Methyltransferase mutant and its application in production of icariin

Abstract

The application provides a methyltransferase mutant and application thereof in production of icariin, and belongs to the technical field of bioengineering. The application solves the problem of low icariin yield in the prior art. The methyltransferase mutant is obtained by mutating any one of the 76th position, the 146th position, the 250th position and the 290th position of the amino acid shown in SEQ ID NO. 2. The methyltransferase mutant is mainly used for efficient methyltransferase and application thereof in synthesis of icariin.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a methyltransferase mutant and its application in the production of icariin. Background Technology

[0002] Icaritin is a natural flavonoid compound mainly derived from plants of the genus Epimedium in the Berberidaceae family (such as Epimedium brevicornu and Epimedium sagittatum). Its chemical structure is a deglycosylated derivative of 8-isopentenylkaempferol-3-rhamnoside, possessing a unique isopentenyl substitution and polyhydroxy structure. Modern research indicates that icariin has broad biological activities, including antitumor activity (such as inhibiting the proliferation of liver and lung cancer cells), anti-inflammatory effects, improving bone metabolism (treating osteoporosis), and cardiovascular protection. It is an important material basis for the traditional Chinese medicine Epimedium's effects of "tonifying the kidneys and strengthening yang, and fortifying muscles and bones."

[0003] Currently, the main production methods for icariin include plant extraction, chemical synthesis, and biosynthesis (enzyme catalysis / microbial fermentation). Plant extraction is the traditional and still widely used method. It uses dried leaves of *Epimedium* plants as raw material, extracting total flavonoids with organic solvents such as ethanol and methanol, followed by purification by column chromatography (e.g., macroporous resin, silica gel column) to obtain icariin. Because the product is naturally extracted, it retains the natural configuration of the compound and has good biocompatibility, making it suitable for pharmaceuticals, health products, and other fields. The disadvantages of plant extraction are that the raw material depends on wild or cultivated *Epimedium* plants, making it highly susceptible to changes in origin, season, and climate, resulting in unstable resource supply. Furthermore, the icariin content in plants is extremely low (usually only 0.01%-0.1%), leading to low extraction rates, high production costs, and the need for large amounts of organic solvents during extraction, which can cause environmental pollution. Additionally, the purification process is complex, and impurities are difficult to completely remove. Chemical synthesis can modify the structure of simple precursors (such as flavonoids like kaempferol and isopentenyl reagents) through methods like isopentenylation, deglycosylation, and methylation to synthesize icariin. While chemical synthesis is not limited by plant resources, can increase yield through large-scale production, and offers controllable raw material costs and high purity, icariin's structure contains multiple hydroxyl and isopentenyl groups, requiring high stereoselectivity, and involves complex synthesis steps (typically 5-8 steps). The reaction process also necessitates the use of heavy metal catalysts, strong acids, and strong bases, easily generating toxic byproducts and causing severe environmental pollution. Compared to these methods, biosynthesis is a relatively economical and environmentally friendly approach to realizing greater market value for icariin. Therefore, identifying components that can efficiently produce icariin is of great research significance. Summary of the Invention

[0004] The purpose of this invention is to propose a methemotransferase mutant and its application in the production of icariin, providing a valuable component for constructing high-yielding icariin strains.

[0005] The present invention provides a methyltransferase mutant, which is obtained by mutating any one of the 76th, 146th, 250th and 290th positions, starting from the amino acid shown in SEQ ID NO.2.

[0006] This invention provides a methyltransferase mutant, wherein the methyltransferase mutant is any one of the following:

[0007] (1) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the lysine at position 76 with glutamic acid;

[0008] (2) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the proline at position 146 with alanine;

[0009] (3) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the aspartic acid at position 250 with alanine;

[0010] (4) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the serine at position 290 with glycine.

[0011] The present invention provides a gene encoding the above-mentioned methyltransferase mutant.

[0012] The present invention provides a recombinant vector containing the above-mentioned genes.

[0013] To further specify, the launch vehicle is the pACYCDuet series.

[0014] The present invention provides a host cell containing the above-mentioned genes.

