Glucan synthase mutants with improved enzyme activity, preparation thereof and application in drug screening

Abstract

The present invention relates to a mutant of glucan synthase with improved enzyme activity and its preparation and application in drug screening, belonging to the technical fields of bioengineering, microbial fermentation technology and drug screening technology. The present invention provides a recombinant β-1,3-glucan synthase mutant with improved enzyme activity, its construction method, product determination method and application in drug screening. The present invention mutated the gene sequence of natural β-1,3-glucan synthase (FKS1), and on this basis, obtained a recombinant β-1,3-glucan synthase mutant FKS1-S643P of yeast, whose ability to synthesize glucan was significantly improved after the addition of caspofungin. In addition, compared with natural FKS1, the FKS1-S643P mutant is tolerant to the echinocandin antifungal drug caspofungin but highly sensitive to the novel modified echinocandin antifungal drug micafungin. Therefore, this mutant can also be applied to the research and development of novel antifungal drugs.

Description

Technical Field

[0001] The present invention belongs to the technical fields of bioengineering, microbial fermentation technology and drug screening technology. More specifically, it relates to a glucan synthase mutant with improved enzyme activity, and its preparation and application in drug screening. Background Art

[0002] β-glucan is a natural dietary fiber that exists in various organisms such as plants, fungi, bacteria, and algae. Although β-glucan uses glucose as the condensation unit, it is different from common polysaccharides (such as starch, dextrin, etc.). The main difference lies in the different bond connection methods: the macromolecules of common polysaccharides are bound by α-1,4-glycosidic bonds, while β-glucan is mainly composed of β-1,3-glycosidic bonds and contains some branches of β-1,6-glycosidic bonds or β-1,4-glycosidic bonds. Studies have found that β-glucan has functions such as regulating blood lipids, reducing cholesterol, maintaining intestinal flora, skin moisturizing, and wound repair. Therefore, it can be widely used in industries such as medicine, health food, cosmetics, and animal feed.

[0003] In the food industry, β-1,3-glucan produced by yeast has high viscosity, water-holding capacity, emulsification stability and other properties, and is often used as a thickener, water-holding agent, binder and emulsification stabilizer in the food industry. Since β-1,3-glucan is difficult to be digested in the human digestive organs, it can be used as a non-calorie food additive. Moreover, β-1,3-glucan can promote cholesterol excretion and increase intestinal peristalsis, so it is also a high-quality health food additive. The Interpretation of Document No. 5 in 2021 on the official website of the National Health Commission of China, "Announcement on 6 'New Foods' such as β-1,3 / α-1,3-glucan" (No. 5 in 2021), has included β-1,3-glucan produced by biological fermentation in the new food raw materials.

[0004] In the field of medical beauty, high-purity β-1,3-glucan produced by yeast can form a barrier on the skin surface due to its large molecular weight. Moreover, there are specific binding sites for β-1,3-glucan on cells such as human Langerhans cells, which promotes the binding of β-1,3-glucan to intracellular receptors (Dectin-1) to improve the activity of T cells and B cells and maintain the balance of the skin immune system. Therefore, it can be applied to promote skin wound healing.

[0005] In the aspects of animal breeding and feed industry, yeast β-1,3-glucan is a good natural immune enhancer. Adding β-1,3-glucan as an antibiotic substitute to the daily diets of livestock and poultry such as pigs and chickens can significantly improve the humoral immunity and cellular immunity functions of livestock and poultry. Therefore, it is widely used in the animal breeding and feed industry.

[0006] Currently, the main methods for industrial extraction of yeast β-1,3-glucan are hot water process, hot alkali process, and enzymatic hydrolysis process. The yields of β-1,3-glucan by the above extraction methods are highly dependent. The enzyme that catalyzes the synthesis of β-1,3-glucan in yeast is glucan synthase FKS on its cell membrane, and this enzyme synthesizes the product β-1,3-glucan using UDP-glucose as a substrate. Therefore, improving the enzyme activity of FKS plays an important role in increasing the yield of β-1,3-glucan.

[0007] The research group of this application has established in previous patents (2022105713334) the in vitro preparation and in vitro enzyme activity determination methods of wild-type FKS1. Through research, it is found that adjusting and applying this preparation method and enzyme activity detection method to the FKS1-S643P mutant is beneficial for screening novel echinocandin drugs that are more sensitive to fungi. Summary of the Invention

[0008] The present invention solves the technical problem in the prior art that the enzyme activity of glucan synthase is not high, resulting in a relatively high preparation cost of β-1,3-glucan. The present invention constructs and screens a high-activity mutant FKS1-S643P of glucan synthase. The yield of the product of this mutant after 24 hours is significantly increased compared to the wild-type glucan synthase without an inducer after adding a drug such as caspofungin. This method provides a broad application prospect for increasing the yield of industrial products and reducing the preparation cost of β-1,3-glucan.

