A recombinant strain of Pichia pastoris expressing dextranase and its application

Abstract

The present invention discloses a recombinant strain of Pichia pastoris expressing dextranase and its application. The present invention successfully achieves high-level extracellular expression of dextranase by systematically modifying promoters, signal peptides and co-expression molecular chaperones, and the extracellular enzyme activity reaches 159.68U / mL, which is 2.1 times higher than that of the unmodified starting strain. Further, by amplifying fermentation on a 5L tank, the enzyme activity reaches 3257.23U / mL.

Description

Technical Field

[0001] The invention relates to a Pichia pastoris recombinant strain expressing dextranase and application thereof, belonging to the technical field of genetic engineering. Background Art

[0002] Dextran, also known as α-glucan, is a type of glucan macromolecule mainly composed of α-1,6 glycosidic bonds, interspersed with α-1,3 and α-1,4 glycosidic bonds. It is obtained by fermenting sucrose with Enterobacter mesenteroides. Its molecular weight varies with the degree of polymerization.

[0003] Dextranase can specifically cut α-1,6 glycosidic bonds and hydrolyze high molecular weight dextran into low molecular weight dextran, isomaltose, etc., so it is widely used in the sugar industry, dental caries prevention and medical fields. In the sugar industry, dextran is obtained from sucrose in the raw materials by bacterial fermentation, so the main applications of dextranase in the sugar industry are: 1) reducing raw material loss; 2) preventing interference with material balance; 3) reducing the viscosity increase caused by the production of dextran; 4) improving the quality of sucrose crystals. In the prevention and treatment of dental caries, dextranase can degrade bacterial plaque produced by the growth of dental bacteria. In medicine, low molecular weight dextran can be used as a blood volume supplement.

[0004] There are many strains that can produce dextranase in nature, including various prokaryotes and eukaryotes. Its eukaryotic sources include Penicillium, Micrococcus velutipes, and Steinberg's liposomal yeast. In recent years, it has also been reported to be found in marine microorganisms. Prokaryotic sources are mainly Streptococcus, Bacteroidetes, etc. At present, the main commercial dextranases are mainly derived from Chaetomium and Penicillium. Domestic strain screening for dextranase mainly comes from marine flora, mainly Pseudoalteromonas tetraodonis, Catenovulum sp.DP03, Arthrobacter sp., Arthrobacter oxydans, and Bacillus megatherium. Most of them have low enzyme activity, and only the dextranase from Pseudoalteromonas has reached 270.1U / mL after optimization.

[0005] At present, commercial dextranase still comes from wild bacteria. Through natural screening, further mutagenesis is used to increase enzyme production, thereby obtaining high-yield strains with excellent shape. However, when using wild bacteria to produce dextranase, secondary metabolites will also be produced, which brings certain difficulties to separation, so there are certain limitations in application. In recent years, the heterologous expression of dextranase has also been widely studied. Therefore, the construction of engineered bacteria for heterologous expression of dextranase, the simplification of enzyme expression and purification processes, and the optimization of costs have received more and more attention. The heterologous expression of dextranase has been studied at home and abroad for more than 20 years, and the main heterologous expression systems are divided into prokaryotes and eukaryotes. In the prokaryotic expression system, the main hosts are Escherichia coli and Bacillus subtilis. In 2019, Wang Leyi et al. used Bacillus subtilis as a host to heterologously express the dextranase gene from Chaetomium gracilis, and the final enzyme activity was 21.25U / mL. Liu Le and others heterologously expressed the modified dextranase gene from Arthrobacter marine oxidans in Escherichia coli, and the final enzyme activity reached 50U / mL. The prokaryotic expression system has a relatively shorter cycle than the eukaryotic expression system, but for the dextranase gene of eukaryotic origin, the prokaryotic expression system has the problem of being unable to perform post-translational modifications. In addition, the large intestine system is difficult to achieve secretory expression, and the bacteria need to be broken to obtain the target protein, which is cumbersome to operate, and there is also the problem of endotoxin production. Therefore, the exogenous expression of dextranase in the eukaryotic expression system has received widespread attention.

