Aureobasidium pullulans CBH II gene knockout mutant strain as well as construction method and application thereof

Abstract

The invention provides an aureobasidium pullulans CBH II gene knockout mutant strain and a construction method and application thereof, and belongs to the field of microbial metabolism engineering.The mutant strain is obtained by knocking out all or part of coding genes of CBH II genes in an aureobasidium pullulans strain and replacing the coding genes with a hygromycin B resistance gene cassette; all coding gene sequences of the CBH II gene are as shown in SEQ ID NO. 1; the invention also provides a construction method of the mutant strain, and the aureobasidium pullulans CBH II gene knockout mutant strain constructed by the invention shows obvious advantages in the aspects of increasing the yield of beta-glucan and the like.

Description

Technical Field

[0001] The invention belongs to the field of microbial metabolic engineering, and specifically relates to a budding Aureobasidium pullulans CBH II gene knockout mutant strain and a construction method and application thereof. Background Art

[0002] β-glucan is a polysaccharide with important biological activity, which is widely used in medicine, food, cosmetics and other fields. Its immunomodulatory, antioxidant and film-forming properties have led to its continued growth in market demand. At present, β-glucan is mainly produced by microbial fermentation, among which Aureobasidium pullulans has become the main production strain due to its high polysaccharide production characteristics. However, the competitive utilization of carbon sources by strains in traditional fermentation processes (such as cellulose decomposition) limits the efficiency of β-glucan synthesis, making it difficult to further increase production.

[0003] Cellobiohydrolase II (CBH II) is a key enzyme in the cellulose degradation pathway (belonging to the glycoside hydrolase family), which is usually responsible for hydrolyzing the β-glycosidic bonds in cellulose, hydrolyzing cellulose to produce cellobiose, and promoting the flow of carbon sources towards energy metabolism.

[0004] About Aureobasidium pullulans Aureobasidium pullulans ), the targeted knockout of CBH II gene and its effect on β-glucan synthesis have not been reported. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a CBH II gene knockout mutant of Aureobasidium pullulans and a construction method and application thereof.

[0006] The technical solution of the present invention is as follows: A budding Aureobasidium pullulans CBH II gene knockout mutant strain, wherein all or part of the coding gene of the CBH II gene in the budding Aureobasidium pullulans strain is knocked out and replaced with a hygromycin B resistance gene cassette; The entire coding gene sequence of the CBH II gene is shown in SEQ ID NO.1.

[0007] Preferably, according to the present invention, the mutant strain is obtained by knocking out the coding gene between positions 33 and 1353 of the CBH II gene in the Aureobasidium pullulans strain and replacing it with a hygromycin B resistance gene cassette.

[0008] Preferably according to the present invention, the mutant strain, the budding Aureobasidium pullulans is budding Aureobasidium pullulans ATCC15233.

[0009] According to the preferred embodiment of the present invention, the mutant strain, the hygromycin B resistance gene cassette comprises: a filamentous fungus promoter 3-phosphoglyceraldehyde dehydrogenase gPDA promoter from Aspergillus nidulans and a hygromycin B resistance gene Hyg connected to a trpC terminator; The sequence of the filamentous fungus promoter 3-phosphoglyceraldehyde dehydrogenase gPDA promoter from Aspergillus nidulans is shown in SEQ ID NO.2; The hygromycin B resistance gene Hyg and trpC terminator sequences are shown in SEQ ID NO.3.

[0010] The method for constructing the above-mentioned Aureobasidium pullulans CBH II gene knockout mutant comprises the following steps: (1) pUC57-Asp.gPDA-Hyg Construction of knockout vector: Linearized pUC57 vector, upstream homology arm of CBH II gene, gPDA promoter, Hyg resistance gene and trpC terminator, downstream homology arm of CBH II gene were seamlessly cloned and connected, and the connection product was transformed into Escherichia coli competent cells. After transformation, positive clones were screened and verified to obtain recombinant plasmids. pUC57-Asp.gPDA-Hyg ; The sequence of the linearized pUC57 vector is shown in SEQ ID NO.6; The sequence of the upstream homology arm of the CBH II gene is shown in SEQ ID NO.4; The sequence of the downstream homology arm of the CBH II gene is shown in SEQ ID NO.5; The sequence of the gPDA promoter is shown in SEQ ID NO.2; The sequences of the Hyg resistance gene and trpC terminator are shown in SEQ ID NO.3; (2) Genetic transformation of Aureobasidium pullulans: PCR amplification of the primers CBH II upstream-F and CBH II downstream-R was performed in step (1). pUC57-Asp.gPDA-Hyg Recombinant plasmid to obtain linear fragment to be transferred Asp.gPDA-Hyg ; Prepare the protoplasts of budding Aureobasidium pullulans and transfer the linear fragment to be transferred Asp.gPDA-Hyg The resultant product was transferred into protoplasts, and transformants were screened and verified using a resistant medium to obtain a knockout mutant strain of the CBH II gene of Aureobasidium pullulans. The sequence of the CBH II upstream-F is shown in SEQ ID NO.7; The sequence of the CBH II downstream-R is shown in SEQ ID NO.10.