[0009] According to the first aspect of the present invention, there is provided a gene expressing a mutant of β-1,3-glucan synthase, characterized in that the base sequence of the gene is as shown in SEQ ID NO:1.

[0010] According to another aspect of the present invention, there is provided a genetically engineered yeast containing the gene expressing the mutant of β-1,3-glucan synthase.

[0011] According to another aspect of the present invention, there is provided a mutant of β-1,3-glucan synthase, which is expressed by the genetically engineered yeast, and the amino acid sequence of the mutant is as shown in SEQ ID NO:2.

[0012] According to another aspect of the present invention, there is provided an in vitro preparation method of the mutant of β-1,3-glucan synthase, comprising the following steps:

[0013] (1) Culturing the genetically engineered yeast and collecting the cells, lysing the cells and collecting the cell membrane;

[0014] (2) Dissolve the cell membrane obtained in step (1) in a membrane-lysing buffer containing N-dodecyl-β-D-maltoside and cholesteryl hemisuccinate, and centrifuge to collect the supernatant.

[0015] (3) Purify the β-1,3-glucan synthase mutant using anti-FLAG M2 affinity gel and elute it with an elution buffer containing FLAG peptide, where the elution buffer contains diosgenin glycoside, thus obtaining the β-1,3-glucan synthase mutant.

[0016] According to another aspect of the present invention, there is provided a method for in vitro establishing the synthesis of β-1,3-glucan product by fungal glucan synthase, comprising the following steps:

[0017] (1) Add the fungal glucan synthase or the mutant thereto into a reaction system containing cofactor GTP-γ-S and Rho1; subsequently add uridine diphosphate-glucose substrate and conduct an enzymatic activity reaction.

[0018] (2) After the reaction, centrifuge. The milky white precipitate is β-1,3-glucan. The more the milky white precipitate is, the higher the yield of β-1,3-glucan; or after the reaction, add aniline blue solution, incubate in the dark, and the brighter the fluorescence under a fluorescence microscope, the higher the yield of β-1,3-glucan.

[0019] Preferably, the reaction system further contains an inducer for inducing an increase in the yield of β-1,3-glucan.

[0020] Preferably, the inducer is caspofungin.

[0021] According to another aspect of the present invention, there is provided a method for a drug-induced synthesis of glucan product by genetically engineered yeast, comprising the following steps:

[0022] (1) Culture the genetically engineered yeast until the adaptation phase, add an inducer, then culture until the exponential phase, and centrifuge to collect yeast cells.

[0023] (2) After suspending the cells obtained in step (1) with a buffer, add an alkali solution, incubate in a water bath to dissolve the glucan synthesized by the cells; then add aniline blue solution for incubation.

[0024] (3) Use a fluorescence microplate reader to read the fluorescence value. Take the fluorescence value measured from the wild-type yeast without inducer as the standard value of 100. The ratio of the fluorescence value measured from the mutant after adding the inducer to the fluorescence value measured from the wild-type yeast without inducer is defined as the relative activity of the mutant after adding the inducer. When the relative activity is higher than 100, it means that the yield of glucan synthesized by the genetically engineered yeast increases; when the relative activity is equal to 100, the yield of glucan synthesized by the genetically engineered yeast remains unchanged; when the relative activity is lower than 100, the yield of glucan synthesized by the genetically engineered yeast decreases.

[0025] According to another aspect of the present invention, there is provided the use of the β-1,3-glucan synthase mutant for screening antifungal drugs, including the following steps:

[0026] (1) Incubate the drug to be screened with the β-1,3-glucan synthase mutant, and then add it to the reaction system. The reaction system contains cofactor GTP-γ-S and Rho1; subsequently, add uridine diphosphate-glucose substrate for enzymatic activity reaction; add a fluorescence chromogenic reagent, and use a microplate reader to read the fluorescence value;

[0027] (2) Take the fluorescence value measured from the in vitro enzymatic activity of the wild-type β-1,3-glucan synthase before adding the drug as the standard value of 10. The ratio of the fluorescence value measured from the mutant after adding the drug to the fluorescence value measured from the wild-type before adding the drug is defined as its relative activity. If the relative activity value is lower than 3, the β-1,3-glucan synthase mutant is highly sensitive to the drug; if the relative activity value is between 3 and 10, the β-1,3-glucan synthase mutant is sensitive to the drug; if the relative activity value is between 10 and 30, the β-1,3-glucan synthase mutant is insensitive to the drug; if the relative activity value is above 30, the β-1,3-glucan synthase mutant is tolerant to the drug.

