4-hydroxybenzoic acid hydroxylase mutants and their use in the synthesis of gallic acid or salts thereof

Abstract

The application belongs to the field of enzyme engineering and biotechnology, and discloses a 4-hydroxybenzoic acid hydroxylase mutant and application thereof in synthesis of gallic acid or a salt thereof. The 4-hydroxybenzoic acid hydroxylase mutant is obtained through directional evolution modification and high-throughput screening, and has an increased protocatechuate hydroxylase activity by more than 4 times. The 4-hydroxybenzoic acid hydroxylase mutant is expressed by using E. coli, and the obtained recombinant E. coli can efficiently catalyze the conversion of protocatechuate into gallic acid. The 4-hydroxybenzoic acid hydroxylase mutant is expressed in the recombinant E. coli for producing protocatechuate, and the obtained recombinant E. coli can de novo synthesize gallic acid from glucose as a substrate, and the yield reaches 58.65 g / L, and the residual amount of protocatechuate is only 0.33 g / L. The application solves the problem of a large amount of residual protocatechuate in the fermentation process of the existing strains, and can provide an efficient enzyme element for the creation of gallic acid industrial strains.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of enzyme engineering and biotechnology, and relates to 4-hydroxybenzoic acid hydroxylase mutants and their application in synthesizing gallic acid or a salt thereof, in particular to mutants with protocatechuate hydroxylase activity obtained by molecular modification of 4-hydroxybenzoic acid hydroxylase, recombinant Escherichia coli, and application of the mutants or recombinant Escherichia coli in synthesizing gallic acid or a salt thereof. BACKGROUND

[0002] Gallic acid (GA) is also known as 3,4,5-trihydroxybenzoic acid, a natural polyphenolic compound with a molecular formula of C7H6O5 and a relative molecular mass of 170.12, containing three ortho-positioned phenolic hydroxyl groups. Gallic acid is weakly acidic and unstable under strong oxidation, high temperature, alkaline and neutral conditions, relatively stable under strong acid conditions, slightly soluble in cold water at room temperature (25℃), easily soluble in boiling water, ethanol and diethyl ether, and turns brown in air due to combination with oxygen. Gallic acid can form a blue-black precipitate with aqueous ferric chloride solution. Gallic acid and its derivatives are distributed in plants such as apples, grapes, aloe and tea leaves. Gallic acid and its derivatives have various biological activities such as antibacterial, anti-inflammatory, antitumor and antioxidant activities, and can be applied in the fields of food, medicine, chemical industry and cosmetics.

[0003] The traditional production method of gallic acid is mainly plant extraction. Although this method dominates industrial production, it has problems such as high energy consumption, high pollution, low yield and harm to the environment. Biological synthesis technology is green, efficient and sustainable, which can not only reduce environmental pollution, but also reduce production cost and improve product quality. Escherichia coli is a typical engineering strain and has been reported to be used for producing gallic acid.

[0004] However, Escherichia coli lacks a natural pathway for efficiently synthesizing gallic acid. It has been reported that 4-hydroxybenzoic acid hydroxylase derived from Pseudomonas aeruginosa or Pseudomonas fluorescens has the ability to catalyze the synthesis of gallic acid from protocatechuic acid after the tyrosine at position 385 is mutated to phenylalanine, but the catalytic activity is low. In recent years, many studies have focused on the screening of high-efficiency 4-hydroxybenzoic acid hydroxylase mutants. However, the enzyme activity of 4-hydroxybenzoic acid hydroxylase still cannot meet the needs of industrialized high-yield gallic acid production. In summary, in order to remove the key limiting step of gallic acid biosynthesis, there is an urgent need for a 4-hydroxybenzoic acid hydroxylase mutant with higher catalytic activity for protocatechuic acid. Therefore, obtaining a 4-hydroxybenzoic acid hydroxylase mutant with high catalytic activity and stability is of great significance for promoting the industrialized production of gallic acid biosynthesis. SUMMARY

[0005] Therefore, the present application provides a 4-hydroxybenzoic acid hydroxylase mutant and its application to solve the problem of low activity of 4-hydroxybenzoic acid hydroxylase in the prior art.

[0006] The present application provides a 4-hydroxybenzoic acid hydroxylase (PobA) mutant, wherein the parent amino acid sequence of the 4-hydroxybenzoic acid hydroxylase is shown in SEQ ID NO: 1.

[0007] The 4-hydroxybenzoic acid hydroxylase in the present application is derived from Pseudomonas aeruginosa (PobA) (GenBank: AYF 12673.1). Pseudomonas aeruginosa ).

[0008] In an embodiment, the high-throughput screening of the PobA mutant is based on color screening and gallic acid content. The present application first observes the fermentation broth of the mutant strain, retains the mutant strain with dark gray fermentation broth, and then quantifies the production of gallic acid and protocatechuic acid using high performance liquid chromatography, taking the gallic acid content of the control strain as 1 to calculate the relative yield of gallic acid of the mutant strain.

