An Aureobasidium pullulans Kxk gene knockout mutant strain and its construction method and application

Abstract

The present invention provides a Aureobasidium pullulans Kxk gene knockout mutant strain, its construction method and application, belonging to the field of microbial metabolic engineering. Specifically, it provides an Aureobasidium pullulans Kxk gene knockout mutant strain, in which all or part of the coding genes of the Kxk gene in the Aureobasidium pullulans strain are knocked out and replaced with a hygromycin B resistance gene cassette; the entire coding gene sequence of the Kxk gene is as shown in SEQ ID NO.1; and the construction method of this mutant strain. The Aureobasidium pullulans Kxk gene knockout mutant strain constructed by the present invention shows significant 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 Aureobasidium pullulans Kxk gene knockout mutant strain, and a construction method and application thereof. Background Art

[0002] β-glucan is a polysaccharide with important biological activities, and is widely used in the fields of food, medicine, cosmetics, etc. Its unique molecular structure endows it with functions such as immune regulation, cholesterol reduction, and antioxidant properties. The market demand continues to grow. It is an ideal health food, and can also be used as a food additive in the food industry, with good application value and development prospects, and has currently become a research hotspot at home and abroad. At present, the production of β-glucan mainly relies on microbial fermentation, and Aureobasidium pullulans has become a commonly used production strain due to its high polysaccharide production characteristics. However, problems such as low carbon source utilization efficiency and by-product accumulation in traditional fermentation processes limit the further improvement of production. The unique structural characteristics of β-glucan endow it with various special physiological functions such as cholesterol reduction, blood sugar regulation, and immune enhancement.

[0003] Hexokinase (Putative hexokinase Kxk) is a key enzyme in sugar metabolism, which catalyzes the phosphorylation of hexoses such as glucose into glucose-6-phosphate (G-6-P), and promotes glycolysis and the tricarboxylic acid cycle.

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

[0005] Aiming at the deficiencies of the prior art, the present invention provides a Aureobasidium pullulans Kxk gene knockout mutant strain, and a construction method and application thereof.

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

[0007] A Aureobasidium pullulans Kxk gene knockout mutant strain is obtained by knocking out all or part of the coding genes of the Putative hexokinase Kxk gene in the Aureobasidium pullulans strain and replacing them with a hygromycin B resistance gene cassette;

[0008] The entire coding gene sequence of the Putative hexokinase Kxk gene is shown in SEQ ID NO.1.

[0009] Preferably according to the present invention, the mutant strain is obtained by knocking out the coding gene between the 82nd and 1557th positions of the Putative hexokinase Kxk gene in the Aureobasidium pullulans strain and replacing it with a hygromycin B resistance gene cassette.

[0010] Preferably according to the present invention, for 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: the strong promoter phosphoglycerate kinase PGK promoter in Aureobasidium pullulans, the hygromycin B resistance gene Hyg, and the trpC terminator are ligated;

[0012] The sequence of the strong promoter phosphoglycerate kinase PGK promoter in Aureobasidium pullulans is shown in SEQ ID NO.2;

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

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

[0015] (1) pUC57-Asp.PGK-Hyg Construction of the knockout vector: The linearized pUC57 vector, the upstream homologous arm of the Putativehexokinase Kxk gene, the PGK promoter, the Hyg resistance gene and the trpC terminator, and the downstream homologous arm of the Putativehexokinase Kxk 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.PGK-Hyg ;

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

[0017] The sequence of the upstream homologous arm of the Putative hexokinase Kxk gene is shown in SEQ ID NO.4;

[0018] The sequence of the downstream homologous arm of the Putative hexokinase Kxk gene is shown in SEQ ID NO.5;

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

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

[0021] (2)Genetic transformation of Aureobasidium pullulans: The recombinant plasmid obtained in step (1) was subjected to PCR amplification using primers Kxk upstream-F and Kxk downstream-R to obtain a linear fragment to be transferred. pUC57-Asp.PGK-Hyg ; Protoplasts of Aureobasidium pullulans were prepared, and the linear fragment to be transferred Asp.PGK-Hyg was transferred into the protoplasts. Transformants were screened using a resistant medium, verified, and an Aureobasidium pullulans Kxk gene knockout mutant was obtained; Asp.PGK-Hyg

[0022] The sequence of the Kxk upstream-F is shown in SEQ ID NO.7;

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

[0024] Preferably according to the present invention, in step (1), after transformation, positive clone screening was performed: The bacterial solution was 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 was transferred into the protoplasts by the PEG method. Asp.PGK-Hyg

[0026] Preferably according to the present invention, in step (2), transformants were screened using a resistant medium: The transformed protoplasts were 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 was extracted and amplified and verified using primers Kxk upstream-F and Kxk downstream-R.

