Phenylpyruvate decarboxylase mutant and application thereof in production of tyrosol

Abstract

The invention discloses a phenylpyruvate decarboxylase mutant and application of the phenylpyruvate decarboxylase mutant in production of tyrosol, and belongs to the technical field of bioengineering. The purpose of the present invention is to increase the yield of tyrosol. The phenylpyruvate decarboxylase mutant provided by the invention is obtained by mutating any one of the 56th site, the 92th site, the 93th site, the 106th site, the 115th site, the 153th site, the 178th site, the 188th site, the 214th site, the 220th site, the 234th site, the 242th site, the 273th site, the 285th site, the 371th site, the 416th site, the 581th site and the 621th site by taking amino acid as shown in SEQ ID NO.2 as a starting sequence. The phenylpyruvate decarboxylase is mainly used for high-efficiency phenylpyruvate decarboxylase and application thereof in de novo synthesis of tyrosol.

Description

Technical Field

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

[0002] Tyrosol is a natural active compound with a polyphenol structure, widely found in natural products such as olive oil, wine, and traditional Chinese medicine. Due to its significant antioxidant, anti-inflammatory, neuroprotective, and cardiovascular protective functions, it has high application value in the pharmaceutical, health product, and cosmetic fields. In the pharmaceutical field, tyrosol, as an antioxidant, can scavenge free radicals and inhibit lipid peroxidation, and is used to prevent neurodegenerative diseases such as Alzheimer's and Parkinson's. It also shows potential in the adjunctive treatment of cardiovascular diseases (such as atherosclerosis) by regulating the release of inflammatory factors. In the health product and cosmetic fields, it can be added to dietary supplements and skincare products as a natural antioxidant to alleviate skin oxidative damage and delay aging, meeting consumers' preference for "natural and safe" ingredients. The global natural antioxidant market continues to expand, and tyrosol, as a highly active natural polyphenol, is projected to see an annual market demand growth rate exceeding 15% in 2024. However, the inefficiency of traditional production methods keeps its price high, necessitating a breakthrough in low-cost, large-scale production technology.

[0003] Currently, tyrosol production mainly relies on natural extraction and chemical synthesis. However, with increasing market demand, the limitations of traditional extraction and chemical synthesis methods are becoming increasingly apparent. Both methods have insurmountable shortcomings and cannot meet industrial demands. Natural extraction uses olive oil byproducts (such as olive leaves and pomace) or wine lees as raw materials, separating and purifying them through solvent extraction and column chromatography. However, the tyrosol content in the raw materials is extremely low (usually <0.1%), resulting in low extraction efficiency, high cost, and significant dependence on the origin and season of the raw materials, leading to unstable yields. Chemical synthesis uses p-hydroxybenzaldehyde, acetaldehyde, etc., as raw materials, synthesizing them through multiple chemical reactions such as condensation and reduction. This method suffers from problems such as reliance on fossil resources, harsh reaction conditions (such as high temperature and pressure), and the potential generation of toxic byproducts (such as heavy metal residues), which does not conform to green production principles, and the product purity is difficult to meet pharmaceutical-grade standards.

[0004] Synthetic biology techniques using microorganisms as cell factories are gradually becoming the core direction for the large-scale production of tyrosol. By modifying the metabolic pathways of microorganisms, inexpensive carbon sources (such as glucose and sucrose) can be converted into tyrosol, offering advantages such as low cost and environmental friendliness. Phenyleukate decarboxylase ARO10 is the rate-limiting enzyme in tyrosol synthesis. This study proposes a phenylpyleukate decarboxylase mutant and its application in tyrosol production, providing a valuable component for increasing tyrosol yield. Summary of the Invention

[0005] The present invention aims to provide a phenylpyruvate decarboxylase mutant and its application in the production of tyrosol, and to provide a highly efficient phenylpyruvate decarboxylase and its application in the de novo synthesis of tyrosol, so as to solve the problem of low tyrosol yield in the prior art.