[0015] Further specifying, this includes methyltransferase genes containing site-directed mutations. MpOMT4 Mut 1. Shortened and optimized pentenyltransferase mutant gene EpPTtru60 Isopentenyl pyrophosphate isomerase gene idi Isopentenyl kinase gene AtIPK and choline kinase gene ScCK The overexpression plasmid was transferred into Escherichia coli BL21(DE3) competent cells to obtain genetically engineered bacteria.

[0016] The present invention provides the application of the above-mentioned methyltransferase mutant, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned host cell in the production of icariin.

[0017] This invention provides a method for producing epimedium, the steps of which are as follows: fermenting the above-mentioned genetically engineered bacteria at 25°C for 48 hours.

[0018] Further specifying, the fermentation medium consists of 20 g / L glucose, 4 mL / L glycerol, 12 g / L peptone, 16.4 g / L K2HPO4·3H2O, and 2.3 g / L KH2PO4.

[0019] Beneficial Effects: This invention provides a methyltransferase mutant for the efficient synthesis of icariin. Through co-evolutionary analysis and substrate binding analysis of the catalytic cavity modification, site-directed mutagenesis was performed on the methyltransferase mutant MpOMT4 at positions 76 (Lys), 117 (His), 146 (Pro), 250 (Asp), 289 (Phe), and 290 (Ala), respectively, yielding seven isopentenyltransferase mutants, namely MpOMT4. K76E MpOMT4 H117Q MpOMT4 P146A MpOMT4 D250A MpOMT4 F289A and MpOMT4 S290G Icariin (MpOMT4) was obtained by shake-flask fermentation of genetically engineered bacteria overexpressing unmutated methyltransferases and seven genetically engineered bacteria overexpressing site-directed methyltransferases. P146A As the optimal mutant, after 48 h of shake-flask fermentation, the icariin yield was 9.56 mg / L, which was 24% higher than that of the control strain, providing a valuable component for improving icariin yield. Attached Figure Description

[0020] Figure 1 A is the liquid chromatography chromatogram of icariin standard, with a retention time of 22.079 min.

[0021] Figure 1 B contains MpOMT4 P146A The liquid phase diagram of the fermentation broth of the mutant strain shows that the retention time of icariin in the fermentation broth is 22.084 min, which is consistent with the retention time of the icariin standard. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other, and the described embodiments are only some embodiments of the present invention, not all embodiments.

[0023] The seed culture system consisted of 10 mL of LB medium in a 100 mL Erlenmeyer flask containing 10 mL of culture medium, which was composed of 5 g / L yeast extract, 10 g / L NaCl, and 10 g / L peptone.

[0024] The fermentation system for shake-flask fermentation consisted of 250 mL Erlenmeyer flasks containing 50 mL of culture medium, the composition of which was 20 g / L glucose, 4 mL / L glycerol, 12 g / L peptone, 16.4 g / L K2HPO4·3H2O, and 2.3 g / L KH2PO4.

[0025] Example 1. Obtaining a methyltransferase mutant

[0026] This invention provides a methyltransferase mutant for the efficient synthesis of icariin. Through co-evolutionary analysis and substrate binding analysis of the catalytic cavity modification, site-directed mutagenesis was performed on the methyltransferase mutant MpOMT4 at positions 76 (Lys), 117 (His), 146 (Pro), 250 (Asp), 289 (Phe), and 290 (Ala), respectively, yielding six isopentenyltransferase mutants, namely MpOMT4. K76E MpOMT4 H117Q MpOMT4 P146A MpOMT4 D250A MpOMT4 F289A and MpOMT4 S290G The site-directed mutation's parental sequence is derived from peppermint (Mentha haplocalyx). Mentha The methyltransferase MpOMT4, with its nucleotide sequence shown in SEQ ID NO: 1 and its amino acid sequence shown in SEQ ID NO: 2; the isopentenyl pyrophosphate isomerase IDI, with its nucleotide sequence shown in SEQ ID NO: 3 and its amino acid sequence shown in SEQ ID NO: 4; the truncated and optimized isopentenyl transferase EpPTtru60, with its nucleotide sequence shown in SEQ ID NO: 5 and its amino acid sequence shown in SEQ ID NO: 6; the isopentenyl kinase AtIPK, with its nucleotide sequence shown in SEQ ID NO: 7 and its amino acid sequence shown in SEQ ID NO: 8; and the choline kinase ScCK, with its nucleotide sequence shown in SEQ ID NO: 9 and its amino acid sequence shown in SEQ ID NO: 10. (Reference: Biotransformation of Kaempferol to Icaritinin Engineered) Saccharomyces cerevisiae )