[0028] Preferably, the drug to be screened is an echinocandin antifungal drug.

[0029] Preferably, the echinocandin antifungal drug is micafungin.

[0030] Generally speaking, compared with the prior art through the above technical solutions conceived by the present invention, the following technical advantages are mainly achieved:

[0031] (1) The present invention constructs and screens a highly active mutant FKS1-S643P of glucan synthase FKS. The yield of the product of this mutant after 24 hours of adding an inducer such as caspofungin is significantly higher than that of the wild-type FKS1 without adding the inducer. This method provides a broad application prospect for improving the yield of industrial products and reducing the preparation cost of β-1,3-glucan, and lays a theoretical and practical foundation for the large-scale production of glucan by genetic engineering methods.

[0032] (2) FKS1 is also the target of echinocandin antifungal drugs (including the first-generation drug caspofungin and the second-generation drug micafungin). Echinocandin antifungal drugs belong to semi-synthetic lipopeptides, which can non-competitively inhibit β-1,3-glucan synthase, interfere with the synthesis of β-1,3-glucan in fungal cells, cause changes in the permeability of the fungal cell wall, and then cause fungal cell lysis and death. Since human cells do not contain cell walls, echinocandin antifungal drugs can directly act on the components of the fungal cell wall, so this type of drug has low toxicity to the human body and is the safest class of antifungal drugs to date. Through secondary structure alignment, we found that the yeast S643 site corresponds to the FKS1-S645 site of Candida Albicans, a common pathogenic fungus in humans. This site is a common drug-resistant mutation site in Candida Albicans, but the significance of the mutation at the S643 site in yeast has not been reported, and there is no in vitro screening method for the antifungal drug activity using the purified Saccharomyces cerevisiae FKS1-S643P mutant (and corresponding site mutants in other fungal FKSs) as the detection target. In this invention, through the establishment of an in vitro enzyme activity detection method, it was found that the FKS1-S643P mutant is tolerant to the echinocandin antifungal drug caspofungin and highly sensitive to the new improved antifungal drug micafungin compared with the wild-type FKS1. Through the structural research of our research group, it was found that the mutation at the S643 site changes the lipid environment of FKS1 binding to the cell membrane and thus changes the binding state with lipid-containing drugs. This change may provide a more favorable environment for FKS1 to synthesize glucan. Therefore, the FKS1-S643P mutant can also be used for the research and development of new antifungal drugs.

[0033] (3) Preferably, in the preparation of the β-1,3-glucan synthase mutant, the membrane-lysing buffer is a membrane-lysing buffer for maintaining protein stability, containing N-dodecyl-β-D-maltoside (DDM) and cholesteryl hemisuccinate (CHS) to promote the solubility of membrane proteins and maintain the interaction between proteins and lipids; preferably, the elution buffer contains glycodiosgenin (GDN) to maintain the stability of membrane proteins. Description of the Drawings

[0034] Figure 1 : Wild-type FKS1 and mutant FKS1-S643P proteins purified in vitro.

[0035] Figure 2 : Detection of in vitro synthesized β-1,3-glucan products by direct observation method.

[0036] Figure 3 : Detection of in vitro synthesized β-1,3-glucan products by fluorescence observation method.

[0037] Figure 4 : The level of β-1,3-glucan product synthesized by genetically engineered yeast under the induction of CAS.

[0038] Figure 5 : The changes in enzyme activity of FKS1 and FKS1-S643P under the action of CAS or MFG. Detailed implementation manners

[0039] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0040] The objective of the present invention is to provide a β-1,3-glucan synthase mutant FKS1-S643P with increased enzyme activity after drug induction, its construction method and product determination method; the mutant of the present invention has high enzyme activity and high substrate conversion rate, and has great industrial application potential.

[0041] The present invention also provides a method for expressing and purifying a β-1,3-glucan synthase mutant FKS1-S643P.

[0042] The present invention also provides an in vitro enzyme activity determination method for a β-1,3-glucan synthase mutant.