[0006] This invention provides a phenylpyruvate decarboxylase mutant, wherein the phenylpyruvate decarboxylase mutant is any one of the following: (1) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace K at position 92 with R; (2) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the I at position 93 with V; (3) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A; (4) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the S at position 153 with T; (5) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace L at position 178 with F; (6) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace L at position 220 with A; (7) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace Y at position 234 with F; (8) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace D at position 242 with E; (9) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace F at position 273 with L; (10) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace N at position 285 with Q; (11) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace T at position 581 with S.

[0007] The present invention provides a gene encoding the above-mentioned phenylpyruvate decarboxylase mutant.

[0008] The present invention provides a recombinant vector containing the above-mentioned genes.

[0009] Further specifying, the launch vehicle is the pRSFDuet series.

[0010] This invention provides a recombinant microbial cell containing the above-mentioned genes.

[0011] The present invention provides a recombinant Escherichia coli expressing the above-mentioned phenylpyruvate decarboxylase mutant.

[0012] Further specifying, the alcohol dehydrogenase gene ADH6 The aroG gene, containing a mutant 3-deoxy-D-arabinohepeptulose-7-phosphate (DAHP) synthase. fbr Cyclohexadiene dehydrogenase gene tyrC L-amino acid ammonia-lyase gene LAAD The overexpression plasmid was transferred into Escherichia coli BL21(DE3) competent cells.

[0013] The present invention provides the application of the above-mentioned phenylpyruvate decarboxylase mutant, the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant microbial cell in the production of tyrosol and products containing tyrosol.

[0014] The present invention provides a method for producing tyrosol, the steps of which are as follows: fermenting the above-mentioned recombinant Escherichia coli at 30°C for 72 hours.

[0015] Further specified, the fermentation medium consisted of 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH4)2SO4, 3 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 2.0 g / L MgSO4·7H2O, 1.0 g / L citric acid monohydrate, 0.1 g / L vitamin B1, and 7 g / L yeast extract, with 10 g / L CaCO3 used to maintain the pH of the fermentation medium. The culture medium was supplemented with 0.1% by volume of trace elements, consisting of: 2.0 g / L Al2(SO4)3·18H2O, 0.75 g / L CoSO4·7H2O, 2.5 g / L CuSO4·5H2O, 0.5 g / L H3BO3, 24 g / L MnSO4·H2O, 2.5 g / L NiSO4·6H2O, and 15 g / L ZnSO4·7H2O. The pH was adjusted to 7.0 using NaOH.

[0016] Beneficial Effects: This invention provides a phenylpyruvate decarboxylase mutant for efficient synthesis of tyrosol. Through co-evolutionary analysis and substrate binding analysis of the catalytic cavity modification, site-directed mutagenesis was performed on the following positions of the phenylpyruvate decarboxylase ARO10: Glu at position 56, Lys at position 92, Ile at position 93, Ser at position 106, Phe at position 115, Ser at position 153, Leu at position 178, Val at position 188, Ser at position 214, Leu at position 220, Tyr at position 234, Asp at position 242, Phe at position 273, Asn at position 285, Phe at position 371, Gly at position 416, Thr at position 581, and Lys at position 621. This yielded 18 mutant phenylpyruvate decarboxylases, namely ARO10... E56Q ARO10 K92RARO10 I93V ARO10 S106A ARO10 F115W ARO10 S153T ARO10 L178F ARO10 V188I ARO10 S214A ARO10 L220A ARO10 Y234F ARO10 D242E ARO10 F273L ARO10 N285Q ARO10 F371Y ARO10 G416A ARO10 T581S ARO10 K621A Overexpression originates from Saccharomyces cerevisiae (Saccharomyces cerevisiae) Saccharomyces cerevisiae Tyrosol was obtained by shake-flask fermentation of wild-type phenylpyruvate decarboxylase and 18 genetically engineered bacteria that overexpress mutant phenylpyruvate decarboxylases. The phenylpyruvate decarboxylase ARO10 was also used. L220A As the dominant mutant, it increased tyrosol production by 50% compared to the control strain after 72 h of shake-flask fermentation. Attached Figure Description