[0006] As a eukaryotic expression system, Pichia pastoris is considered a GRAS strain with excellent exogenous protein expression ability, which allows its secreted proteins to be purified quickly and efficiently. In addition, using Pichia pastoris as an expression host, recombinant proteins can be post-translationally modified from the endoplasmic reticulum to the Golgi apparatus, which is very important for the rapid development of recombinant protein expression using Pichia pastoris as a host in recent years. The dextranase recombinant Pichia pastoris derived from Lipomyces starkeyi constructed by Kang et al. reached an enzyme activity of 134U / mL in a 10L fermenter.

[0007] In general, the research on microbial dextranase at home and abroad has achieved certain results, but it still cannot meet the needs of industrial production. How to construct a recombinant strain that expresses dextranase at a high level has become a current research hotspot. Summary of the invention

[0008] To solve the above technical problems, the present invention provides a recombinant strain of Pichia pastoris expressing dextranase. The present invention achieves efficient secretory expression of dextranase through signal peptide, promoter modification and co-expression of molecular chaperones, and provides a recombinant plasmid comprising the gene of the present invention and a host cell comprising the recombinant plasmid.

[0009] The first object of the present invention is to provide a recombinant Pichia pastoris strain expressing dextranase, wherein the recombinant Pichia pastoris strain uses Pichia pastoris as a host, uses α(Δ57-70)-HL28 as a signal peptide, and uses a promoter DAS2 to heterologously express dextranase.

[0010] Furthermore, the amino acid sequence of the dextranase is shown in SEQ ID NO.4.

[0011] Furthermore, the nucleotide sequence of the signal peptide is shown in SEQ ID NO.1.

[0012] Furthermore, the nucleotide sequence of the promoter DAS2 is shown in SEQ ID NO.2.

[0013] Furthermore, the recombinant Pichia pastoris also co-expresses a molecular chaperone.

[0014] Furthermore, the molecular chaperone is Kar2, Hac1, Ero1, Cne1, Ubc1, Hrd1, Sec1, Bmh2, Sly1, Vps33, Vps45, Sbh1, Sso2, Sec53 or Ssa4.

[0015] Furthermore, the molecular chaperone uses plasmid pGAPZA as a vector.

[0016] Furthermore, the sequence of the recombinant plasmid for co-expressing the molecular chaperone Ssa4 is shown in SEQ ID NO.3.

[0017] Furthermore, the Pichia pastoris is Pichia pastoris GS115.

[0018] Furthermore, the recombinant Pichia pastoris strain uses plasmid pPIC9K as an expression vector.

[0019] The second object of the present invention is to provide a method for producing dextranase by fermentation using the Pichia recombinant strain, comprising the following steps:

[0020] The seed liquid of the recombinant strain of Pichia pastoris is inoculated into the fermentation medium at an inoculation rate of 5-10%. After culturing for 12-24 hours, 30%-50% glycerol is added as feed volume until the wet weight of the bacteria reaches 200-250g / L. The feeding is stopped, the bacteria are starved for 1-3 hours, and the inducer is added to induce enzyme production.

[0021] Furthermore, the inducer is prepared by mixing sorbitol and methanol in a mass-to-volume ratio of 1:15-20.

[0022] Furthermore, the fermentation medium contains 60%-85% H3PO4 26.7mL / L, CaSO4·2H2O0.5-1.0g / L, K2SO4 15-20g / L, MgSO4·2H2O 10-20g / L, KOH 1-10g / L, glycerol 20-100g / L and PTM1 1-10mL / L.

[0023] Furthermore, the seed solution is obtained by inoculating the recombinant strain of Pichia pastoris into a seed culture medium and culturing the culture medium at 25-30° C. and 200-220 rpm until the logarithmic growth phase.

[0024] Furthermore, the seed culture medium contains: 10-20 g / L yeast powder, 20-40 g / L peptone and 10-30 g / L glucose.