[0011] Preferably, in step (1), positive clones are screened after transformation: the bacterial solution is spread on a LB solid culture medium plate containing 100 μg / mL ampicillin, and cultured overnight to obtain positive clones; In step (2), the linear fragment to be transferred is transferred using the PEG method. Asp.gPDA-Hyg Transfer into protoplasts.

[0012] Preferably, in step (2), transformants are screened using a resistant culture medium: the transformed protoplasts are inoculated onto a double-layer culture medium for culture to obtain positive transformants; The composition of the double-layer culture medium included: the bottom layer was a seed solid culture medium containing 1.2 M sorbitol, the top layer was 0.7% agar, and each layer of the culture medium contained 200 μg / mL of Hyg; The components of the seed solid culture medium include: 4 g / L yeast extract powder, 2 g / L peptone, 6 g / L glucose, pH 6.0, and 20 g / L agar; In step (2), positive transformant verification: extract genomic DNA of positive transformants and perform amplification verification using primers CBH II upstream-F and CBH II downstream-R.

[0013] Use of the above-mentioned Aureobasidium pullulans CBH II gene knockout mutant or the Aureobasidium pullulans CBHII gene knockout mutant constructed by the above-mentioned construction method in the preparation of β-glucan.

[0014] A method for preparing β-glucan comprises the following steps: inoculating the above-mentioned Aureobasidium pullulans CBH II gene knockout mutant or the Aureobasidium pullulans CBH II gene knockout mutant constructed by the above-mentioned construction method into a seed culture medium to prepare a seed liquid, transferring the seed liquid into a fermentation culture medium, and fermenting to prepare β-glucan.

[0015] Preferably, according to the present invention, the method for preparing the seed solution comprises the following steps: The CBH II gene knockout mutant of Aureobasidium pullulans was inoculated into a seed culture medium, and the culture conditions were 28° C. and 200 r / min for 24 hours to prepare a seed solution; The seed culture medium components included: 4 g / L yeast extract powder, 2 g / L peptone, 6 g / L glucose, pH 6.0; The inoculation amount of the seed liquid into the fermentation medium is 5% by volume; The fermentation medium components included: peptone 2 g / L, glucose 112 g / L, NaCl 2.1 g / L, (NH4)2SO40.7 g / L, K2HPO42.1 g / L, MgSO40.4 g / L, pH 6.0; The seed liquid was transferred to the fermentation medium and the fermentation conditions were 28°C and 200 r / min for 144 hours.

[0016] The beneficial effects of the present invention include at least the following: The CBH II gene knockout mutant of Aureobasidium pullulans constructed by the present invention shows obvious advantages in improving the yield of β-glucan, laying a solid foundation for the large-scale industrial application of Aureobasidium pullulans. The mutant has been verified by multiple rounds of experiments, and its genetic stability is good, and its trait performance is excellent. It is expected to play a key role in the fields of food, medicine, and chemical industry, and greatly promote the innovative development of related industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The CBH II gene knockout of the present invention is pUC57-Asp.gPDA-Hyg PCR electrophoresis diagram of recombinant plasmid construction; In the figure, M is DNA ladder 5000 bp; 1. A. pullulans 1. The PCR product of E (ATCC15233) genome as template and CBH II upstream-F / CBH II upstream-R as primer pair; 2. The PCR product of pBARGPE1-Hygro plasmid as template and gPDA promoter-F / gPDA promoter-R as primer pair; 3. The PCR product of pBARGPE1-Hygro-EGFP plasmid as template to amplify the Hyg resistance sequence and trpC terminator and Hyg-F / Hyg-R as primer pair; 4. The PCR product of pUC57 plasmid as template and pUC57 reverse-F / pUC57 reverse-R as primer pair; 5. A. pullulans E (ATCC15233) genome was used as template and PCR product was generated using CBH II downstream-F / CBH II downstream-R as primer pair.

