An Aureobasidium pullulans CBH II gene knockout mutant strain, and its construction method and application

Abstract

The present invention provides a Aureobasidium pullulans CBH II gene knockout mutant strain, a construction method thereof and an application thereof, belonging to the field of microbial metabolic engineering. The mutant strain is obtained by knocking out all or part of the coding genes of the CBH II gene in the Aureobasidium pullulans strain and replacing them with a hygromycin B resistance gene cassette; the entire coding gene sequence of the CBH II gene is as shown in SEQ ID NO.1; and a construction method of the mutant strain is provided. The Aureobasidium pullulans CBH II gene knockout mutant strain constructed by the present invention shows obvious advantages in aspects such as improving the yield of β-glucan.

Description

Technical Field

[0001] The present invention belongs to the field of microbial metabolic engineering, and particularly relates to a mutant strain of Aureobasidium pullulans with a knocked-out CBH II gene, and a construction method and application thereof. Background Art

[0002] β-glucan is a polysaccharide with important biological activities and is widely used in fields such as medicine, food, and cosmetics. Its immunomodulatory, antioxidant, and film-forming properties have led to a continuous increase in market demand. Currently, β-glucan is mainly produced by microbial fermentation, and Aureobasidium pullulans has become the main production strain due to its high polysaccharide production characteristics. However, in the traditional fermentation process, the competitive utilization of carbon sources by the strain (such as cellulose catabolism) limits the synthesis efficiency of β-glucan, and it is difficult to further increase the yield.

[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 bond in cellulose, hydrolyzing cellulose to produce cellobiose, and promoting the flow of carbon sources towards energy metabolism.

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

[0005] In view of the deficiencies of the prior art, the present invention provides a mutant strain of Aureobasidium pullulans with a knocked-out CBH II gene, and a construction method and application thereof.

[0006] The technical solution of the present invention is as follows:

[0007] A mutant strain of Aureobasidium pullulans with a knocked-out CBH II gene is obtained by knocking out all or part of the coding gene of the CBH II gene in the Aureobasidium pullulans strain and replacing it with a hygromycin B resistance gene cassette;

[0008] The entire coding gene sequence of the CBH II gene is shown as SEQ ID NO.1.

[0009] Preferably according to the present invention, in the mutant strain, the coding gene between the 33rd and 1353rd positions of the CBH II gene in the Aureobasidium pullulans strain is knocked out and replaced with a hygromycin B resistance gene cassette.

[0010] Preferably according to the present invention, in the mutant strain, the Aureobasidium pullulans is Aureobasidium pullulans ATCC15233.

[0011] Preferably according to the present invention, for the mutant strain, the hygromycin B resistance gene cassette comprises: a filamentous fungal promoter glyceraldehyde-3-phosphate dehydrogenase gPDA promoter from Aspergillus nidulans, a hygromycin B resistance gene Hyg, and a trpC terminator connected thereto;

[0012] The sequence of the filamentous fungal promoter glyceraldehyde-3-phosphate dehydrogenase gPDA promoter from Aspergillus nidulans is as shown in SEQ ID NO.2;

[0013] The sequences of the hygromycin B resistance gene Hyg and the trpC terminator are as shown in SEQ ID NO.3.

[0014] The method for constructing the above-mentioned Aureobasidium pullulans CBH II gene knockout mutant strain comprises the following steps:

[0015] (1) pUC57 - Asp.gPDA - Hyg Construction of the knockout vector: The linearized pUC57 vector, the upstream homologous arm of the CBH II gene, the gPDA promoter, the Hyg resistance gene and the trpC terminator, and the downstream homologous arm of the CBH II gene are subjected to seamless cloning and ligation. The ligation product is transformed into Escherichia coli competent cells, and positive clone screening and verification are carried out after transformation to obtain a recombinant plasmid pUC57 - Asp.gPDA - Hyg ;

[0016] The sequence of the linearized pUC57 vector is as shown in SEQ ID NO.6;

[0017] The sequence of the upstream homologous arm of the CBH II gene is as shown in SEQ ID NO.4;

[0018] The sequence of the downstream homologous arm of the CBH II gene is as shown in SEQ ID NO.5;

[0019] The sequence of the gPDA promoter is as shown in SEQ ID NO.2;

[0020] The sequences of the Hyg resistance gene and the trpC terminator are as shown in SEQ ID NO.3;