[0027] SEQ ID NO: 1 (nucleotide sequence of the methyltransferase gene MpOMT4):

[0028]

[0029] SEQ ID NO: 2 (Amino acid sequence of methyltransferase gene MpOMT4):

[0030] MVADEEVRVRAEAWNNAFGYIKPTAVATAVELGLPDILENHDGPMSLLELSAATDCPAEPLHRLMRFLVFHGIFKKTAKPPLSNEAVYYARTALSRLFTRDELGDFMLLQTGPLSQHPAGLTASSLRTGKPQFIRSVNGEDSWTDPVNGYHMKVFSDAMAAHARETTAAIVR YCPAAFEGIGTVVDVGGRHGVALEKLVAAFPWVRGISFDLPEIVAKAPPRPGIEFVGGSFFESVPKGDLVLLMWILHDWSDESCIEIMKKCKEAIPTSGKVMIVDAIVDEDGEGDDFAGARLSLLDLIMMAVLARGKERTYREWEYLLREAGFTKFVVKNINTVEFVIEAYP*;

[0031] SEQ ID NO: 3 (nucleotide sequence of isopentenyl pyrophosphate isomerase IDI):

[0032] ATGCAAACGGAACACGTCATTTTATTGAATGCACAGGGAGTTCCCACGGGTACGCTGGAAAAGTATGCCGCACACACGGCAGACACCCGCTTACATCTCGCGTTCTCCAGTTGGCTGTTTAATGCCAAAGGACAATTATTAGTTACCCGCCGCGCACTGAGCAAAAAAGCATGGCCTGGCGTGTGGACTAACTCGGTTTGTGGGCACCCACAACTGGGAGAAAGCAACGAAGACGCAGTGATCCGCCGTTGCCGTTATGAGCTTGGCGTGGAAATTACGCCTCCTGAATCTATCTATCCTGACTTTCGCTACCGCGCCACCGATCCGAGTGGCATTGTGGAAAATGAAGTGTGTCCGGTATTTGCCGCACGCACCACTAGTGCGTTACAGATCAATGATGATGAAGTGATGGATTATCAATGGTGTGATTTAGCAGATGTATTACACGGTATTGATGCCACGCCGTGGGCGTTCAGTCCGTGGATGGTGATGCAGGCGACAAATCGCGAAGCCAGAAAACGATTATCTGCATTTACCCAGCTTAAATAA;

[0033] SEQ ID NO: 4 (Amino acid sequence of isopentenyl pyrophosphate isomerase IDI):

[0034] MQTEHVILLNAQGVPTGTLEKYAAHTADTRLHLAFSSWLFNAKGQLLVTRRALSKKAWPGVWTNSVCGHPQLGESNEDAVIRRCRYELGVEITPPESIYPDFRYRATDPSGIVENEVCPVFAARTTSALQINDDEVMDYQWCDLADVLHGIDATPWAFSPWMVMQATNREARKRLSAFTQLK*;

[0035] SEQ ID NO: 5 (Nucleotide sequence of truncated and optimized isopentenyl transferase EpPTtru60):