[0018] Figure 2 For seamless cloning of recombinant products pUC57-Asp.gPDA-Hyg PCR electrophoresis diagram of PCR identification; In the figure, M is the DNA ladder 15,000 bp; 1.2.3.4. Linear fragments to be transferred Asp.gPDA-Hyg PCR products.

[0019] Figure 3 A comparison chart of the microscopic examination of the budding short-stalked mold body and protoplast preparation; In the figure: A is the fungus of Aureobasidium pullulans; B is the protoplast of Aureobasidium pullulans.

[0020] Figure 4 This is the validation diagram of the knockout strain; In the figure: M is a DNA ladder of 15,000 bp; 1. Using the DNA of the knockout strain as a template, the CBH II gene was amplified using primers CBH II upstream-F and CBH II downstream-R; 2. A. pullulans E (ATCC15233) genome was used as a template, and the CBH II gene was amplified using primers CBH II upstream-F and CBH II downstream-R.

[0021] Figure 5 This is a comparison chart of β-glucan production between the starting strain and the knockout strain. DETAILED DESCRIPTION

[0022] The present invention will be described in further detail below, but the protection scope of the present invention is not limited thereto.

[0023] The contents without specific conditions in the examples were carried out under conventional conditions; the reagents or instruments used without indicating the manufacturer were all common commercially available products.

[0024] Sources The pBARGPE1-Hygro-EGFP plasmid was purchased from Wuhan Miaoling Biotechnology Co., Ltd.; the pBARGPE1-Hygro fungal expression plasmid was purchased from Shanghai Qincheng Biotechnology Co., Ltd.; the plasmid pUC57 Escherichia coli glycerol bacteria was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; and the Lysing Enzymes were purchased from Sigma-Aldrich.

[0025] The starting strain for knockout was Aureobasidium pullulans ( Aureobacidium pullulans ) strain was purchased from ATCC strain collection through Beijing Bomai Biological Company, with specific number Aureobasidium pullulans var. melanigenum (ATCC 15233).

[0026] In the subsequent description of the present invention, "budding short stalk mold ( Aureobacidium pullulans )" is recorded as " A. pullulans The strain to be knocked out is recorded as A. pullulans E (ATCC15233).

[0027] Example 1 pUC57-Asp.gPDA-Hyg Construction of knockout vector (1) Extraction of pUC57 plasmid Inoculate E. coli containing pUC57 plasmid into LB liquid medium containing ampicillin (100 μg / mL) and culture overnight at 37°C and 200 r / min. Use a plasmid extraction kit to extract pUC57 plasmid. Specific operations are performed according to the instructions of the kit. Dissolve the extracted plasmid with sterile water and store at -20°C for later use.

[0028] (2) PCR amplification of each functional element Aureobasidium pullulans A. pullulans E (ATCC15233) genomic DNA was used as a template, and PCR amplification was performed using the designed upstream homology arm primers. CBH II gene upstream homology arm: amplify the 750 kb sequence upstream of the CBH II gene, as shown in SEQ ID NO. 4. CBH II gene downstream homology arm: amplify the 720 kb sequence downstream of the CBH II gene, as shown in SEQ ID NO. 5.

[0029] The primers are as follows: CBH II upstream-F is shown in SEQ ID NO.7, and CBH II upstream-R is shown in SEQ ID NO.8; CBH II downstream-F is shown in SEQ ID NO.9, and CBH II downstream-R is shown in SEQ ID NO.10.

[0030] According to the sequence design of SEQ ID NO.6, the pUC57 plasmid was used as a template, and the pUC57 vector was linearly amplified by inverse PCR; the sequence of the pUC57 vector linearized by inverse PCR is shown in SEQ ID NO.6.

[0031] The primers for the pUC57 vector are as follows: pUC57 reverse-F is shown as SEQ ID NO.11, and pUC57 reverse-R is shown as SEQ ID NO.12.

[0032] Using pBARGPE1-Hygro-EGFP plasmid as a template, PCR linearly amplified the Hyg resistance gene and trpC terminator sequence. The detailed sequence is shown in SEQ ID NO.3.

[0033] The primers are as follows: Hyg-F is shown in SEQ ID NO.13, and Hyg-R is shown in SEQ ID NO.14.