[0021] (2) Genetic transformation of Aureobasidium pullulans: The recombinant plasmid obtained in step (1) is subjected to PCR amplification using primers CBH II upstream-F and CBH II downstream-R to obtain a linearized fragment to be transferred pUC57 - Asp.gPDA - Hyg ; Protoplasts of Aureobasidium pullulans are prepared, and the linearized fragment to be transferred Asp.gPDA - Hyg is transferred into the protoplasts, and transformants are screened using a resistance medium and verified to obtain an Aureobasidium pullulans CBH II gene knockout mutant strain; Asp.gPDA - Hyg

[0022] The sequence of the CBH II upstream-F is as shown in SEQ ID NO.7;​

[0023] The sequence of CBH II downstream-R is shown in SEQ ID NO. 10.

[0024] Preferably according to the present invention, in step (1), after transformation, positive clone screening is carried out: the bacterial liquid is spread on an LB solid medium plate containing 100 μg / mL ampicillin and cultured overnight to obtain positive clones;

[0025] In step (2), the linear fragment to be transferred Asp.gPDA - Hyg is transferred into protoplasts by the PEG method.

[0026] Preferably according to the present invention, in step (2), transformants are screened using a resistant medium: the transformed protoplasts are inoculated onto a double-layer medium for culture to obtain positive transformants;

[0027] The composition of the double-layer medium includes: the bottom layer is a seed solid medium containing 1.2 M sorbitol, and the upper layer is 0.7% agar. Each layer of the medium contains 200 μg / mL Hyg;

[0028] The components of the seed solid medium include: 4 g / L yeast extract powder, 2 g / L peptone, 6 g / L glucose, pH 6.0, and 20 g / L agar;

[0029] In step (2), verification of positive transformants: genomic DNA of positive transformants is extracted and amplified and verified using primers CBH II upstream-F and CBH II downstream-R.

[0030] 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.

[0031] A method for preparing β-glucan, comprising 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 medium to prepare a seed liquid, and transferring the seed liquid into a fermentation medium for fermentation to prepare β-glucan.

[0032] Preferably according to the present invention, the method for preparing the seed liquid includes the following:

[0033] Inoculate the Aureobasidium pullulans CBH II gene knockout mutant into a seed medium, and the culture conditions are 28 °C and 200 r / min for 24 hours to obtain a seed liquid;

[0034] The components of the seed medium include: 4 g / L yeast extract powder, 2 g / L peptone, 6 g / L glucose, pH 6.0;

[0035] The inoculation amount of the seed liquid into the fermentation medium is 5% by volume fraction;

[0036] The components of the fermentation medium include: 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;

[0037] Transfer the seed liquid into the fermentation medium, and the fermentation conditions are culturing at 28°C and 200 r / min for 144 hours.

[0038] The beneficial effects of the present invention at least include the following:

[0039] The Aureobasidium pullulans CBH II gene knockout mutant strain constructed by the present invention shows obvious advantages in improving the yield of β-glucan and other aspects, laying a solid foundation for the large-scale industrial application of Aureobasidium pullulans. This mutant strain has been verified by multiple rounds of experiments, with good genetic stability and excellent trait performance, and is expected to play a key role in the fields of food, medicine, chemical industry, etc., greatly promoting the innovative development of related industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 For the knockout of the CBH II gene of Aureobasidium pullulans described in the present invention pUC57 - Asp.gPDA - Hyg PCR electrophoresis diagram of the recombinant plasmid construction;

[0041] In the figure, M is the DNA ladder 5000 bp; 1. The PCR product with the genomic DNA of E (ATCC15233) as the template and the primer pair of CBH II upstream-F / CBH II upstream-R; 2. The PCR product with the pBARGPE1-Hygro plasmid as the template and the primer pair of gPDA promoter-F / gPDA promoter-R; 3. The PCR product for amplifying the Hyg resistance sequence and the trpC terminator with the pBARGPE1-Hygro-EGFP plasmid as the template and the primer pair of Hyg-F / Hyg-R; 4. The PCR product with the pUC57 plasmid as the template and the primer pair of pUC57 reverse-F / pUC57 reverse-R; 5. The PCR product with the genomic DNA of A. pullulans E (ATCC15233) as the template and the primer pair of CBH II downstream-F / CBH II downstream-R. A. pullulans E (ATCC15233) genomic DNA as the template, and the PCR product with the primer pair of CBH II downstream-F / CBH II downstream-R.