[0030] Use of the above-mentioned Aureobasidium pullulans Kxk gene knockout mutant or the Aureobasidium pullulans Kxk gene knockout mutant constructed by the above method in the preparation of β-glucan.

[0031] A method for preparing β-glucan, comprising the following steps: inoculating the Aureobasidium pullulans Kxk gene knockout mutant into a seed medium to prepare a seed solution, and transferring the seed solution into a fermentation medium for fermentation to prepare β-glucan.

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

[0033] The Aureobasidium pullulans Kxk gene knockout mutant strain was inoculated into the seed medium and cultured at 28°C and 200 r / min for 24 hours to obtain the 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 inoculated into the fermentation medium is 5% by volume fraction.

[0036] 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.

[0037] The fermentation conditions are 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 Putative hexokinase Kxk gene knockout mutant strain constructed by the present invention shows significant advantages in improving the yield of β-glucan and other aspects, laying a solid foundation for the large-scale industrial application of Aureobasidium pullulans. After multiple rounds of experimental verification, the mutant strain has 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. Description of the Drawings

[0040] Figure 1 For the PCR electrophoresis diagram of the construction of the recombinant plasmid of the Aureobasidium pullulans Putative hexokinase Kxk gene knockout described in the present invention pUC57- Asp.PGK-Hyg Recombinant plasmid construction

[0041] In the figure, M is the DNA ladder 5000 bp; 1. Using A. pullulans The genome of E (ATCC15233) as a template, and the PCR product using Kxk upstream-F / Kxk upstream-R as the primer pair; 2. Using A. pullulansPCR products using the genome of E (ATCC15233) as a template and PGK promoter-F / PGK promoter-R as primer pairs; 3. Amplify the Hyg resistance sequence and the trpC terminator using the pBARGPE1-Hygro-EGFP plasmid as a template, and PCR products using Hyg-F / Hyg-R as primer pairs; 4. PCR products using the pUC57 plasmid as a template and pUC57 reverse-F / pUC57 reverse-R as primer pairs; 5. Using A.pullulans the genome of E (ATCC15233) as a template and PCR products using Kxk downstream-F / Kxk downstream-R as primer pairs.

[0042] Figure 2 For seamless cloning and recombinant products pUC57-Asp.PGK-Hyg PCR electrophoresis diagram for PCR identification;

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

[0044] Figure 3 Microscopic examination comparison diagram of Aureobasidium pullulans cells and protoplast preparation;

[0045] In the figure: A is Aureobasidium pullulans cells; B is Aureobasidium pullulans protoplasts.

[0046] Figure 4 Verification diagram of the knockout strain;

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

[0048] Figure 5 Comparison diagram of β-glucan production between Aureobasidium pullulans strains and Kxk knockout mutants. Detailed implementation methods

[0049] The following will further elaborate on the present invention, 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, conduct according to conventional conditions; for reagents or instruments without indicating the manufacturer, they are all ordinary commercially available products.

[0051] Material sources

[0052] The pBARGPE1-Hygro-EGFP plasmid was purchased from Wuhan Miaoling Biotechnology Co., Ltd.; the plasmid pUC57 Escherichia coli glycerol bacteria were purchased from Sangon Biotech (Shanghai) Co., Ltd.; Lysing Enzymes were 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.PGK-Hyg Construction of knockout vector

[0057] (1)Extraction of pUC57 plasmid

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

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

[0060] Using A.pullulans the genomic DNA of E (ATCC15233) as a template, PCR amplification was carried out using the designed upstream homologous arm primers. Upstream homologous arm of the Kxk gene: Amplify the 733 kb sequence upstream of the Kxk gene, as shown in SEQ ID NO.4. Downstream homologous arm of the Kxk gene: Amplify the 716 kb sequence downstream of the Kxk gene, as shown in SEQ ID NO.5.

[0061] The primers are as follows:

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

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

[0064] Designed according to the sequence of SEQ ID NO.6, using the pUC57 plasmid as a template, the pUC57 vector was amplified linearly by inverse PCR; the sequence of the linearly amplified pUC57 vector by inverse PCR is shown in SEQ ID NO.6.