[0034] The pBARGPE1-Hygro plasmid was used as a template to amplify the gPDA promoter and express the Hyg resistance gene. The sequencing result is shown in SEQ ID NO.2.

[0035] The primers are as follows: gPDA promoter-F is shown as SEQ ID NO.15, and gPDA promoter-R is shown as SEQ ID NO.16.

[0036] PCR reaction conditions and procedures, upstream primer 2 μL, downstream primer 2 μL, budding Aureobasidium A. pullulans E(ATCC15233)DNA 25μL, 2×Phanta Max Master Mix (Dye Plus) 2μL, dd H2O19μL.

[0037] Initial denaturation: 95°C, 5 minutes; 30 cycles; denaturation: 95°C, 30 seconds; annealing: temperature set according to primer Tm temperature, 30 seconds; extension: 72°C, 1 minute; final extension: 72°C, 5 minutes; storage: 4°C.

[0038] (3) PCR product purification After the PCR reaction is completed, 5 μL of PCR product is taken for agarose gel electrophoresis to confirm the size and purity of the amplified fragment. Figure 1 As shown. After the fragment is cut out and recovered, the PCR product is purified using a DNA gel recovery kit. The specific operation is carried out according to the instructions of the kit. The purified PCR product is dissolved in sterile water and stored at -20℃ for future use.

[0039] (4) Seamless cloning, connection and transformation Use the seamless cloning kit to gently mix the purified linearized pUC57 vector (SEQ ID NO.6), CBH II gene upstream homology arm (SEQ ID NO.4), gPDA promoter (SEQ ID NO.2), Hyg resistance gene and trpC terminator (SEQ ID NO.3), and CBH II gene downstream homology arm (SEQ ID NO.5) according to the ratio in the seamless cloning kit instructions, centrifuge briefly, incubate at 37℃ for 30 min, and immediately cool on ice for 5 min. Take 100 μL of Escherichia coli DH5α competent cells and thaw on ice. Add 10 μL of seamless cloning ligation product, mix gently, and ice bath for 30 min. Heat shock at 42℃ for 90 s, quickly return to ice bath for 2 min, add 900 μL of LB liquid medium without antibiotics, and culture at 37℃ and 150 r / min for 1 h to revive the bacteria. Take 100-200 μL of bacterial solution and spread it on LB solid culture medium plate containing ampicillin (100 μg / mL), and culture it upside down at 37℃ overnight.

[0040] (5) Screening and verification of positive clones Pick a single colony from the plate and inoculate it into LB liquid medium containing ampicillin (100 μg / mL), and culture overnight at 37°C and 200 r / min. Use a plasmid extraction kit to extract the plasmid from the culture solution, and follow the instructions of the kit. The extracted recombinant plasmid pUC57-Asp.gPDA-Hyg Perform whole plasmid sequencing.

[0041] Example 2 Genetic transformation of Aureobasidium pullulans Strains: A.pullulans E (ATCC15233) (1) Obtaining the linear fragment to be converted Use plasmid extraction kit to extract recombinant plasmid pUC57-Asp.gPDA-Hyg Specific operations were performed according to the instructions of the kit. PCR amplification was performed using primers CBH II upstream-F and CBH II downstream-R pUC57-Asp.gPDA-Hyg The recombinant plasmid and the obtained linear fragment nucleic acid gel image are as follows Figure 2 As shown, the fragment gel was recovered and the PCR product was purified using a DNA gel recovery kit to obtain a linear fragment to be transferred. Asp.gPDA-Hyg .

[0042] (2) Preparation of protoplasts of Aureobasidium pullulans Pick a single colony and inoculate it into 3 mL seed culture medium (4 g / L yeast extract, 2 g / L peptone, 6 g / L glucose), and culture at 28℃ with shaking for 48 h. Transfer 100 μL of bacterial solution to 50 mL seed culture medium, culture until OD600=1.0-1.2, transfer the cultured bacterial solution to a 50 mL centrifuge tube, centrifuge at 4℃, 5000 r / min for 10 min, and discard the supernatant. Collect the bacteria by centrifugation (4000×g, 4℃, 10 min), wash twice with pretreatment buffer (10 mM Tris-HCl pH7.5, 1.2 M sorbitol), resuspend in enzymatic solution (1.2 M sorbitol 20 mg / mL Lysing Enzymes), and treat with gentle shaking (80 r / min) at 30℃ for 90-120 min. Observe under a microscope (such as Figure 3 ) When >90% of the cells were spherical, the protoplasts were collected by centrifugation (2000×g, 4℃, 10 min).