[0042] Figure 2 PCR electrophoresis diagram for the PCR identification of the seamless cloning recombinant product pUC57 - Asp.gPDA - Hyg ;

[0043] In the figure, M is the DNA ladder of 15,000 bp; 1, 2, 3, 4 are the linear fragments to be transferred. Asp.gPDA - Hyg PCR products.

[0044] Figure 3 It is a comparison diagram of the microscopic examination of Aureobasidium pullulans cells and protoplast preparation;

[0045] In the figure: A is the cell of Aureobasidium pullulans; B is the protoplast of Aureobasidium pullulans.

[0046] Figure 4 It is a verification diagram of the knockout strain;

[0047] In the figure: M is the DNA ladder of 15,000 bp; 1. Using the DNA of the knockout strain as a template, amplifying the CBH II gene with primers CBH II upstream-F and CBH II downstream-R; 2. Using A. pullulans the genome of E (ATCC15233) as a template, amplifying the CBH II gene with primers CBH II upstream-F and CBH II downstream-R.

[0048] Figure 5 It is a comparison diagram of the β-glucan yields of the starting strain and the knockout strain. Detailed implementation manners

[0049] The present invention will be further described in detail below, but the scope of protection required by the present invention is not limited thereto.

[0050] For the content where specific conditions are not specified in the examples, they are carried out according to conventional conditions; for the reagents or instruments whose manufacturers are not specified, they are all ordinary commercially available products.

[0051] Source of materials

[0052] 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 were purchased from Sangon Biotech (Shanghai) Co., Ltd.; Lysing Enzymes was purchased from Sigma-Aldrich.

[0053] The starting strain Aureobasidium pullulans ( Aureobacidium pullulans ) for knockout was purchased from the ATCC strain collection through Beijing Bomei Biotechnology Co., Ltd., and the specific number is Aureobasidium pullulans var. melanigenum (ATCC 15233).

[0054] In the subsequent description of the present invention, "Aureobasidium pullulans ( Aureobacidium pullulans )" will be denoted as "A. pullulans ”. The strain to be knocked out is denoted as A. pullulans E (ATCC15233).

[0055] Example 1

[0056] pUC57 - Asp.gPDA - Hyg Construction of knockout vector

[0057] (1) Extraction of pUC57 plasmid

[0058] Inoculate Escherichia coli containing pUC57 plasmid into LB liquid medium containing ampicillin (100 μg / mL), and culture it overnight at 37°C with shaking at 200 r / min. Use a plasmid miniprep kit to extract the pUC57 plasmid, and the specific operation is carried out according to the kit instructions. The extracted plasmid is dissolved in sterile water and stored at -20°C for later use.

[0059] (2) PCR amplification of each functional element

[0060] Using the genomic DNA of Aureobasidium pullulans A. pullulans E (ATCC15233) as a template, use the designed upstream homologous arm primers for PCR amplification. Upstream homologous arm of CBH II gene: Amplify the 750 kb sequence upstream of the CBH II gene, as shown in SEQ ID NO.4. Downstream homologous arm of CBH II gene: Amplify the 720 kb sequence downstream of the CBH II gene, as shown in SEQ ID NO.5.

[0061] The primers are as follows:

[0062] CBH II upstream-F is as shown in SEQ ID NO.7, and CBH II upstream-R is as shown in SEQ ID NO.8;

[0063] CBH II downstream-F is as shown in SEQ ID NO.9, and CBH II downstream-R is as shown in SEQ ID NO.10.

[0064] Designed according to the SEQ ID NO.6 sequence, using the pUC57 plasmid as a template, perform inverse PCR linear amplification of the pUC57 vector; the sequence of the inverse PCR linearized pUC57 vector is as shown in SEQ ID NO.6.

[0065] The primers of the pUC57 vector are as follows:

[0066] pUC57 reverse-F is as shown in SEQ ID NO.11, and pUC57 reverse-R is as shown in SEQ ID NO.12.

[0067] Using the pBARGPE1-Hygro-EGFP plasmid as a template, the Hyg resistance gene and the trpC terminator sequence were linearly amplified by PCR. The detailed sequence is shown in SEQ ID NO.3.

[0068] The primers are as follows:

[0069] Hyg-F is shown in SEQ ID NO.13, and Hyg-R is shown in SEQ ID NO.14.

[0070] Using the pBARGPE1-Hygro plasmid as a template, the gPDA promoter was amplified to express the Hyg resistance gene. The sequencing result is shown in SEQ ID NO.2.