[0043] The present invention also provides a method for in vitro synthesizing a β-1,3-glucan product, and a method for in vitro detecting this product.

[0044] The present invention also provides a genetically engineered Saccharomyces cerevisiae expressing a β-1,3-glucan synthase mutant at the genomic level, and a method for detecting the β-1,3-glucan product synthesized by the genetically engineered yeast.

[0045] The present invention also provides a method for significantly increasing the level of β-1,3-glucan product synthesized by a genetically engineered yeast expressing a β-1,3-glucan synthase mutant at the genomic level under the induction of the antifungal drug caspofungin.

[0046] The present invention also provides a β-1,3-glucan synthase mutant that is tolerant to the traditional echinocandin antifungal drug caspofungin and highly sensitive to the new improved antifungal drug micafungin, providing a specific target for screening new echinocandin drugs and a basis for the research and development of antifungal drugs.

[0047] The present invention provides the application of a mutant of β-1,3-glucan synthase that is tolerant to the traditional echinocandin antifungal drug caspofungin and highly sensitive to the novel modified antifungal drug micafungin by using an in vitro enzyme activity detection method.

[0048] Example 1: Provide the amino acid sequence of the FKS1-S643P mutant protein

[0049] (1) The FKS1-S643P mutant was obtained by homologous recombination to obtain a mutant at the yeast genome level.

[0050] (2) The amino acid sequence of the FKS1-S643P mutant protein is as follows: namely SEQ ID NO:2.

[0051] Example 2: In vitro preparation method of FKS1 and FKS1-S643P mutant proteins

[0052] (1) Using the method of site-directed mutagenesis by PCR, the bases TCT at positions 2927-2929 of the wild-type fks1 gene were mutated to CCA, which is the sequence shown in SEQ ID NO:1, so that the serine S at position 643 of the expressed mutant was changed to proline P.

[0053] (2) Using a PCR-based gene recombination method, a 3×FLAG tag was ligated to the C-terminus of the fks1 gene sequence of the Saccharomyces cerevisiae strain chromosome or the gene sequence after mutation of the bases at positions 2927-2929.

[0054] (3) The strain was cultured in YPD medium at 30 °C for 20 hours. Then the cells were collected by centrifugation and resuspended in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM MgCl2 (supplemented with 1 mM PMSF)), and then lysed by high-pressure fragmentation method.

[0055] (4) Centrifuge at 15,000×g for half an hour to remove cell debris. Then ultracentrifuge the supernatant at 100,000×g for 1 hour to collect the cell membrane.

[0056] (5) The collected membrane was dissolved in membrane lysis buffer (50 mM Tris-HCl pH 7.4, 500 mM NaCl, 2 mM MgCl2, 10% (v / v) glycerol, 1 mM PMSF, 1.5% (w / v) DDM, 0.15% (w / v) CHS), gently stirred for 2 hours, and then the supernatant was collected by centrifugation at 15,000×g for half an hour.

[0057] (6) Purify FKS1 and FKS1-S643P proteins using anti-FLAG M2 affinity gel (Sigma), and elute with elution buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM MgCl2, 0.04% GDN) containing 150 μg / ml 3×FLAG peptide.

[0058] (7) The eluted protein was further eluted through a molecular sieve (Superose 6 10 / 300GL column) with elution buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM MgCl2, 0.04% GDN).

[0059] (8) Detect the purification purity of FKS1 and FKS1-S643P mutant proteins by SDS-PAGE. As Figure 1 shown, it can be seen from Figure 1 that the purification purity of FKS1 and FKS1-S643P mutant proteins with a molecular weight of about 200 KD is above 90% after purification.

[0060] Example 3: Establishment of an in vitro synthesis system for β-1,3-glucan products catalyzed by fungal glucan synthase FKS1 and an observation method for in vitro synthesis

[0061] (1) The in vitro synthesis reaction of fungal β-1,3-glucan is as follows: Add 0.02 mg / ml FKS1 to initiate the reaction in 100 μL reaction buffer (50 mM Tris-HCl pH 7.4, 33% glycerol, 1 mM EDTA, 6 μg / ml Rho1, 0.2% CHAPS, 0.04% CHS, 4 μM GTP[γ]S, 20 mM KF) containing 2.5 mM substrate UDP-glucose, and the reaction temperature is 30°C.