[0043] (3) Knockout fragment transfer into protoplasts Mix 100 μL of protoplast suspension (10 7 cells / mL) and 10 μg of the linear fragment to be transferred Asp.gPDA-HygAn equal volume of PEG-CaT solution (40% PEG4000, 50 mM CaCl2, 10 mM Tris-HCl pH7.5) was added, mixed gently and placed on ice for 20 min, then allowed to stand at 25°C for 5 min, heat-shocked in a 42°C water bath for 90 s, immediately placed on ice for 2 min to terminate the reaction, and then centrifuged at 4000×g, 4°C for 10 min.

[0044] (4) Screening of transformants using resistance culture medium The transformed protoplasts obtained by centrifugation in the above operation were washed with STC buffer (1.2 M sorbitol, 10 mM Tris-HCl pH7.5, 50 mM CaCl2), and the operation was repeated three times. The protoplasts were washed three times with 1 mol / L sorbitol solution, and 1 mL of seed culture medium containing 1 mol / L sorbitol was added. The cells were revived at 28°C for 3 h, and coated with a double-layer culture medium containing 200 μg / mL Hyg (hygromycin): the bottom layer was a seed solid culture medium containing 1.2 M sorbitol (4 g / L yeast extract, 2 g / L peptone, 6 g / L glucose, pH6.0, and 20 g / L agar), and the upper layer was 0.7% agar (0.7 g agar was added to 100 mL water). Each layer of the culture medium contained 200 μg / mL Hyg. The cells were cultured at 28°C in the dark for 5-7 days to obtain positive transformants.

[0045] Example 3 Validation of knockout strains (1) Use the Fungal Genome Extraction Kit (OMEGA) to extract the DNA of the strain before and after the CBH II gene was knocked out, and use the primers CBH II upstream-F and CBH II downstream-R to amplify the CBH II gene region.

[0046] (2) If Figure 4 Agarose electrophoresis showed that the amplified fragment of the knockout strain was 4.6 kb (including the hyg gene), while that of the starting strain was 2.7 kb, indicating that the knockout was successful.

[0047] Example 4 Fermentation and yield determination The initial strain (ATCC15233) and the knockout mutant were inoculated into the seed medium, and then transferred to the fermentation medium for fermentation determination of β-glucan. The components of the seed medium include: 4 g / L yeast extract, 2 g / L peptone, 6 g / L glucose, pH 6.0. The strain was inoculated into the seed medium, and the culture conditions were 28℃ and 200 r / min for 24 hours to obtain the seed liquid. Then the seed liquid was inoculated into the fermentation medium at an inoculum volume fraction of 5%. The fermentation medium components include: peptone 2 g / L, glucose 112 g / L, NaCl 2.1 g / L, (NH4)2SO40.7g / L, K2HPO42.1g / L, MgSO40.4g / L, pH 6.0.

[0048] The starting strain and the CBH II knockout strain were fermented in 5L fermenters at 28°C and 200 r / min, and the fermentation was stopped at 144h. After 144h of fermentation, samples were taken and the β-glucan content in the fermentation broth was detected using a β-glucan detection kit (Megazyme, Bray, Ireland).

[0049] The results are shown in Figure 5 The β-glucan production of the knockout strain reached 3.25 g / L, which was significantly improved compared with the starting strain's production of 2.93 g / L and can be used in actual production.

Claims

1. A CBH II gene knockout mutant of Aureobasidium pullulans, characterized in that: The method is to knock out all or part of the coding gene of the CBHII gene in the Aureobasidium pullulans strain and replace it with a hygromycin B resistance gene cassette; The entire coding gene sequence of the CBH II gene is shown in SEQ ID NO.

1.

2. The mutant strain according to claim 1, characterized in that The coding gene between positions 33 and 1353 of the CBH II gene in the budding Aconitum bacillus strain is knocked out and replaced with a hygromycin B resistance gene cassette.

3. The mutant strain according to claim 1, characterized in that The budding Aureobasidium pullulans is Aureobasidium pullulans ATCC15233.