[0025] The beneficial effects of the present invention are:

[0026] The present invention successfully achieved high-level extracellular expression of dextranase by systematically modifying the promoter, signal peptide and co-expression molecular chaperone, with the extracellular enzyme activity reaching 159.68U / mL, which was 2.1 times higher than that of the unmodified starting strain. Further, through amplified fermentation in a 5L tank, the enzyme activity reached 3257.23U / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the pPIC9K-DAS2-α(Δ57-70)-HL28-DEX plasmid map;

[0028] Figure 2 is the pGAPZA-Ssa4 plasmid map;

[0029] Figure 3 It is the enzymatic activity test result of dextranase under the co-expression of expression elements and molecular chaperones; among them, 1: GS115-pPIC9K-DEX, 2: GS115-pPIC9K-DAS2-DEX, 3: GS115-pPIC9K-DA S2-α(Δ57-70)-HL28-DEX, 4: GS115-pPIC9K-DAS2-α(Δ57-70)-HL28-DEX-pGAP ZA-Ssa4;

[0030] Figure 4 It is the SDS-PAGE spectrum of dextranase under the transformation of expression elements; wherein, M: protein standard marker, 1: GS115, 2: GS115-pPIC9K, 3: GS115-GS115-pPIC9K-DAS2-α(Δ57-70)-HL28-DEX-pGAPZA-Ssa4;

[0031] Figure 5 It is the fermentation tank enzyme activity-time curve. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0033] Determination of dextranase activity

[0034] Preparation of 2% dextran T2000 substrate: 2 g of dextran T2000 was dissolved in 100 ml of phosphate buffer (pH=6.0) to prepare a 2% dextran T2000 substrate solution.

[0035] Determination method: The reducing sugar content produced by the dextranase decomposition of dextran T2000 was determined according to the 3,5-dinitrohydrochloric acid (DNS) colorimetric method. The fermentation broth and dextran T2000 solution were used as the experimental group, and the fermentation broth and water were used as the control group. The reaction was carried out at 50°C for 10 minutes, and the DNS reagent was added. The reaction was terminated in a boiling water bath. After cooling, the absorbance was measured at 540nm using an enzyme reader. Glucose solutions of different concentrations were prepared to establish a glucose standard curve.

[0036] Enzyme activity calculation method: Enzyme activity (1U) is defined as the amount of enzyme required to produce 1 μmol of reducing sugar per minute under the above experimental conditions. The determination was repeated 3 times. This is used as the standard determination method for dextranase activity.

[0037] Embodiment 1:

[0038] The invention provides a dextranase comprising 606 amino acids, and the amino acid sequence is shown in SEQ ID NO.4.

[0039] The invention provides a recombinant plasmid for expressing dextranase, comprising a dextranase sequence, a signal peptide α(Δ57-70)-HL28 for extracellular expression of the dextranase, and a promoter DAS2 for expressing the dextranase, wherein the nucleotide sequence of the signal peptide α(Δ57-70)-HL28 is shown in SEQ ID NO.1, has a total length of 309 nucleotides, and encodes 103 amino acids; the nucleotide sequence of the promoter DAS2 is shown in SEQ ID NO.2, has a total length of 1099 nucleotides.

[0040] The present invention also provides a recombinant plasmid for expressing a molecular chaperone, wherein the molecular chaperone in this embodiment is Ssa4, and the nucleotide sequence of the recombinant plasmid containing Ssa4 is shown in SEQ ID NO.3, with a total length of 4714 nucleotides.

[0041] Embodiment 2:

[0042] This example provides a method for constructing a recombinant plasmid pPIC9K-DEX and expressing it in Pichia pastoris. The specific steps are as follows:

[0043] (1) Amplification of dextranase coding sequence

[0044] Using the recombinant plasmid pET-28a(+)-DEX containing the dextranase encoding gene as a template, primers (P1, P2) were designed to amplify the dextranase encoding sequence:

[0045] Primer P1: 5′-ACGACGACGACAAGGAATTCATGGGTACGACTAATAACACACATTGT-3′

[0046] Primer P2: 5′-GCGAATTAATTCGCGGCCGCTCAGGAGATTTGCCACTCTCCC-3′

[0047] PCR amplification reaction was carried out in 40 μL system, and 20 μL was added to the reaction system. Max, 15 μL ddH2O, 1 μL template DNA, 2 μL upstream and downstream primers. The reaction conditions were: 95°C denaturation for 3 min, followed by cycling: 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s, 30 cycles in total; 72°C final extension for 5 min. PCR products were identified by nucleic acid electrophoresis and purified by gel cutting.