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

[0066] pUC57 reverse-F is shown in SEQ ID NO.11, and pUC57 reverse-R is 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 amplified linearly by PCR, and 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 A.pullulans the genomic DNA of E (ATCC15233) as a template, the strong promoter of Aspergillus brasiliensis PGK 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] PGK promoter-F is shown in SEQ ID NO.15, and PGK 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, 25 μL of Aspergillus brasiliensis A. pullulans E (ATCC15233) DNA, 2 μL of 2×Phanta Max Master Mix (Dye Plus), 19 μL of dd H2O.

[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 detect the size and purity of the amplified fragment. The nucleic acid gel diagrams of each recombinant element are as Figure 1As shown, the fragment was cut off and recovered, and the PCR product was purified using a DNA gel extraction 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, the purified linearized pUC57 vector (SEQ ID NO.6), the upstream homologous arm of the Putativehexokinase Kxk gene (SEQ ID NO.4), the PGK promoter (SEQ ID NO.2), the Hyg resistance gene and the trpC terminator (SEQ ID NO.3), and the downstream homologous arm of the Putative hexokinase Kxk gene (SEQ IDNO.5) were gently mixed according to the ratio in the seamless cloning kit instructions, centrifuged briefly, incubated at 37°C for 30 min, and immediately placed 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 antibiotic-free LB liquid medium, 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 a single colony on the plate and inoculate it into an LB liquid medium containing ampicillin (100 μg / mL), and shake culture at 37°C and 200 r / min overnight. Use a plasmid miniprep kit to extract the plasmid from the cultured bacterial solution, and the operation is carried out according to the kit instructions. The extracted recombinant plasmid pUC57-Asp.PGK-Hyg was 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 transformed

[0085] Use a plasmid miniprep kit to extract the recombinant plasmid pUC57-Asp.PGK-Hyg , and the specific operation is carried out according to the kit instructions. Perform PCR amplification with primers Kxk upstream-F and Kxk downstream-R pUC57-Asp.PGK-HygThe recombinant plasmid, and the nucleic acid gel image of the linear fragment to be transferred is as shown in Figure 2 the figure. After gel recovery of the fragment, the PCR product was purified using a DNA gel recovery kit to obtain the linear fragment to be transferred Asp.PGK-Hyg .

[0086] (2)Preparation of protoplasts of Aureobasidium pullulans

[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 shake culture at 28 °C for 48 h. Transfer 100 μL of the bacterial solution to 50 mL of seed medium, and 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 bacteria (4000×g, 4 °C, 10 min), wash twice with the pretreatment buffer (10 mM Tris-HCl pH7.5, 1.2 M sorbitol), and resuspend in the enzyme solution (1.2 M sorbitol 20 mg / mL Lysing Enzymes). Incubate at 30 °C with gentle shaking (80 r / min) for 90 - 120 min, and observe under the microscope (as shown in Figure 3 ) > 90% of the bacteria are spheroidized. Centrifuge (2000×g, 4 °C, 10 min) to collect the protoplasts.

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

[0089] Mix 100 μL of the protoplast suspension (10 7 cells / mL) with 10 μg of the linearized vector Asp.PGK-Hyg Add an equal volume of PEG-CaT solution (40% PEG4000, 50 mM CaCl2, 10 mM Tris-HCl pH7.5), gently mix, and incubate on ice for 20 min. Then, let it stand at 25 °C for 5 min, perform a 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)Screening for transformants on the resistance medium

[0091] Wash the transformed protoplasts obtained by centrifugation in the above operation with STC buffer (1.2 M sorbitol, 10 mM Tris-HCl pH 7.5, 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, 2 g / L peptone, 6 g / L glucose, pH 6.0, 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 of Hyg. Incubate in the dark at 28 °C for 5 - 7 days to obtain positive transformants.

[0092] Example 3

[0093] Verification of the knockout strain

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

[0095] (2) As Figure 4 , agarose gel electrophoresis shows that the amplified fragment of the knockout strain is 5.6 kb (containing the hyg gene), while that of the starting strain is 2.9 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 to ferment and determine 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. 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] The starting strain and the Kxk knockout strain were respectively fermented and cultured in a 5L fermenter under the fermentation conditions of 28°C and 200 r / min, and the fermentation was stopped at 144 h. Starting from 48 h of fermentation, samples were taken every 24 h and the content of β-glucan in the fermentation broth was detected using a β-glucan detection kit (Megazyme, Bray, Ireland).