[0062] (2) Direct observation method for in vitro synthesis of β-1,3-glucan: After reacting for 48 hours, centrifuge the sample at 5000×g for 5 minutes, and observe the formed milky white product under visible light. As Figure 2 shown, it can be seen from Figure 2 that the more milky white precipitate, the higher the yield of β-1,3-glucan.

[0063] (3) Fluorescent observation method for in vitro synthesis of β-1,3-glucan: After reacting the above reaction system for 24 hours, add an equal volume of 0.03% aniline blue solution (0.03% aniline blue, 0.18 M HCl, 0.49 M glycine-NaOH, pH 9.5), incubate at room temperature in the dark for 20 minutes, then load each sample into a capillary tube, and observe the product level under a fluorescence microscope. As Figure 3As shown by Figure 3 it can be seen that when observing the brightness under a fluorescence microscope, the brighter the fluorescence, the higher the yield of β-1,3-glucan.

[0064] Example 4: Method for increasing the yield of the synthesis product of the glucan synthase FKS1-S643P mutant induced by caspofungin

[0065] (1) The β-1,3-glucan product synthesis system catalyzed by the corresponding FKS1 mutant refers to Example 3. For example, in the β-1,3-glucan product synthesis system catalyzed by the FKS1-S643P mutant, the 0.02 mg / ml FKS1 to be added is replaced with the in vitro prepared 0.02 mg / ml FKS1-S643P protein.

[0066] (2) Before the reaction starts, add 200 μM caspofungin to the reaction system. After 24 hours, use the direct observation method and fluorescence observation method in Example 3 to detect the level of the product induced by caspofungin. As Figure 2 and Figure 3 shown, it can be known that under the induction of caspofungin, the glucan synthase FKS1-S643P in the corresponding centrifuge tube (i.e., Figure 2 the tube labeled S643P+CAS) and the corresponding capillary tube (i.e., Figure 3 the tube labeled S643P+CAS) produce the highest glucan yield. ([[]] Figure 2 is the in vitro synthesized β-1,3-glucan product detected by the direct observation method in the centrifuge tube under visible light; Figure 3 is the in vitro synthesized β-1,3-glucan product detected by observing in the capillary tube under a fluorescence microscope.)

[0067] Example 5: Detection method for the synthesis of glucan product and the increase of the product by genetically engineered yeast induced by caspofungin

[0068] (1) Culture the FKS1-S643P genetically engineered strain with mutations at the genomic level in YPD medium until OD = 0.3, add the inducer caspofungin to 0.5 μg / mL, and then culture until the exponential phase (OD = 0.5). Centrifuge the cells of different strains at 5,000×g for 30 minutes to collect them. Subsequently, wash them twice with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA).

[0069] (2) Suspend the precipitated cells in 0.5 ml of TE buffer, and then add 0.1 ml of 6 M NaOH. Incubate in a water bath at 80 °C for 30 minutes to dissolve the glucan synthesized by the bacteria. Then add 0.03% aniline blue solution (0.03% aniline blue, 0.18 M HCl, 0.49 M glycine-NaOH, pH 9.5). After mixing evenly, incubate at 50 °C for 30 minutes, and then incubate at 24 °C for another 30 minutes.

[0070] (3) Use a fluorescence microplate reader ( Plus, BMG Labtech) to read the fluorescence value at an excitation wavelength of 400 nm and an emission wavelength of 460 nm. Take the fluorescence value measured from wild-type yeast without inducer as the standard value of 100, and define the ratio of the fluorescence value measured from the mutant or the mutant plus inducer to the fluorescence value measured from wild-type yeast without inducer as the relative activity of the mutant or the mutant plus inducer. When the relative activity is higher than 100, the level of glucan product synthesized by the corresponding genetically engineered yeast increases. As Figure 4 shown, the level of glucan product synthesized by the FKS1-S643P genetically engineered strain increases, and the level of glucan product synthesized by the FKS1-S643P genetically engineered strain is higher under the induction of caspofungin.

[0071] Example 6: Application of in vitro enzyme activity assay to analyze the activity of FKS1-S643P mutant

[0072] (1) Add 2.5 μL of FKS1-S643P to the reaction system (30 μL of elution buffer added with Rho1, 4 μM GTP-γ-S, 20 mM KF). Then add UDP-glucose substrate to a final concentration of 2.5 mM, and start the reaction at 37 °C.

[0073] (2) After reacting for 60 min, add 30 μL of the fluorescence chromogenic reagent of the glycosyltransferase detection kit (Promega), and react at room temperature for 30 min.

