 respectively. -4 , 10 -5 , 10 -6 100 μL of three gradient dilutions were evenly spread on a PDA plate medium, the composition of which was: potato flour 6.0 g / L; glucose 20.0 g / L; chloramphenicol 0.1 g / L; agar 20.0 g / L; the pH of the PDA plate medium at 25°C was 5.8-6.2. After culturing at 30°C for 3 days, a single brown colony was obtained (see Figure 1 (a)). Single colonies were selected and streaked multiple times to isolate pure bacteria. Finally, the isolated single bacteria were stored in a -80℃ refrigerator with 30% sterilized glycerol.

[0047] Pick the above strain cells of mung bean size from the fresh solid culture medium into a centrifuge tube, extract the strain DNA with a fungal genome extraction kit, and use ITS universal primers ITS1 and ITS4 for PCR amplification, wherein the primer sequence of ITS1 is shown in SEQ ID NO: 1, specifically: 5'-TCCGTAGGTGAACCTGCGG-3', and the primer sequence of ITS4 is shown in SEQ ID NO: 2, specifically: 5'-TCCTCCGCTTATTGATATGC-3'. The PCR product was sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the sequenced ITS DNA sequence was spliced ​​using Bioedit software, and then uploaded to the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), and BLAST sequence alignment and homology analysis were performed. The phylogenetic tree was constructed by the neighbor-joining method using MEGAX software. The results are shown in Figure 2. Figure 2 The results showed that the above strains had 100% homology with the sequence of Aspergillus aculeatinus CBS 121060.

[0048] The above-mentioned Aspergillus aculeatinus SF1 was sent to the China Center for Type Culture Collection for preservation on July 7, 2023. The address of the preservation unit is Wuhan University; classification name: Aspergillus aculeatinus SF1, preservation number: CCTCC NO.M 20231229.

[0049] Example 2

[0050] A method for preparing flavonols by using Aspergillus aculeatinus SF1, the specific steps are as follows:

[0051] Fermentation: The culture medium used for fermentation in this experiment is PDB culture medium. An appropriate amount of bacterial solution is selected from the strain storage tube with the storage number CCTCC M 20231229 and spread on the PDA flat culture medium. It is cultured in a constant temperature incubator at 30°C for 3-5 days to obtain a single colony. Pick a single colony and place it in a PDB liquid culture medium (20% potato extract, 2% glucose, natural pH, sterilized at 115°C for 20 minutes). Culture it at 30°C and 200rpm for 4 days to obtain a bacterial solution in the logarithmic growth phase. Then inoculate the bacterial solution into the PDB culture medium at a 1% inoculation rate and culture it for 7 days to obtain the fermentation liquid. The culture temperature is 28°C and the rotation speed is 180rpm. The morphology of the strain Aspergillus aculeatinus SF1 in the PDB liquid culture medium is as follows Figure 1 As shown in (b), it turns light yellow when cultured in liquid culture medium to the stable growth phase.

[0052] Extraction of fermentation broth: 200 mL of fermentation broth was placed on a cell disruptor, and the total ultrasonic time was set to 2 h, where the program was ultrasonic for 2 s and 3 s intermittently to obtain a fermentation broth with completely broken bacteria, so as to release the target substances that may exist in the bacterial cells; the fermentation broth after the crushing treatment was mixed with ethyl acetate in a ratio of 1:1, shaken to make them fully contact, and after standing, the lower organic phase was separated; the fermentation broth after the initial extraction was fully mixed with ethyl acetate again in a ratio of 2:1, and the upper organic phase was taken after standing. The organic phases collected from the two extractions were mixed; 300 mL of ethyl acetate extract was subjected to rotary evaporation, and the rotary distillation flask was washed with 5 mL of methanol three times (1, 2, 2 mL), and 5 mL of methanol washing liquid was collected. After the concentrated methanol washing liquid was filtered through a 0.22 μm filter membrane, an appropriate amount was taken and placed in a brown high-performance liquid sample bottle.