[0017] Figure 1 This is an HPLC chromatogram showing the tyrosol content in the fermentation broth of the strain according to Example 3 of the present invention, wherein... Figure 1 A shows the HPLC chromatogram of the tyrosol standard; Figure 1 B shows the genetically engineered bacterium ARO10. L220A HPLC chromatogram of tyrosol production after 72 h of fermentation. Detailed Implementation

[0018] 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.

[0019] 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. The fermentation system for shake-flask fermentation consisted of 250 mL Erlenmeyer flasks containing 50 mL of culture medium. The culture medium consisted of 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH4)2SO4, 3 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 2.0 g / L MgSO4·7H2O, 1.0 g / L citric acid monohydrate, 0.1 g / L vitamin B1, and 7 g / L yeast extract. The pH of the fermentation medium was maintained using 10 g / L CaCO3. The culture medium was supplemented with 0.1% by volume of trace elements, the composition of which was: 2.0 g / L Al2(SO4)3·18H2O, 0.75 g / L CoSO4·7H2O, 2.5 g / L CuSO4·5H2O, 0.5 g / L H3BO3, 24 g / L MnSO4·H2O, 2.5 g / L NiSO4·6H2O, and 15 g / L ZnSO4·7H2O. The pH was adjusted to 7.0 using NaOH.

[0020] Example 1. Obtaining a phenylpyruvate decarboxylase mutant This invention provides a mutant phenylpyruvate decarboxylase for efficient synthesis of tyrosol. Through co-evolutionary analysis and substrate binding analysis of the catalytic cavity modification, site-directed mutagenesis was performed on the following positions of the phenylpyruvate decarboxylase ARO10: Glu (56), Lys (92), Ile (93), Ser (106), Phe (115), Ser (153), Leu (178), Val (188), Ser (214), Leu (220), Tyr (234), Asp (242), Phe (273), Asn (285), Phe (371), Gly (416), Thr (581), and Lys (621). This yielded 18 mutant phenylpyruvate decarboxylases, namely ARO10... E56Q ARO10 K92R ARO10 I93V ARO10 S106A ARO10 F115W ARO10 S153T ARO10 L178F ARO10 V188I ARO10 S214A ARO10 L220A ARO10 Y234F ARO10 D242E ARO10 F273L ARO10 N285Q ARO10 F371Y ARO10 G416A ARO10 T581S ARO10K621A The wild-type phenylpyruvate decarboxylase gene enzyme ARO10 is derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae Its nucleotide sequence is shown in SEQ ID NO.1; its amino acid sequence is shown in SEQ ID NO.2.

[0021] SEQ ID NO.1 (nucleotide sequence of wild-type phenylpyruvate decarboxylase ARO10): SEQ ID NO.2 (Amino acid sequence of wild-type phenylpyruvate decarboxylase ARO10): *

[0022] Example 2: Construction of genetically engineered bacteria containing different phenylpyruvate decarboxylase mutants Overexpression derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) Saccharomyces cerevisiae ) phenylpyruvate decarboxylase gene ARO10 and alcohol dehydrogenase gene ADH6, Derived from Escherichia coli ( Escherichia coli The mutant 3-deoxy-D-arabinohepeptulose-7-phosphate (DAHP) synthase gene aroG fbr Derived from motile fermentation monosporus ( Zymomonas mobilis ) cyclohexadiene dehydrogenase gene tyrC, Derived from Proteus mirabilis ( Proteus mirabilisL-amino acid ammonia-lyase gene LAAD recombinant plasmid pRSFDuet -ARO10-ADH6 - aroG fbr -tyrC-LAAD Synthesized by Kinwids Sequencing Company; pRSFduet- ARO10-ADH6 and pETDuet- aroG fbr -tyrC-AtUGT85A1 Published in the references Engineering Escherichia coli for Efficient De Novo Synthesis of Salidroside), pETDuet- aroG fbr -tyrC-AtUGT85A1 The carrier is connected to pRSFduet- ARO10-ADH6 In, and connect to LAAD Gene (SEQ ID NO.4), the obtained recombinant plasmid pRSFDuet -ARO10-ADH6 - aroG fbr -tyrC-LAAD.