4. The mutant strain according to claim 1, characterized in that The hygromycin B resistance gene cassette comprises: a filamentous fungus promoter 3-phosphoglyceraldehyde dehydrogenase gPDA promoter from Aspergillus nidulans and a hygromycin B resistance gene Hyg connected to a trpC terminator; The sequence of the filamentous fungus promoter 3-phosphoglyceraldehyde dehydrogenase gPDA promoter from Aspergillus nidulans is shown in SEQ ID NO.2; The sequences of the hygromycin B resistance gene Hyg and the trpC terminator are shown in SEQ ID NO.

3.

5. The method for constructing a mutant according to any one of claims 1 to 4, characterized in that The steps include: (1) pUC57-Asp.gPDA-Hyg Construction of knockout vector: Linearized pUC57 vector, upstream homology arm of CBH II gene, gPDA promoter, Hyg resistance gene and trpC terminator, downstream homology arm of CBH II gene were seamlessly cloned and connected, and the connection product was transformed into Escherichia coli competent cells. After transformation, positive clones were screened and verified to obtain recombinant plasmids. pUC57-Asp.gPDA-Hyg ; The sequence of the linearized pUC57 vector is shown in SEQ ID NO.6; The sequence of the upstream homology arm of the CBH II gene is shown in SEQ ID NO.4; The sequence of the downstream homology arm of the CBH II gene is shown in SEQ ID NO.5; The sequence of the gPDA promoter is shown in SEQ ID NO.2; The sequences of the Hyg resistance gene and trpC terminator are shown in SEQ ID NO.3; (2) Genetic transformation of Aureobasidium pullulans: PCR amplification of the primers CBH II upstream-F and CBH II downstream-R was performed in step (1). pUC57-Asp.gPDA-Hyg Recombinant plasmid to obtain linear fragment to be transferred Asp.gPDA-Hyg ; Prepare the protoplasts of budding Aureobasidium pullulans and transfer the linear fragment to be transferred Asp.gPDA-Hyg The resultant product was transferred into protoplasts, and transformants were screened and verified using a resistant medium to obtain a knockout mutant strain of the CBH II gene of Aureobasidium pullulans. The sequence of the CBH II upstream-F is shown in SEQ ID NO.7; The sequence of the CBH II downstream-R is shown in SEQ ID NO.

10.

6. The construction method according to claim 5, characterized in that: In step (1), positive clones are screened after transformation: the bacterial solution is spread on a LB solid culture medium plate containing 100 μg / mL ampicillin, and cultured overnight to obtain positive clones; In step (2), the linear fragment to be transferred is transferred using the PEG method. Asp.gPDA-Hyg Transfer into protoplasts.

7. The construction method according to claim 5, characterized in that: In step (2), transformants are screened using a resistance culture medium: the transformed protoplasts are inoculated onto a double-layer culture medium and cultured to obtain positive transformants; The composition of the double-layer culture medium included: the bottom layer was a seed solid culture medium containing 1.2 M sorbitol, the top layer was 0.7% agar, and each layer of the culture medium contained 200 μg / mL of Hyg; The components of the seed solid culture medium include: 4 g / L yeast extract powder, 2 g / L peptone, 6 g / L glucose, pH 6.0, and 20 g / L agar; In step (2), positive transformant verification: genomic DNA of positive transformants was extracted and amplified using primers CBH II upstream-F and CBH II downstream-R for verification.

8. Use of the mutant strain according to any one of claims 1 to 4 or the mutant strain constructed by the construction method according to claim 5 in the preparation of β-glucan.

9. A method for preparing β-glucan, characterized in that: The method comprises the following steps: inoculating the mutant strain according to any one of claims 1 to 4 or the mutant strain constructed by the construction method according to claim 5 into a seed culture medium to prepare a seed liquid, transferring the seed liquid into a fermentation culture medium, and fermenting to prepare β-glucan.

10. The method according to claim 9, characterized in that The preparation method of the seed solution comprises the following steps: The mutant strain was inoculated into the seed culture medium and cultured at 28°C and 200 r / min for 24 hours to obtain the seed solution; The seed culture medium components included: 4 g / L yeast extract powder, 2 g / L peptone, 6 g / L glucose, pH 6.0; The inoculation amount of the seed liquid into the fermentation medium is 5% by volume; The fermentation medium components included: peptone 2 g / L, glucose 112 g / L, NaCl 2.1 g / L, (NH4)2SO4 0.7 g / L, K2HPO4 2.1 g / L, MgSO4 0.4 g / L, pH 6.0; The seed liquid was transferred to the fermentation medium and the fermentation conditions were 28°C and 200 r / min for 144 hours.

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