[0053] Preparation of standard samples: Accurately weigh 0.020g of 3,5,7,4'tetrahydroxy-8-methoxyflavone (sexangularetin), dissolve it in methanol and dilute it to 50mL to prepare a mother solution with a concentration of 0.2mg / mL. Dilute the mother solution to prepare a series of standard solutions of flavonoid standards, the concentration series is 0.100mg / mL, 0.050mg / mL, 0.025mg / mL, 0.0125mg / mL, 0.00625mg / mL. Pass the standard solution through a 0.22μm filter membrane and place it in a brown high-performance liquid phase sample bottle (stored at -20℃).

[0054] The detection conditions were set as follows: the autosampler was set to an injection volume of 10 μL, the total flow rate was 1.0 mL / min (A:B = 1:1); the column temperature was 35 °C; the detector was set to a detection wavelength of 360 nm; the extraction and elution program was set to stop at 0-10 min. Mobile phase A: acetonitrile, mobile phase B: double distilled water; chromatographic column: YMC-TriartC18 (250 mm × 4.6 mm, 5 μm).

[0055] Cleaning the chromatographic column: Before measuring the sample, use phase A and phase B to clean the chromatographic column for more than half an hour until no peak appears.

[0056] The above methanol washing solution and the standard product were subjected to HPLC detection. The test results were as follows Figure 3 shown.

[0057] Figure 3 The present invention is a chromatogram of a methanol washing liquid after extraction of a fermentation liquid of Aspergillus aculeatinus SF1. In the figure, it can be seen that an obvious peak is present at about 7.5 minutes, indicating that the bacteria produces 3,5,7,4'-tetrahydroxy-8-methoxyflavone (sexangularetin) by fermentation. According to calculation, after 7 days of fermentation, the yield of 3,5,7,4'-tetrahydroxy-8-methoxyflavone (sexangularetin) in the experimental group of Aspergillus aculeatinus SF1 is 5.21 mg / L, and the bacteria has the ability to naturally produce 3,5,7,4'-tetrahydroxy-8-methoxyflavone (sexangularetin), and is feasible for the fermentation of 3,5,7,4'-tetrahydroxy-8-methoxyflavone (sexangularetin).

[0058] Example 3

[0059] A method for constructing a gene editing system of Aspergillus pseudoacanthus SF1 strain

[0060] (1) Prepare strains and culture medium:

[0061] Strain: Aspergillus pseudoacanthus SF1;

[0062] Culture medium:

[0063] PDB liquid medium: 20% potato extract, 2% glucose, natural pH, sterilized at 115℃ for 20min;

[0064] Upper isotonic screening MM medium: 1.2M sorbitol, 20×Salts solution 2%, 1000×trace elements solution 0.1%, glucose 1%, agar 1%, supplemented with 100ug / ml hygromycin, natural pH, sterilized at 115℃ for 20min;

[0065] Lower isotonic screening MM medium: 1.2M sorbitol, 20×Salts solution 2%, 1000×trace elements solution 0.1%, glucose 1%, agar 1.5%, supplemented with 100ug / ml hygromycin, natural pH, sterilized at 115℃ for 20min;

[0066] STC solution: 1.2 M sorbitol, 50 mM calcium chloride, 50 mM Tris-HCl.

[0067] (2) Plasmid and DNA

[0068] The plasmid p19ACas9g used was constructed by combining the pUC19 plasmid and the fungal plasmid cloned fragment AMA1 plus the Cas9 gene and sgRNA fragment, with the hygromycin gene as the homologous insert.

[0069] (3) Preparation of protoplasts of Aspergillus pseudoacanthus

[0070] 1) Inoculate the fresh activated strain SF1 into 100 mL PDB liquid medium and culture at 30°C for 5-7 days;

[0071] 2) Use 0.1% Tween 80 solution to wash the surface of the bacteria, collect the spores, then centrifuge and wash twice with sterile water;

[0072] 3) Inoculate spores into 100 ml PDB liquid medium, control the spore concentration to 10 to the 8th power, 37°C, 180 rpm, 6.5 h;

[0073] 4) When the spores germinate to 2 to 3 times their length, centrifuge at 8000 rpm, 4°C, 15 min, collect the spores in a sterile conical flask, suspend them in 10 mL of 3% lysozyme + 2% yatalase solution + 1% cellulase enzymatic solution that has been sterilized by filtration, and perform enzymatic hydrolysis;