[0023] SEQ ID NO.3 (Amino acid sequence of L-amino acid ammonia-lyase LAAD) MNISRRKLLLGVGAAGVLAGGAATLVPMVRRDGKFVESSKSRALFVESTEGALPSESDVVIIGGGIQGIMTAINLAERGMSVTILEKGEVAGEQSGRAYSQIISYQTSPEIFPLHHYGK ILWRGMNEKIGADTSYRTQGRVEALADEKALDRAQEWIKTAKETAGFDVPLNTRIIKGEELSNRLVGAQTPWTVAAFEEDSGSVDPETGTPTLARYAKQIGVKIYTHCAVRGIETAGGK ISDVVTEKGAIRTSNVVLAGGIWSRLFMGNMGVDLPTLNVYLSQQRVSGVPGAPRGNVHLPNGIHFREQADGTYAVAPRIFTSSIVKDSFLLGPKFMHLLGGGELPLEFSIGEDLFNSF KMPTSWKLDEKSPFEQYRIATATQNTEHLDAVFQRMKTEFPVFEKSQIVERWGAVVSPTFDELPIISEVKEYPGLVINTATVWGMTEGPAAGEVTADIVTGKKPVIDPTPFSLDRFKK; SEQ ID NO. 4 (nucleotide sequence of L-amino acid ammonia-lyase LAAD) overexpression plasmid pRSFDuet -ARO10-ADH6 - aroG fbr -tyrC-LAAD The wild-type phenylpyruvate decarboxylase ARO10 gene fragment was replaced with the mutant phenylpyruvate decarboxylase gene fragment (mutant phenylpyruvate decarboxylase ARO10). E56Q Mutant phenylpyruvate decarboxylase ARO10 K92R Mutant phenylpyruvate decarboxylase ARO10 I93V Mutant phenylpyruvate decarboxylase ARO10 S106A Mutant phenylpyruvate decarboxylase ARO10 F115W Mutant phenylpyruvate decarboxylase ARO10 S153T Mutant phenylpyruvate decarboxylase ARO10 L178F Mutant phenylpyruvate decarboxylase ARO10 V188I Mutant phenylpyruvate decarboxylase ARO10 S214A Mutant phenylpyruvate decarboxylase ARO10 L220A Mutant phenylpyruvate decarboxylase ARO10 Y234F Mutant phenylpyruvate decarboxylase ARO10 D242E Mutant phenylpyruvate decarboxylase ARO10 F273L Mutant phenylpyruvate decarboxylase ARO10 N285Q Mutant phenylpyruvate decarboxylase ARO10 F371Y Mutant phenylpyruvate decarboxylase ARO10 G416A Mutant phenylpyruvate decarboxylase ARO10 T581S Mutant phenylpyruvate decarboxylase ARO10 K621A (from the gene), to obtain the recombinant plasmid: pRSFDuet -ARO10 E56Q -ADH6 - aroG fbr -tyrC-LAAD ; pRSFDuet -ARO10 K92R -ADH6 - aroG fbr -tyrC-LAAD ; pRSFDuet -ARO10 I93V -ADH6 - aroG fbr -tyrC-LAAD ; pRSFDuet -ARO10S106A - ADH6 - aroG fbr -tyrC-LAAD ; pRSFDuet -ARO10 F115W -ADH6 - aroG fbr -tyrC-LAAD ; pRSFDuet -ARO10 S153T -ADH6 - aroG fbr -tyrC-LAAD ; pRSFDuet -ARO10 L178F -ADH6 - aroG fbr -tyrC-LAAD ; pRSFDuet -ARO10 V188I -ADH6 - aroG fbr -tyrC-LAAD ; pRSFDuet -ARO10 S214A -ADH6 - aroG fbr -tyrC-LAAD ; pRSFDuet -ARO10 L220A -ADH6 - aroG fbr -tyrC-LAAD ; pRSFDuet -ARO10 Y234F -ADH6 - aroG fbr -tyrC-LAAD ; pRSFDuet -ARO10 D242E -ADH6 - aroG fbr -tyrC-LAAD ; pRSFDuet -ARO10 F273L -ADH6 - aroG fbr -tyrC-LAAD ; pRSFDuet-ARO10 N285Q -ADH6 - aroG fbr -tyrC-LAAD ; pRSFDuet -ARO10 F371Y -ADH6 - aroG fbr -tyrC-LAAD ; pRSFDuet-ARO10 G416A -ADH6-aroGfbr-tyrC-LAAD ; pRSFDuet -ARO10 T581S -ADH6-aroGfbr-tyrC-LAAD ; pRSFDuet -ARO10 K621A -ADH6- aroG fbr -tyrC-LAAD .