[0009] The present application provides a 4-hydroxybenzoic acid hydroxylase mutant, wherein the amino acid sequence of the 4-hydroxybenzoic acid hydroxylase mutant is based on the amino acid sequence shown in SEQ ID NO: 1 and one or more of the following amino acid mutations occur: D37E, M65T, T356A, A379T, A10V, Q19L, M65T, T241E, A125C, I336V; preferably, only the following mutations D37E / T356A / A379T, D37E / T356A / A379T / Q19L / M65T exist.

[0010] The numbering of the above-mentioned mutation sites 37, 65, etc. in the present application is numbered according to the parent amino acid sequence of 4-hydroxybenzoic acid hydroxylase (SEQ ID NO: 1), for example, D37E represents that the aspartic acid at position 37 is mutated to glutamic acid.

[0011] Preferably, the present application uses the COF04 strain (patent application number 202410963207.2) with excess NADPH as the chassis strain for screening the 4-hydroxybenzoic acid hydroxylase mutant.

[0012] In an embodiment, the catalytic efficiency of the 4-hydroxybenzoic acid hydroxylase mutant is measured by whole cell reaction with protocatechuic acid as the substrate, and then detecting the product peak area by high performance liquid chromatography.

[0013] The present application also provides a nucleic acid encoding the 4-hydroxybenzoic acid hydroxylase mutant.

[0014] Also provided are recombinant vectors containing the coding nucleic acid, and in an embodiment of the present application, the recombinant vector is a pET-30a plasmid or a pET-21a plasmid as an expression vector.

[0015] The present application further provides a recombinant genetically engineered bacterium containing the recombinant Escherichia coli containing the coding nucleic acid. Preferably, the chassis cell expressing the 4-hydroxybenzoic acid hydroxylase is a protocatechuate-producing strain (Patent Application No. 202410963207.2).

[0016] Preferably, the coding nucleic acid is integrated into the genome.

[0017] The present application provides the use of the 4-hydroxybenzoic acid hydroxylase mutant, the coding nucleic acid, or the recombinant genetically engineered bacterium in the synthesis of gallic acid or a salt thereof.

[0018] The present application also provides a method for synthesizing gallic acid or a salt thereof, which is synthesized by using the recombinant Escherichia coli expressing the coding nucleic acid through whole-cell catalysis with protocatechuic acid as a substrate, or by using the recombinant Escherichia coli expressing the coding nucleic acid through aerobic fermentation with glucose as a substrate.

[0019] In an embodiment of the present application, the whole-cell catalysis system of the 4-hydroxybenzoic acid hydroxylase mutant comprises, but is not limited to, 500 μL of M9 medium, 1-20 g / L of protocatechuic acid, a reaction temperature of 25-37 °C, and a reaction time of 36-60 h. After fermentation, the content of gallic acid and the residual amount of protocatechuic acid are detected.

[0020] In an embodiment, the M9 medium comprises 5-20 g / L of Na2PO4 x 12H2O, specifically 5 g / L, 10 g / L, 15 g / L, or 20 g / L; 1-5 g / L of KH2PO4, specifically 1 g / L, 2 g / L, 3 g / L, or 5 g / L; 0.1-0.6 g / L of NaCl, specifically 0.1 g / L, 0.3 g / L, 0.5 g / L, or 0.6 g / L; 0.2-1 g / L of NH4Cl, specifically 0.2 g / L, 0.5 g / L, or 1 g / L; 0.1-0.5 g / L of MgSO4 x 7H2O, specifically 0.1 g / L, 0.2 g / L, 0.4 g / L, or 0.5 g / L; 0.01-0.02 g / L of CaCl2 x 2H2O, specifically 0.01 g / L, 0.015 g / L, or 0.02 g / L; and 2-10 g / L of glucose, specifically 2 g / L, 4 g / L, 6 g / L, 8 g / L, or 10 g / L.

[0021] The components and their contents, fermentation temperature, and fermentation time of the M9 culture medium can all be adjusted as needed.

[0022] In one embodiment of the present invention, the aerobic fermentation culture method used by the recombinant Escherichia coli to biosynthesize gallic acid using glucose as a substrate is shown below.

[0023] The components, their contents, inoculum size, fermentation temperature, and fermentation time in the seed culture medium and fermentation medium mentioned in this invention can all be adjusted as needed, for example:

[0024] Seed culture medium: yeast extract content is 1-5 g / L, specifically 1 g / L, 2 g / L, 3 g / L, 4 g / L, or 5 g / L, etc.; tryptone content is 2-10 g / L, specifically 2 g / L, 4 g / L, 6 g / L, 8 g / L, or 10 g / L, etc.; NaCl content is 2-10 g / L, specifically 2 g / L, 4 g / L, 6 g / L, 8 g / L, or 10 g / L, etc.