[0074] 5) Place the above solution in a constant temperature shaking incubator, set to 30°C and 90 rpm, incubate for 2.5-4 hours, and observe every half an hour;

[0075] 6) After the protoplasts are observed to be hydrolyzed under a microscope, they are rinsed with 1.2 M sorbitol solution;

[0076] 7) Filter the above solution through a falcon cell strainer to remove residual mycelium and collect the protoplast fluid;

[0077] 8) Place the above protoplast fluid in a centrifuge at 5000 rpm, room temperature, for 10 min;

[0078] 9) After centrifugation, remove the supernatant and suspend the protoplasts in STC solution to adjust the concentration to 10 7 / ml, and the obtained protoplasts were observed under an electron microscope. The electron microscope image is as follows Figure 4 As shown by Figure 4 It can be seen that the protoplasts of Aspergillus pseudoacanthus SF1 strain can be successfully prepared.

[0079] (4) Transformation of Aspergillus pseudoacanthus protoplasts

[0080] 1) Add 10ug of plasmid to 100uL of protoplasts, mix well by pipetting, and let stand on ice for 50min;

[0081] 2) Add 1.5 mL of PEG buffer, shake gently to mix, and let stand at room temperature for 20 min;

[0082] 3) Add 4 mL of STC solution, mix the system with the upper isotonic screening MM medium, and spread it on the lower isotonic screening MM medium plate containing 100 ug / ml hygromycin. After the upper medium solidifies, seal it with a sealing film and culture it at 37°C for 2 days.

[0083] (5) PCR identification and sequencing verification of transformants

[0084] 1) The protoplasts of Aspergillus pseudoacanthus SF1 from which the cell wall was removed were cultured in an isotonic screening MM medium (the upper layer was an isotonic screening MM medium, and the lower layer was a lower isotonic screening MM medium) for 2 days until they recovered into a small colony. The cells cultured for 2 days were dipped with a sterile bamboo stick, streaked on a screening medium (isotonic screening MM medium), and cultured again for 2 days.

[0085] 2) Scrape a small amount of growing mycelium, place it in a 0.5 mL EP tube, add 20 ul of sterile water, mix well, and place in a 90°C water bath for 5 min. After cooling, use it as a template.

[0086] Using the above mycelium as a template, primers were designed for sequences at both ends of the target gene for PCR amplification, and the amplified fragment was about 5000 bp.

[0087] hph-F: 5'-CATCGCCGAGTGAGTAGGTC-3'SEQ ID NO: 3

[0088] hph-R: 5'-TGCCCCCTGATCCCAATTTC-3'SEQ ID NO: 4

[0089] PCR system:

[0090]

[0091] PCR reaction procedure:

[0092]

[0093] Loop 32 times

[0094] PCR products were detected by 1% agarose gel electrophoresis, and observed and photographed using a UV gel imaging system. Figure 5 As shown by Figure 5 It can be seen that colony PCR products can be obtained, indicating that the gene editing system of Aspergillus pseudoacanthus SF1 strain was successfully constructed.

[0095] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. An Aspergillus aculeatinus SF1 strain, characterized in that The deposit number of this strain is CCTCCNO.M20231229.

2. A use of Aspergillus pseudoacanthus as claimed in claim 1, characterized in that: The application is to prepare flavonols using the Aspergillus pseudoacanthus SF1 strain described in claim 1.

3. The use according to claim 2, characterized in that: The application comprises the following steps: Step 1: adding Aspergillus pseudoacanthus SF1 bacterial liquid into liquid culture medium for cultivation to obtain fermentation liquid; Step 2: The fermentation liquid is placed on a cell disruptor to be disrupted to obtain a fermentation liquid with completely disrupted cells; Step 3: The fermented liquid after the crushing process is extracted and concentrated using an organic solvent to obtain a flavonol concentrate.

4. The use according to claim 3, characterized in that: In step 1, the amount of Aspergillus pseudoacanthus SF1 bacterial solution added is 1-2wt% of the liquid culture medium, the liquid culture medium is PDB liquid culture medium, the culture time is 7-10 days, the culture temperature is 28-30°C, and the culture rotation speed is 180-200rpm.