[0048] (2) Construction of recombinant plasmid pPIC9K-DEX

[0049] The pPIC9K plasmid was double-digested with EcoR I and Not I, and then digested at 37°C for 1 hour, and then the nucleic acid electrophoresis was performed to verify and the target gene and target plasmid were recovered by cutting the gel. The recovered products were ligated with homologous recombinase at 37°C for 30 minutes, and the ligated products were transformed into E. coli JM 109 competent cells, spread on solid LB medium containing ampicillin resistance (100 mg / L), cultured overnight, and multiple transformants were picked and transferred to LB liquid medium (Amp r ), culture at 37°C, 220 rpm for 10-12 h, extract the plasmid and perform PCR verification.

[0050] (3) Transformation of recombinant plasmid pPIC9K-DEX into P. pastoris GS115

[0051] The recombinant plasmid in step (2) was sent to a sequencing company for sequence determination. The recombinant plasmid with the correct sequence was named pPIC9K-DEX, linearized with Sal I, recovered and purified, and transformed into P. pastoris GS115. The recombinant strain was cultured in the culture medium for 144 hours. The fermentation broth was centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was collected for activity determination. The enzyme activity of the recombinant strain P. pastoris GS115 / pPIC9K-DEX was 51.48 U / mL.

[0052] Embodiment 3:

[0053] This example provides a method for constructing a promoter-modified recombinant plasmid pPIC9K-DAS2-DEX and expressing it in Pichia pastoris. The specific steps are as follows:

[0054] (1) Using the P. pastoris GS115 genome as a template, primers (P3, P4) were designed to amplify the DAS2 promoter sequence:

[0055] Primer P3: 5'-ATTGGGCTTGATTGGAGCTCAATGATATTTGAGGGTGTTAGTTACT-3'

[0056] Primer P4: 5'-AAATCTCATCGTTTGGATCCttttgatgtttgATAGTTTGATAAGAGTGAAC-3'

[0057] PCR amplification reaction was carried out in 40 μL system, and 20 μL was added to the reaction system. Max, 15 μL ddH2O, 1 μL template DNA, 2 μL each of upstream and downstream primers. The reaction conditions were: 95℃ pre-denaturation for 3 min, followed by cycling: 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 10 s, 30 cycles in total; 72℃ final extension for 5 min. PCR products were identified by nucleic acid electrophoresis and purified by gel cutting.

[0058] (2) The plasmid pPIC9K-DEX constructed in Example 2 was double-digested with Sac I and BamH I. After digestion at 37° C. for 1 h, nucleic acid electrophoresis was performed to verify and the target gene and target plasmid were recovered by gel tapping. The product was recovered.

[0059] (3) Construction of recombinant plasmid pPIC9K-DAS2-DEX

[0060] Add 0.2 pmol and 0.06 pmol of the products recovered in steps (1) and (2) to the PCR tube, and add the homologous recombination enzyme. After mixing, connect at 37°C for 30 minutes. Transform the ligation product into E. coli JM 109 competent cells and culture overnight on solid LB medium containing ampicillin resistance (100 mg / L). Pick a number of transformants and transfer them to LB liquid medium (Amp r ), culture at 37°C, 220 rpm for 10-12 h, extract the plasmid and perform PCR verification.

[0061] (4) Transformation of P. pastoris GS115 with the recombinant plasmid pPIC9K-DAS2-DEX

[0062] The recombinant plasmid in step (3) was sent to a sequencing company for sequence determination. The recombinant plasmid with the correct sequence was named pPIC9K-DAS2-DEX. The recombinant plasmid was linearized with Sal I and then recovered and purified and transformed into P. pastoris GS115. The recombinant strain was cultured in the culture medium for 144 hours. The fermentation broth was centrifuged at 12000 rpm for 10 minutes at 4°C to collect the supernatant for activity determination. The enzyme activity of the recombinant strain P. pastoris GS115 / pPIC9K-DAS2-DEX was 94.42 U / mL, which was 83.41% higher than that of the strain P. pastoris GS115 / pPIC9K-DEX.