[0071] The primers are as follows:

[0072] gPDA promoter-F is shown in SEQ ID NO.15, and gPDA promoter-R is shown in SEQ ID NO.16.

[0073] PCR reaction conditions and procedures: 2 μL of upstream primer, 2 μL of downstream primer, Aureobasidium pullulans A. pullulans E (ATCC15233) DNA 25 μL, 2×Phanta Max Master Mix (Dye Plus) 2 μL, dd H2O 19 μL.

[0074] Initial denaturation: 95°C, 5 minutes; 30 cycles; denaturation: 95°C, 30 seconds; annealing: temperature set according to the primer Tm temperature, 30 seconds; extension: 72°C, 1 minute; final extension: 72°C, 5 minutes; storage: 4°C.

[0075] (3)Purification of PCR products

[0076] After the PCR reaction, 5 μL of the PCR product was taken for agarose gel electrophoresis to confirm the size and purity of the amplified fragment. The nucleic acid gel diagrams of each recombinant element are as Figure 1 shown. After cutting the fragment, it was recovered, and the PCR product was purified using a DNA gel recovery kit. The specific operation was carried out according to the kit instructions. The purified PCR product was dissolved in sterile water and stored at -20°C for later use.

[0077] (4)Seamless cloning ligation and transformation

[0078] Using a seamless cloning kit, gently mix the purified linearized pUC57 vector (SEQ ID NO.6), the upstream homologous arm of the CBH II gene (SEQ ID NO.4), the gPDA promoter (SEQ ID NO.2), the Hyg resistance gene and the trpC terminator (SEQ ID NO.3), and the downstream homologous arm of the CBH II gene (SEQ ID NO.5) according to the ratio in the seamless cloning kit instructions. Briefly centrifuge and incubate at 37 °C for 30 min, then immediately place on ice to cool for 5 min. Take 100 μL of Escherichia coli DH5α competent cells and thaw them on ice. Add 10 μL of the seamless cloning ligation product, gently mix, and incubate on ice for 30 min. Heat shock at 42 °C for 90 s, quickly return to the ice bath for 2 min, add 900 μL of LB liquid medium without antibiotics, and shake culture at 37 °C and 150 r / min for 1 h to resuscitate the bacteria. Take 100 - 200 μL of the bacterial solution and spread it on an LB solid medium plate containing ampicillin (100 μg / mL), and incubate it upside down at 37 °C overnight.

[0079] (5)Positive clone screening and verification

[0080] Pick single colonies on the plate and inoculate them into LB liquid medium containing ampicillin (100 μg / mL), and shake culture overnight at 37 °C and 200 r / min. Use a plasmid miniprep kit to extract the plasmid from the cultured bacterial solution, and operate according to the kit instructions. The extracted recombinant plasmid pUC57 - Asp.gPDA - Hyg is subjected to whole plasmid sequencing.

[0081] Example 2

[0082] The genetic transformation part of Aureobasidium pullulans

[0083] Strain: A.pullulans E (ATCC15233)

[0084] (1)Obtaining the linear fragment to be transferred

[0085] Use a plasmid miniprep kit to extract the recombinant plasmid pUC57 - Asp.gPDA - Hyg , and the specific operation is carried out according to the kit instructions. Perform PCR amplification of the recombinant plasmid with primers CBH II upstream - F and CBH II downstream - R pUC57 - Asp.gPDA - Hyg The nucleic acid gel diagram of the linear fragment to be transferred obtained is as Figure 2 shown. After gel recovery of the fragment, use a DNA gel recovery kit to purify the PCR product to obtain the linear fragment to be transferred Asp.gPDA - Hyg .

[0086] (2)Preparation of Aureobasidium pullulans protoplasts

[0087] Pick a single colony and inoculate it into 3 mL of seed medium (4 g / L yeast extract powder, 2 g / L peptone, 6 g / L glucose), and culture it with shaking at 28°C for 48 h. Transfer 100 μL of the bacterial solution to 50 mL of seed medium, culture until OD600 = 1.0 - 1.2, transfer the cultured bacterial solution to a 50 mL centrifuge tube, centrifuge at 4°C and 5000 r / min for 10 min, and discard the supernatant. Centrifuge to collect the cells (4000×g, 4°C, 10 min), wash twice with pretreatment buffer (10 mM Tris-HCl pH7.5, 1.2 M sorbitol), and resuspend in the enzymatic hydrolysis solution (1.2 M sorbitol, 20 mg / mL Lysing Enzymes), and incubate with gentle shaking (80 r / min) at 30°C for 90 - 120 min, and observe under the microscope (such as Figure 3 ). >90% of the cells are spheroidized, and centrifuge (2000×g, 4°C, 10 min) to collect the protoplasts.