[0036] ATGCACACCCATGAAAAAGAACTGCTGTTTAAAGACAAGAACCCGACCCGCGAAAATCCGTGCCCGTCAGCAACCAGCAGCGAAAATGCACCGCTGAGTTTTAGCACCAAACTGGATATGTTTATCAAGTTCGTGCGTCCGTATGCAACCATTGGTATTATTGGTAATACCATCTGCATGTGCATCCTGCCGGTTCAGACAATGGCTGATCTGAGCCCGAAATTTTTCATTGGTGTGGCCCAGGCAATTGCCAGTATGGTTCTGATGAATCTGTTTAATGTGGCAGTGAATCAGGTTTATGACGTTGAACTGGATAAGGTTAACAAGCCGTATCTGCCGCTGGCAAGCGGTGGTGTTAGCATGACCAGTGCAACCCTGTTTACCATTCTGACCGCCGCCCTGAGTATTGCACTGGGTTATTTTAGCAGCCCGGCACTGTTTTATGGCAGTATTGCCTTTTTCCTGAGCGCAAGCGCATATAGTGTTAATTTTCCGCTGCTGCGCTGGAAAAATAATGCCCTGGGCGCAATTATTAGTCTGATGCTGTGGGGTATTAGCCTGCAGACCGGTGTGTTTTTCCATATTCAGCAGTATGTGCTGGGTAAACCGATGGTTCTGAAAAATAGTTTCATCTACGCGATCATCTTCCAGAGCCTGTTTAGCATTGTTGTTGCCACCCTGAAAGATCTGCCGGATGTTGAAGGCGATAAAGCCAATGGCAGTACCAATCTGACCATTCTGATTGGCAAAGAAAAGGTGTTTTGGGGTTGCACCAGCCTGATGCTGGCCACATATATTGGCACCGCAGCATTTGGCGCAACCCTGCCTATTCTGAAAAATAAGCTGGTTACAATGGTTGCCCATAGCGCCCTGGCCGTTTTTCTGTGGCTGCAGGCTAAACAGATTGATCTGGCAGATGATGCAAGCACCCAGAGCTATTATCTGCTGATGTGGAAACTGTGCAATATTGAATACCTGCTGATCCCGTTTGTTGGC;

[0037] SEQ ID NO: 6 (Truncation of the optimized amino acid sequence of isopentenyltransferase EpPTtru60):

[0038] MHTHEKELLFKDKNPTRENPCPSATSSSENAPLSFSTKLDMFIKFVRPYATIGIIGNTICMCILPVQTMADLSPKFFIGVAQAIASMVLMNLFNVAVNQVYDVELDKVNKPYLPLASGGVSMTSATLFTILTAALSIALGYFSSPALFYGSIAFFLSASAYSVNFPLL RWKNNALGAIISLMLWGISLQTGVFFHIQQYVLGKPMVLKNSFIYAIIFQSLFSIVVATLKDLPDVEGDKANGSTNLTILIGKEKVFWGCTSLMLATYIGTAAFGATLPILKNKLVTMVAHSALAVFLWLQAKQIDLADDASTQSYYLLMWKLCNIEYLLIPFVG*;

[0039] SEQ ID NO: 7 (nucleotide sequence of isopentenyl kinase AtIPK):

[0040] ATGGAACTGAATATTAGCGAAAGTCGCAGCCGCAGCATTCGCTGTATTGTTAAACTGGGTGGCGCAGCCATTACCTGCAAAAATGAACTGGAAAAAATCCACGACGAGAATCTGGAAGTTGTTGCATGTCAGCTGCGCCAGGCCATGCTGGAAGGTTCAGCACCTAGTAAAGTGATTGGCATGGATTGGAGTAAACGCCCGGGTAGCAGTGAAATTAGTTGTGATGTTGATGACATCGGCGATCAGAAAAGCAGCGAATTTTCAAAATTCGTGGTGGTTCATGGTGCAGGTAGCTTTGGTCATTTTCAGGCCAGCCGTAGCGGTGTGCATAAAGGCGGTCTGGAAAAACCGATTGTGAAAGCCGGCTTTGTTGCCACCCGTATTAGCGTTACCAATCTGAATCTGGAAATCGTTCGCGCACTGGCCCGTGAAGGTATTCCGACAATTGGTATGAGTCCGTTTAGCTGCGGTTGGAGTACCAGTAAACGCGATGTGGCCAGCGCCGATCTGGCAACCGTTGCAAAAACCATTGATAGTGGTTTTGTGCCGGTGCTGCATGGTGACGCAGTTCTGGATAATATTCTGGGCTGTACCATTCTGAGCGGCGATGTTATTATTCGCCATCTGGCCGATCATCTGAAACCGGAATATGTGGTGTTTCTGACCGATGTGCTGGGTGTTTATGATCGTCCGCCGAGCCCGAGCGAACCTGACGCAGTGTTACTGAAAGAAATTGCAGTGGGTGAAGATGGTAGCTGGAAAGTTGTTAATCCGCTGCTGGAACATACCGATAAAAAAGTTGATTACAGCGTGGCAGCCCATGATACCACCGGTGGTATGGAAACCAAAATTAGCGAAGCAGCCATGATTGCAAAACTGGGTGTGGATGTGTATATTGTGAAAGCAGCAACCACCCATAGCCAGCGCGCCTTAAATGGTGACCTGCGCGATAGCGTGCCGGAAGATTGGCTGGGTACCATTATTCGTTTTAGTAAATAA;。