5. The use according to claim 3, characterized in that: In step 2, the crushing temperature is 28-30°C, the end diameter of the amplitude rod used for crushing is 6 mm, and the total crushing time is 20 minutes, wherein each ultrasound is 2 seconds and the interval is 3 seconds.

6. The use according to claim 3, characterized in that: The step 3 is specifically as follows: Step 31: mixing the fermented liquid after the crushing treatment with ethyl acetate, leaving it to stand, and then separating to obtain an upper organic phase for later use; Step 32: the fermentation liquid from which the upper organic phase is separated after the initial extraction is mixed again with ethyl acetate, the upper organic phase is taken after standing, and the organic phases collected from the two extractions are mixed; Step 33: Concentrate the organic phase collected by extraction to obtain a flavonol concentrate.

7. A method for constructing a gene editing system of Aspergillus pseudoacanthus SF1 strain as claimed in claim 1, characterized in that: The following steps are involved: Step a: hydrolyzing Aspergillus pseudoacanthus SF1 with a combined enzyme hydrolyzing solution to prepare protoplasts; wherein the combined enzyme hydrolyzing solution is 3% lytic enzyme + 2% yatalase enzyme solution + 1% cellulase hydrolyzing solution; Step b: adding plasmid p19ACas9g to the protoplasts, mixing, standing, adding PEG buffer, mixing, and standing; wherein the plasmid p19ACas9g is constructed by pUC19 plasmid and fungal plasmid clone fragment AMA1 plus Cas9 gene and sgRNA fragment; Step c: After adding the STC solution, mix the system with the upper isotonic screening MM culture medium, spread it on the lower isotonic screening MM culture medium plate containing hygromycin, and after the upper culture medium solidifies, seal it with a sealing film and culture it at a constant temperature of 37°C for 2 days.

8. The method for constructing the gene editing system of Aspergillus pseudoacanthus SF1 strain according to claim 7, characterized in that: Step a is specifically as follows: Step a1: inoculate Aspergillus pseudoacanthus SF1 into 100 mL of LPDB liquid culture medium and culture at 30° C. for 5-7 days; Step a2: Use 0.1% Tween 80 solution to wash the surface of the bacteria, collect the spores, then centrifuge and wash twice with sterile water; inoculate the spores into 100 ml PDB liquid culture medium and control the spore concentration to 10 8 , 37°C, 180rpm, 6.5h; Step a3: When the spores germinate to 2 to 3 times their length, centrifuge at 8000 rpm, 4°C, 15 min, collect the spores and place them in a sterile conical flask, suspend them in 10 mL of 3% lysozyme + 2% yatalase solution + 1% cellulase enzymatic solution that has been sterilized by filtration, and perform enzymatic hydrolysis; Step a4: placing the above solution in a constant temperature shaking incubator, setting the temperature at 30°C and the rotation speed at 90 rpm, and observing it every half an hour after culturing for 2.5-4 hours; when the protoplasts are observed to be hydrolyzed under a microscope, washing with 1.2M sorbitol solution, and filtering the above solution with a falcon cell strainer to remove residual mycelium, and collecting the protoplast fluid; Step a5: Place the above protoplast fluid in a centrifuge at 5000 rpm, room temperature, for 10 min; after centrifugation, remove the supernatant to obtain protoplasts.

9. The method for constructing the gene editing system of Aspergillus pseudoacanthus SF1 strain according to claim 7, characterized in that: The STC solution includes: 1.2M sorbitol, 50mM calcium chloride, 50mM Tris-HCl; the upper isotonic screening MM culture medium includes: 1.2M sorbitol, 20×Salts solution 2%, 1000×traceelements solution 0.1%, glucose 1%, agar 1%, supplemented with 100ug / ml hygromycin, natural pH, and sterilized at 115°C for 20min; the lower isotonic screening MM culture medium includes: 1.2M sorbitol, 20×Salts solution 2%, 1000×traceelements solution 0.1%, glucose 1%, agar 1.5%, supplemented with 100ug / ml hygromycin, natural pH, and sterilized at 115°C for 20min.

10. The method for constructing the gene editing system of Aspergillus pseudoacanthus SF1 strain according to claim 8, characterized in that: The PDB liquid culture medium comprises: 20% potato extract, 2% glucose, natural pH value, and sterilized at 115° C. for 20 minutes.

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