[0088] (3)Transfer the knockout fragment into the protoplasts

[0089] Mix 100 μL of the protoplast suspension (10 7 cells / mL) with 10 μg of the linear fragment to be transferred Asp.gPDA - Hyg Add an equal volume of PEG-CaT solution (40% PEG4000, 50 mM CaCl2, 10 mM Tris-HCl pH7.5), gently mix and then incubate on ice for 20 min, then let it stand at 25°C for 5 min, perform precise heat shock in a 42°C water bath for 90 seconds, immediately incubate on ice for 2 min to terminate the reaction, and then centrifuge at 4000×g, 4°C for 10 min.

[0090] (4)Screen the transformants on the resistant medium

[0091] Wash the transformed protoplasts obtained by centrifugation in the above operation with STC buffer (1.2 M sorbitol, 10 mM Tris-HCl pH7.5, 50 mM CaCl2), repeat the operation three times, wash three times with 1 mol / L sorbitol solution, add 1 mL of seed medium containing 1 mol / L sorbitol, resuscitate at 28°C for 3 h, and spread on a double-layer medium containing 200 μg / mL Hyg (hygromycin): the bottom layer is a seed solid medium containing 1.2 M sorbitol (4 g / L yeast extract powder, 2 g / L peptone, 6 g / L glucose, pH6.0, 20 g / L agar), the upper layer is 0.7% agar (add 0.7 g of agar to 100 mL of water), and each layer of the medium contains 200 μg / mL Hyg, and culture in the dark at 28°C for 5 - 7 days to obtain positive transformants.

[0092] Example 3

[0093] Verification of knockout strain

[0094] (1)Extract the DNA of the strain before and after knocking out the CBH II gene using a fungal genomic DNA extraction kit (OMEGA), and amplify the CBH II gene region with primers CBH II upstream-F and CBH II downstream-R.

[0095] (2)As Figure 4 , agarose gel electrophoresis showed that the amplified fragment of the knockout strain was 4.6 kb (including the hyg gene), while that of the parental strain was 2.7 kb, indicating successful knockout.

[0096] Example 4

[0097] Fermentation and yield determination

[0098] Inoculate the initial strain (ATCC15233) and the knockout mutant into the seed medium, and then transfer them to the fermentation medium for fermentation to determine β-glucan. The components of the seed medium include: 4 g / L yeast extract powder, 2 g / L peptone, 6 g / L glucose, pH 6.0. Inoculate the strain into the seed medium, and the culture conditions are 28 °C and 200 r / min for 24 hours to obtain the seed liquid. Then inoculate the seed liquid into the fermentation medium at an inoculation volume fraction of 5%. The components of the fermentation medium include: 2 g / L peptone, 112 g / L glucose, 2.1 g / L NaCl, 0.7 g / L (NH4)2SO4, 2.1 g / L K2HPO4, 0.4 g / L MgSO4, pH 6.0.

[0099] Carry out fermentation culture of the parental strain and the CBH II knockout strain in a 5 L fermenter respectively. The fermentation conditions are 28 °C and 200 r / min, and stop fermentation at 144 h. After 144 h of fermentation, sample and use a β-glucan detection kit (Megazyme, Bray, Ireland) to detect the content of β-glucan in the fermentation broth.

[0100] The results are shown in Figure 5 , the β-glucan yield of the knockout strain reached 3.25 g / L, showing a significant increase compared with the yield of the parental strain of 2.93 g / L, and it 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 the coding gene between positions 33 and 1353 of the CBH II gene in the budding Aureobasidium bracteata 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; 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.

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

3. The method for constructing a mutant strain according to any one of claims 1 to 2, 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.

4. The construction method according to claim 3, 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.

5. The construction method according to claim 3, 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.

6. Use of the mutant strain according to any one of claims 1 to 2 or the mutant strain constructed by the construction method according to claim 3 in the preparation of β-glucan.

7. 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 2 or the mutant strain constructed by the construction method according to claim 3 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.

8. The method according to claim 7, 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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