[0074] (3) Use the Pherastar FS microplate reader system (BMG Labtech) to read the fluorescence value and detect the UDP level generated by the reaction.

[0075] Example 7: Application of screening second-generation echinocandin antifungal drugs with FKS1-S643P mutant protein as the target

[0076] (1) Incubate caspofungin (CAS) (final concentration 200 μg / ml) or micafungin (MFG) (final concentration 200 μg / ml) with 2.5 μL (final concentration 0.02 mg / ml) of FKS1-S643P mutant protein at room temperature for 10 minutes respectively, and then add them to the reaction system (30 μL elution buffer added with Rho1, 4 μM GTP-γ-S, 20 mM KF). Subsequently, add UDP-glucose substrate to a final concentration of 2.5 mM and start the reaction at 37°C. The control reactions are: caspofungin (final concentration 200 μg / ml in the system) or micafungin (final concentration 200 μg / ml in the system) are mixed with 2.5 μL (0.02 mg / ml) of wild-type FKS1 protein at room temperature under the same conditions and the enzyme activity reaction is started.

[0077] (2) Read the absolute fluorescence value using a Pherastar FS microplate reader system (BMG Labtech).

[0078] (3) Take the fluorescence value measured by the in vitro enzyme activity of wild-type β-1,3-glucan synthase before adding the drug as the standard value of 10. Define the ratio of the fluorescence value measured before and after adding the drug to wild-type or mutant to the fluorescence value measured by wild-type before adding the drug as its relative activity. A relative activity value lower than 3 is considered that β-1,3-glucan synthase is highly sensitive to the drug, a relative activity value between 3 and 10 is considered that β-1,3-glucan synthase is sensitive to the drug, a relative activity value between 10 and 30 is considered that β-1,3-glucan synthase is insensitive to the drug, and a relative activity value above 30 is considered that β-1,3-glucan synthase is tolerant to the drug.

[0079] (4) As Figure 5 shown, the enzyme activity of FKS1-S643P mutant protein increases significantly after adding the first-generation echinocandin antifungal drugs, and the relative activity value is about 100. Therefore, the FKS1-S643P mutant protein is tolerant to the first-generation echinocandin antifungal drugs; the enzyme activity decreases significantly after adding the second-generation echinocandin antifungal drugs, and the relative activity value is about 2.5. Therefore, the FKS1-S643P mutant protein is highly sensitive to the second-generation echinocandin antifungal drugs. According to the significant difference in its sensitivity to the first-generation and second-generation drugs, this mutant can be used as a target to screen new echinocandin antifungal drugs.

[0080] It is easy for those skilled in the art to understand that the above are only the preferred embodiments of the present invention, and are not used to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for synthesizing β-1,3-glucan products by catalysis of fungal glucan synthase established in vitro, characterized in that, Including the following steps: (1) Add a fungal glucan synthase mutant to a reaction system, the reaction system containing cofactor GTP-γ-S and Rho1; the reaction system further contains an inducer, the inducer being caspofungin; subsequently add uridine diphosphate-glucose substrate and conduct an enzyme activity reaction; The mutant is a β-1,3-glucan synthase mutant, prepared by the following steps: S1: Culture genetically engineered yeast and collect cells, lyse the cells and collect cell membranes; the genetically engineered yeast contains a gene expressing a β-1,3-glucan synthase mutant; the base sequence of the gene expressing the β-1,3-glucan synthase mutant is as shown in SEQ ID NO:1; S2: Dissolve the cell membranes obtained in step (1) in a membrane-lysing buffer, the membrane-lysing buffer containing N-dodecyl-β-D-maltoside and cholesteryl hemisuccinate, centrifuge and collect the supernatant; S3: Purify the β-1,3-glucan synthase mutant using anti-FLAG M2 affinity gel and elute with an elution buffer containing FLAG peptide, the elution buffer containing diosgenin glycoside, thus obtaining the β-1,3-glucan synthase mutant; (2) After the reaction, conduct centrifugation, the milky white precipitate is β-1,3-glucan, the more the milky white precipitate, the higher the yield of β-1,3-glucan; or after the reaction, add aniline blue solution, incubate in the dark, and the brighter the fluorescence under a fluorescence microscope, the higher the yield of β-1,3-glucan.

2. The method for in vitro establishing a method for synthesizing a β-1,3-glucan product by catalyzing fungal glucan synthase as claimed in claim 1, wherein The inducer is used to induce an increase in the yield of β-1,3-glucan.

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