[0041] SEQ ID NO: 8 (Amino acid sequence of isopentenyl kinase AtIPK):

[0042] MELNISESRSRSIRCIVKLGGAAITCKNELEKIHDENLEVVACQLRQAMLEGSAPSKVIGMDWSKRPGSSEISCDVDDIGDQKSSEFSKFVVVHGAGSFGHFQASRSGVHKGGLEKPIVKAGFVATRISVTNLNLEIVRALAREGIPTIGMSPFSCGWSTSKRDVAS ADLATVAKTIDSGFVPVLHGDAVLDNILGCTILSGDVIIRHLADHLKPEYVVFLTDVLGVYDRPPSPSEPDAVLLKEIAVGEDGSWKVVNPLLEHTDKKVDYSVAAHDTTGGMETKISEAAMIAKLGVDVYIVKAATTHSQRALNGDLRDSVPEDWLGTIIRFSK*;

[0043] SEQ ID NO: 9 (nucleotide sequence of choline kinase ScCK):

[0044]

[0045] SEQ ID NO: 10 (Amino acid sequence of choline kinase ScCK):

[0046] *

[0047] Example 2: Construction of genetically engineered bacteria containing different site-directed methyltransferase mutants

[0048] Overexpression originates from Epimedium ( Epimedium The gene containing a truncated and optimized isopentenyltransferase mutant gene EpPTtru60 Derived from Escherichia coli ( Escherichia coli Isopentenyl pyrophosphate isomerase gene idi Derived from mint ( Mentha Methyltransferase genes containing different site-directed mutations MpOMT4 Mut Derived from Arabidopsis thaliana ( Arabidopsis thaliana Isopentenyl kinase gene AtIPK and derived from brewer's yeast ( Saccharomyces cerevisiae choline kinase gene ScCKoverexpression plasmid pACYCDuet- EpPTtru60- idi-MpOMT4 Mut -AtIPK-ScCK (The reference for plasmid construction is Biotransformation of Kaempferolto Icaritinin Engineered) Saccharomyces cerevisiae The genetically engineered bacteria were obtained by transferring the gene into *E. coli* BL21(DE3) competent cells. The above-mentioned methyltransferase genes contain different site-directed mutations. MpOMT4 Mut overexpression plasmid pACYCDuet- EpPTtru60-idi-MpOMT4-AtIPK-ScCK, pACYCDuet- EpPTtru60-idi- MpOMT4 K76E -AtIPK-ScCK, pACYCDuet- EpPTtru60-idi-MpOMT4 H117Q -AtIPK-ScCK, pACYCDuet- EpPTtru60-idi-MpOMT4 P146A -AtIPK-ScCK, pACYCDuet- EpPTtru60 - idi-MpOMT4 D250A - AtIPK-ScCK, pACYCDuet- EpPTtru60-idi-MpOMT4 F289A -AtIPK-ScCK, pACYCDuet- EpPTtru60- idi

[0049] -MpOMT4 S290G -AtIPK-ScCK All plasmids were synthesized by Suzhou Anshengda Company. The eight engineered strains containing different overexpression plasmids are shown in Table 1.