[0024] The above overexpression plasmid and the control overexpression plasmid pRSFDuet were respectively used. -ARO10-ADH6 - aroG fbr -tyrC- LAAD The chassis strain Escherichia coli BL21(DE3) was introduced to obtain 19 engineered strains as shown in Table 1. Table 1 Genetically engineered bacteria containing different phenylpyruvate decarboxylase mutants

[0025] Example 3: Tyrosol fermentation experiment of genetically engineered bacteria containing different phenylpyruvate decarboxylase mutants These 19 genetically engineered bacteria were inoculated into LB broth containing antibiotic resistance. The culture system consisted of 10 mL of medium dispensed into 100 mL Erlenmeyer flasks, with 50 μg / mL kanamycin 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 volume of 2%. The culture system consisted of 50 mL of medium dispensed into 250 mL Erlenmeyer flasks, with 50 μg / mL kanamycin added. The culture conditions were constant temperature shaking incubator at 37°C and 220 rpm. 600 When the concentration reaches 0.6-0.8, add 0.1 mM IPTG to induce fermentation. Fermentate at 30℃ and 220 rpm for 72 h to prepare samples.

[0026] Example 4: Extraction and liquid chromatography of tyrosol Transfer 1 mL of fermentation broth to a 1.5 mL centrifuge tube and centrifuge at 12000 rpm for 5 min. Mix 200 μL of the supernatant with 800 μL of mobile phase A. Dilute the sample 5-fold and analyze the tyrosol yield using a Shimadzu high-performance liquid chromatograph. Quantification was performed using a reversed-phase C18 column. Mobile phase A was water containing 1% trifluoroacetic acid (TFA); mobile phase B was acetonitrile containing 1% TFA. The flow rate was 1 mL / min. -1 The absorbance of the sample was detected at 274 nm. The HPLC program was as follows: 10% B: 0-5 min, 60% B: 5-10 min, 90% B: 10-12 min, 2% B: 12-16 min, 0% B: 16-20 min. Using the unmutated phenylpyruvate decarboxylase strain Control as a control (with a shake-flask fermentation yield as high as 2.02 g / L), the degree of increase in tyrosol yield during shake-flask fermentation of 18 phenylpyruvate decarboxylase mutants with different site-directed mutations was statistically analyzed. The results are shown in Table 2. Among the 18 genetically engineered strains, the phenylpyruvate decarboxylase ARO10 showed the highest yield. L220A As the dominant mutant, after 72 h of shake-flask fermentation, the tyrosol yield reached 3.04 g / L, which is 50% higher than that of the control strain. This study provides a valuable component for improving the yield of de novo tyrosol synthesis.