[0100] The results showed that ( Figure 5 ), the β-glucan yield of the knockout strain reached 4.84 g / L, which was significantly higher than the yield of 2.93 g / L of the starting strain, and it could be used in actual production.

Claims

1. A Aureobasidium pullulans Kxk gene knockout mutant strain, characterized in that, The coding gene between the 82nd and 1557th positions of the Putative hexokinase Kxk gene in the Aureobasidium pullulans strain was knocked out and replaced with a hygromycin B resistance gene cassette; The entire coding gene sequence of the Putative hexokinase Kxk gene is shown in SEQ ID NO.1; The hygromycin B resistance gene cassette includes: the strong promoter phosphoglycerate kinase PGK promoter in Aureobasidium pullulans and the hygromycin B resistance gene Hyg, which is linked to the trpC terminator; The sequence of the strong promoter phosphoglycerate kinase PGK promoter in Aureobasidium pullulans 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, wherein The starting strain of Aureobasidium pullulans is Aureobasidium pullulans ATCC15233.

3. The method for constructing the mutant strain according to any one of claims 1-2, characterized in that, It includes the following steps: (1) pUC57-Asp.PGK-Hyg Construction of knockout vector: The linearized pUC57 vector, the upstream homologous arm of the Putative hexokinase Kxk gene, the PGK promoter, the Hyg resistance gene and the trpC terminator, and the downstream homologous arm of the Putative hexokinase Kxk gene were subjected to seamless cloning and ligation. The ligation product was transformed into Escherichia coli competent cells, and positive clones were screened and verified after transformation to obtain the recombinant plasmid pUC57-Asp.PGK-Hyg ; The sequence of the linearized pUC57 vector is shown in SEQ ID NO.6; The sequence of the upstream homologous arm of the Putative hexokinase Kxk gene is shown in SEQ ID NO.4; The sequence of the downstream homologous arm of the Putative hexokinase Kxk gene is shown in SEQ ID NO.5; The sequence of the PGK promoter is shown in SEQ ID NO.2; The sequences of the Hyg resistance gene and the trpC terminator are shown in SEQ ID NO.3; (2)Genetic transformation of Aureobasidium pullulans: Use primers Kxk upstream-F and Kxk downstream-R to perform PCR amplification on the pUC57-Asp.PGK-Hyg recombinant plasmid obtained in step (1) to obtain a linear fragment to be transferred Asp.PGK-Hyg ; Prepare the protoplasts of Aureobasidium pullulans, and transfer the linear fragment to be transfected Asp.PGK-Hyg into the protoplasts. Screen the transformants using a resistant medium, verify, and obtain the Aureobasidium pullulans Kxk gene knockout mutant strain; The sequence of Kxk upstream-F is shown in SEQ ID NO.7; The sequence of Kxk downstream-R is shown in SEQ ID NO.

10.

4. The construction method according to claim 3, wherein, In step (1), after transformation, positive clone screening was carried out: the bacterial solution was spread on an LB solid medium plate containing 100 μg / mL ampicillin and cultured overnight to obtain positive clones; In step (2), the linear fragment to be transferred is introduced into protoplasts by the PEG method. Asp.PGK-Hyg ​ 5. The construction method according to claim 3, wherein In step (2), transformants were screened using a resistance medium: the transformed protoplasts were inoculated on a double-layer medium for culture to obtain positive transformants; 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; 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; In step (2), verification of positive transformants: Genomic DNA of positive transformants was extracted and amplified and verified using the primers Kxk upstream-F and Kxk downstream-R.

6. Use of the mutant strain according to any one of claims 1-2 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, It includes the following steps: inoculating the mutant strain described in any one of claims 1-2 or the mutant strain constructed by the construction method described in claim 3 into a seed medium to prepare a seed solution, transferring the seed solution to a fermentation medium, and fermenting to prepare β-glucan.

8. The method according to claim 7, wherein The preparation method of the seed solution includes the following: Inoculating the mutant strain into the seed medium, and culturing at 28°C and 200 r / min for 24 hours to obtain the seed solution; The components of the seed medium include: 4 g / L yeast extract powder, 2 g / L peptone, 6 g / L glucose, pH 6.0; The inoculation amount of the seed solution inoculated into the fermentation medium is 5% by volume fraction; 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; Transferring the seed solution to the fermentation medium, and fermenting at 28°C and 200 r / min for 144 hours.

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