[0050] Table 1 Genetically engineered bacteria containing different methyltransferase mutants

[0051]

[0052] Example 3: Fermentation experiment of icariin by genetically engineered bacteria containing different methyltransferase mutants

[0053] Eight genetically engineered bacteria were inoculated into LB broth containing resistant culture medium. The culture system consisted of 10 mL aliquots in 100 mL Erlenmeyer flasks, with 20 μg / mL chloramphenicol added. The culture conditions were constant temperature shaking incubator at 37°C and 220 rpm overnight to obtain seed culture. This seed culture was then inoculated into fresh fermentation medium at a 1% volume inoculation rate. The culture system consisted of 50 mL aliquots in 250 mL Erlenmeyer flasks, with 20 μg / mL chloramphenicol added. The culture conditions were constant temperature shaking incubator at 37°C and 220 rpm. 600 Add 0.3 mM IPTG to a final concentration of 0.8-1.0, induce overnight at 25°C and 220 rpm, add 250 mg / L Kae and 25 mmol / L 3-methyl-3-buten-1-ol to the culture medium and continue fermentation for 48 h to prepare samples.

[0054] Example 4: Extraction and liquid chromatography of icariin

[0055] HPLC was performed using a Shimadzu LC20A system (equipped with an LC20ADXR pump, autosampler, and diode array detector). Flavonoid products were eluted on a Shim-pack XR-ODS column (75 mm x 2.0 mm, 2.2 lm, Shimadzu, Kyoto, Japan). The gradient elution system consisted of 0.01% acetic acid (A) and acetonitrile (B). Separation was performed using the following gradients: 0–2 min (15% B), 2–16 min (15%–70% B), 16–18 min (95% B), with the flow rate maintained at 0.45 mL / min. Icariin was detected at 350 nm.

[0056] Using the strain Control containing a site-directed mutation of isopentenyltransferase as a control (shake-flask fermentation yield of 7.68 mg / L), the yield of icariin in shake-flask fermentation of six genetically engineered bacteria containing methyltransferase mutants with different site-directed mutations was statistically analyzed. The results are shown in Table 2. Among the six genetically engineered bacteria containing isopentenyltransferase mutants with different site-directed mutations, MpOMT4... P146A As the optimal mutant, after 48 h of shake-flask fermentation, the icariin yield was 9.56 mg / L, which was 24% higher than that of the control strain.

[0057] Table 2. Comparison of icariin production by methyltransferase mutant strains and control strains.

[0058]

[0059] Note: - indicates no improvement.

[0060] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: The present invention performs site-directed mutagenesis on the key enzyme methyltransferase MpOMT4 in the synthesis process of icariin, wherein the methyltransferase mutant MpOMT4 P146A Overexpression of icariin in genetically engineered bacteria significantly increased the yield of icariin, as shown in the following results. Figure 1 As shown, this invention provides valuable components for constructing high-yield icariin strains.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A methyltransferase mutant, characterized in that, The methyltransferase mutant is based on the amino acid sequence shown in SEQ ID NO.2, with proline at position 146 replaced by alanine.

2. The gene encoding the methyltransferase mutant of claim 1.

3. A recombinant vector containing the gene described in claim 2.

4. A host cell containing the gene of claim 2.

5. The host cell according to claim 4, characterized in that, The gene described in claim 2, and the shortened and optimized pentenyltransferase mutant gene EpPTtru60 Isopentenyl pyrophosphate isomerase gene idi Isopentenyl kinase gene AtIPK and choline kinase gene ScCK The overexpression plasmid was transformed into E. coli BL21(DE3) competent cells to obtain genetically engineered bacteria; the optimized pentenyltransferase mutant gene was truncated. EpPTtru60 The gene is shown in SEQ ID NO.5; isopentenyl pyrophosphate isomerase gene. idi The gene is shown in SEQ ID NO.3; isopentenyl kinase gene. AtIPK The gene is shown in SEQ ID NO.7; choline kinase gene. ScCK The gene is shown in SEQ ID NO.

9.

6. The use of the methyltransferase mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3, or the host cell of claim 4 or 5 in the production of icariin.

7. A method for producing icariin, characterized in that, The method involves the following steps: fermenting the host cell described in claim 5 at 25°C for 48 hours.

8. The method according to claim 7, characterized in that, The fermentation medium consisted of 20 g / L glucose, 4 mL / L glycerol, 12 g / L peptone, 16.4 g / L K2HPO4·3H2O, and 2.3 g / L KH2PO4.

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