[0027] Table 2. Comparison of tyrosol production by phenylpyruvate decarboxylase mutant strain and control strain

[0028] Note: - indicates no improvement. The formula for the increase in tyrosol production is as follows: Increase (%) = ((Tyrosol production of mutant strain (g / L) - Tyrosol production of control strain (g / L)) / Tyrosol production of control strain (g / L) × 100%.

[0029] HPLC chromatogram as follows Figure 1 As shown, the fermented sample contains tyrosol ( Figure 1 A) Retention time and tyrosol standard ( Figure 1 The retention time is consistent with B) in the above.

[0030] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: The present invention provides a solution for yeast derived from brewing yeast (… Saccharomyces cerevisiae Site-directed mutagenesis was performed on the phenylpyruvate decarboxylase gene ARO10 to create the phenylpyruvate decarboxylase mutant ARO10. L220A Overexpression in genetically engineered bacteria significantly increased tyrosol yield, with shake-flask fermentation levels reaching up to 3.04 g / L, which holds promise for industrial production of tyrosol.

[0031] 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 phenylpyruvate decarboxylase mutant, characterized in that, The phenylpyruvate decarboxylase mutant is any one of the following: (1) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace K at position 92 with R; (2) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace the I at position 93 with V; (3) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 106 with A; (4) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace S at position 153 with T; (5) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace L at position 178 with F; (6) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace L at position 220 with A; (7) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace Y at position 234 with F; (8) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace D at position 242 with E; (9) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace F at position 273 with L; (10) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace N at position 285 with Q; (11) Using the amino acid shown in SEQ ID NO.2 as the starting sequence, replace T at position 581 with S.

2. The gene encoding the phenylpyruvate decarboxylase mutant of claim 1.

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

4. The recombinant vector according to claim 3, characterized in that, The launch carrier is the pRSFDuet series.

5. A recombinant microbial cell containing the gene of claim 2.

6. A recombinant Escherichia coli, characterized in that, The phenylpyruvate decarboxylase mutant of claim 1 is expressed.

7. The recombinant Escherichia coli according to claim 6, characterized in that, alcohol dehydrogenase gene ADH6 Gene containing a mutant 3-deoxy-D-arabinohepeptulose-7-phosphate (DAHP) synthase aroG fbr Cyclohexadiene dehydrogenase gene tyrC L-amino acid ammonia-lyase gene LAAD The overexpression plasmid was transferred into Escherichia coli BL21(DE3) competent cells.

8. The use of the phenylpyruvate decarboxylase mutant of claim 1, the gene of claim 2, the recombinant vector of claim 3 or 4, or the recombinant microbial cell of any one of claims 5-7 in the production of tyrosol and products containing tyrosol.

9. A method for producing tyrosol, characterized in that, The method involves fermenting the recombinant Escherichia coli as described in claim 6 or 7 at 30°C for 72 hours.

10. The method according to claim 9, characterized in that, The fermentation medium consisted of 25 g / L glucose, 10 g / L glycerol, 7.5 g / L (NH4)2SO4, 3 g / L K2HPO4·3H2O, 2 g / L KH2PO4, 2.0 g / L MgSO4·7H2O, 1.0 g / L citric acid monohydrate, 0.1 g / L vitamin B1, and 7 g / L yeast extract. The pH of the fermentation medium was maintained using 10 g / L CaCO3. The culture medium was supplemented with 0.1% by volume of trace elements, the composition of which was: 2.0 g / L Al2(SO4)3·18H2O, 0.75 g / L CoSO4·7H2O, 2.5 g / L CuSO4·5H2O, 0.5 g / L H3BO3, 24 g / L MnSO4·H2O, 2.5 g / L NiSO4·6H2O and 15 g / L ZnSO4·7H2O. The pH was adjusted to 7.0 using NaOH.

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