[0063] Embodiment 4:

[0064] This example provides a method for constructing an expression element-modified recombinant plasmid pPIC9K-DAS2-α(Δ57-70)-HL28-DEX and expressing it in Pichia pastoris. The specific steps are as follows:

[0065] (1) Using pUC18-signal as a template, primers (P5, P6) were designed to amplify the α(Δ57-70)-HL28 signal peptide sequence:

[0066] Primer P5: 5′-acatcaaaaGGATCCAAACGATGAGATTTCCTTCAATTTTTACTGC-3′

[0067] Primer P6: 5'-TTAGTCGTACCCATGAATTCCTTGTCGTCGTCGTCaccGT-3'

[0068] PCR amplification reaction was carried out in 40 μL system, and 20 μL was added to the reaction system. Max, 15 μL ddH2O, 1 μL template DNA, 2 μL each of upstream and downstream primers. The reaction conditions were: 95℃ pre-denaturation for 3 min, followed by cycling: 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 10 s, 30 cycles in total; 72℃ final extension for 5 min. PCR products were identified by nucleic acid electrophoresis and purified by gel cutting.

[0069] (2) The plasmid pPIC9K-DAS2-DEX constructed in Example 3 was double-digested with Sac I and BamH I. After digestion at 37°C for 1 h, nucleic acid electrophoresis was performed to verify and the target gene and target plasmid were recovered by gel tapping. The product was recovered

[0070] (3) Construction of recombinant plasmid pPIC9K-DAS2-α(Δ57-70)-HL28-DEX

[0071] Add 0.2 pmol and 0.06 pmol of the products recovered in steps (1) and (2) to the PCR tube, and add an equal volume of homologous recombination enzyme. After mixing, connect at 37°C for 30 minutes. Transform the ligation product into E. coli JM 109 competent cells and culture overnight on solid LB medium containing ampicillin resistance (100 mg / L). Pick a number of transformants and transfer them to LB liquid medium (Amp r ), culture at 37°C, 220 rpm for 10-12 h, extract the plasmid and perform PCR verification.

[0072] (4) Transformation of P. pastoris GS115 with the recombinant plasmid pPIC9K-DAS2-α(Δ57-70)-HL28-DEX

[0073] The recombinant plasmid in step (4) was sent to a sequencing company for sequence determination. The recombinant plasmid with the correct sequence was named pPIC9K-DAS2-α(Δ57-70)-HL28-DEX. The recombinant plasmid was linearized with Sal I, recovered and purified, and transformed into P. pastoris GS115. The recombinant strain was cultured in the culture medium for 144 hours. The fermentation broth was centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was collected for activity determination. The enzyme activity of the recombinant strain P. pastoris GS115 / pPIC9K-DAS2-α(Δ57-70)-HL28-DEX was 118.08 U / mL, which was 25.06% higher than that of the strain P. pastoris GS115 / pPIC9K-DAS2-DEX.

[0074] Embodiment 5:

[0075] This example provides the construction of a molecular chaperone co-expression recombinant plasmid pGAPZA-Ssa4 and a method for expressing it in Pichia pastoris. The specific steps are as follows:

[0076] (1) Using the P. pastoris GS115 genome as a template, primers (P7, P8) were designed to amplify the Ssa4 gene:

[0077] Primer P7: 5′-ctaattattcgaaacggaattcATGGGTAAATCAATTGGAATTGATT-3′

[0078] Primer P8: 5'-tcaatgatgatgatgatgatgatcgacttcttccacggttggt-3'

[0079] PCR amplification reaction was carried out in 40 μL system, and 20 μL was added to the reaction system. Max, 15 μL ddH2O, 1 μL template DNA, 2 μL each of upstream and downstream primers. The reaction conditions were: 95℃ pre-denaturation for 3 min, followed by cycling: 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 10 s, 30 cycles in total; 72℃ final extension for 5 min. PCR products were identified by nucleic acid electrophoresis and purified by gel cutting.

[0080] (2) The plasmid pGAPZA was double-digested with EcoR I and Sal I. After digestion at 37°C for 1 h, nucleic acid electrophoresis was performed to verify the target gene and target plasmid.

[0081] (3) Construction of recombinant plasmid pGAPZA-Ssa4

[0082] Add 0.2 pmol and 0.06 pmol of the products recovered in steps (1) and (2) to the PCR tube, and add an equal volume of homologous recombination enzyme. After mixing, connect at 37°C for 30 minutes. Transform the ligation product into E. coli JM 109 competent cells and culture overnight on solid LB medium containing ampicillin resistance (100 mg / L). Pick a number of transformants and transfer them to LB liquid medium (Amp r ), culture at 37°C, 220 rpm for 10-12 h, extract the plasmid and perform PCR verification.

[0083] (4) Transformation of recombinant plasmid pGAPZA-Ssa4 into P. pastoris GS115-pPIC9K-DAS2-α(Δ57-70)-HL28-DEX

[0084] The recombinant plasmid in step (4) was sent to a sequencing company for sequence determination. The recombinant plasmid with the correct sequence was named pGAPZA-Ssa4. The recombinant plasmid was linearized with Bln I and then recovered and purified, and transformed into P.pastoris GS115-pPIC9K-DAS2-α(Δ57-70)-HL28-DEX. The recombinant strain was cultured in the culture medium for 144 hours. The fermentation broth was centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was collected for activity determination. The enzyme activity of the recombinant strain P.pastoris GS115-pPIC9K-DAS2-α(Δ57-70)-HL28-DEX-pGAPZA-Ssa4 was 159.68 U / mL, which was 35.23% higher than that of the strain P.pastoris GS115 / pPIC9K-DAS2-α(Δ57-70)-HL28-DEX.

[0085] Embodiment 6:

[0086] This example provides a high-density fermentation of the recombinant strain P. pastoris GS115-pPIC9K-DAS2-α(Δ57-70)-HL28-DEX-pGAPZA-Ssa4 in a 5L tank, and the specific steps are as follows:

[0087] The recombinant strain P. pastoris GS115-pPIC9K-DAS2-α(Δ57-70)-HL28-DEX-pGAPZA-Ssa4 in Example 5 was inoculated into a seed culture medium, cultured at 25-30°C and 200-220rpm to the logarithmic growth phase, and used as a seed solution. The seed solution was then transferred to a fermentation medium BSM at an inoculum amount of 5-10%. After the bacteria grew for 12-24 hours, 30%-50% (v / v) glycerol was added until the wet weight of the bacteria reached 200-250g / L, and the feeding was stopped. The bacteria were starved for 2 hours, and the inducer was added to induce enzyme production. The enzyme activity reached a maximum of 3257.23U / mL at 138 hours, which was 20.4 times the level of the shake flask.

[0088] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A recombinant strain of Pichia pastoris expressing dextranase, characterized in that: The recombinant Pichia pastoris strain is P. pastoris GS115 was used as the host, α(Δ57-70)-HL28 was used as the signal peptide, and the promoter DAS2 was used to heterologously express dextranase, and the molecular chaperone Ssa4 was co-expressed; Among them, the amino acid sequence of the dextranase is shown as SEQ ID NO.4, the nucleotide sequence of the signal peptide is shown as SEQ ID NO.1, the nucleotide sequence of the promoter DAS2 is shown as SEQ ID NO.2, and the nucleotide sequence of the molecular chaperone Ssa4 is shown as SEQ ID NO.

3.

2. A method for producing dextranase by fermentation using the recombinant Pichia pastoris strain according to claim 1, characterized in that: The steps include: The seed liquid of the recombinant strain of Pichia pastoris is inoculated into the fermentation medium at an inoculum rate of 5-10%. After culturing for 12-24 hours, 30%-50% glycerol is added as feed volume until the wet weight of the bacteria reaches 200-250 g / L. The feeding is stopped, the bacteria are starved for 1-3 hours, and the inducer is fed to induce enzyme production.

3. The method according to claim 2, characterized in that The inducer is prepared by mixing sorbitol and methanol in a mass-to-volume ratio of 1:15-20.

4. The method according to claim 2, characterized in that: The fermentation medium contains 60%-85% H3PO426.7 mL / L, CaSO4·2H2O 0.5-1.0 g / L, K2SO4 15-20 g / L, MgSO4·2H2O 10-20 g / L, KOH 1-10 g / L, glycerol 20-100 g / L and PTM1 1-10 mL / L.

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