[0025] Fermentation medium: K₂HPO₄ content is 2.5-12.5 g / L, specifically 2.5 g / L, 5.0 g / L, 7.5 g / L, 10 g / L, or 12.5 g / L, etc.; MgSO₄ content is 1-5 g / L, specifically 1 g / L, 2 g / L, 3 g / L, 4 g / L, or 5 g / L, etc.; (NH₄)₂SO₄ content is 0.8-4.0 g / L, specifically 0.8 g / L, 1.6 g / L, 2.4 g / L, 3.2 g / L, or 4.0 g / L, etc.; FeSO₄×7H₂O content is 0.025-0.125 g / L, specifically 0.025 g / L, 0.050 g / L, 0.075 g / L, 0.100 g / L, or 0.125 g / L, etc.; citric acid monohydrate content is 1-5 g / L. The concentration of sodium hydroxide (Na2SO4) can be 1 g / L, 2 g / L, 3 g / L, 4 g / L, or 5 g / L, etc.; the concentration of sodium hydroxide (ZnSO4) can be 0-50 g / L, specifically 0 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, or 50 g / L; the concentration of sodium hydroxide (ZnSO4) can be 1.5-7.5 mg / L, specifically 1.5 mg / L, 3.0 mg / L, 4.5 mg / L, 6.0 mg / L, or 7.5 mg / L; the concentration of sodium hydroxide (Na2SO4) can be 10-50 mg / L, specifically 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, or 50 mg / L; the concentration of sodium hydroxide (ZnSO4) can be 0.4-6.4 mg / L, specifically 0.4 mg / L, 1.6 mg / L, 3.2 mg / L, or 4.8 mg / L. mg / L or 6.4 mg / L; CoCl2×6H2O content is 1-5 mg / L, specifically 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, or 5 mg / L; CuSO4×5H2O content is 0.2-1.0 mg / L, specifically 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, or 1.0 mg / L.

[0026] In the aforementioned aerobic fermentation culture method, the initial concentration of the substrate glucose is any concentration within the range of 10-60 g / L, specifically 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, or 60 g / L, etc.

[0027] The aerobic fermentation culture method uses a glucose solution as the feeding medium, with a specific concentration of 100 g / L, 200 g / L, 300 g / L, 400 g / L, 500 g / L, 600 g / L, or 700 g / L, etc.

[0028] The aerobic fermentation culture method wherein the fermentation pH is any pH value within the range of 5.0-8.0, specifically 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0, etc.

[0029] The aerobic fermentation culture method wherein the fermentation temperature is any temperature within the range of 25℃-42℃, specifically 25℃, 30℃, 37℃, 40℃, or 42℃, etc.

[0030] The aerobic fermentation culture method has a fermentation cycle of any time within the range of 48h-84h, specifically 48h, 60h, 72h, or 84h, etc.

[0031] The aerobic fermentation culture method can be carried out by shake flask fermentation or by feeding and batch fermentation using fermenters or bioreactors of different sizes or models.

[0032] This invention utilizes error-prone PCR technology to perform directed evolution and high-throughput screening on the amino acid sequence shown in SEQ ID NO:1. The resulting 4-hydroxybenzoic acid hydroxylase mutant exhibits higher catalytic efficiency in practical applications, leading to higher gallic acid yields. Furthermore, by introducing the 4-hydroxybenzoic acid hydroxylase mutant into a protocatechuic acid-producing strain, a highly efficient recombinant *E. coli* strain for gallic acid production was constructed. This invention provides a highly efficient enzyme and engineered strain for gallic acid biosynthesis, which is of significant importance for gallic acid production. Attached Figure Description

[0033] Figure 1 The effect of different 4-hydroxybenzoic acid hydroxylase mutants on gallic acid synthesis. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0035] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0036] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0037] In this application, abbreviations for enzymes, such as PobA, represent 4-hydroxybenzoic acid hydroxylase.

[0038] This invention utilizes high-throughput screening to obtain key amino acid sites in mutant proteins that significantly enhance the catalytic production of gallic acid by catalyzing the synthesis of gallic acid. This invention reveals that mutations at these key amino acid sites can significantly alter their activity, improve conversion rates, and exhibit excellent catalytic performance.

[0039] Among them, the PobA amino acid sequence (derived from) Pseudomonas aeruginosa (SEQ ID NO:1):

[0040] MKTQVAIIGAGPSGLLLGQLLHKAGIDNVILERQTPDYVLGRIRAGVLEQGMVDLLREAGVDRRMARDGLVHEGVEIAFAGQRRRIDLKRLSGGKTVYGQTEVTRDLMEAREACGATTVYQAAEVRLHDLQGERPYVTFERDGERLRLDCDYIAAGCDGFHGISRQSIPAERLKVFERVYPFGWLGLLADTPPVSH ELIYANHPRGFALCSQRSATRSRYYVQVPLSEKVEDWSDERFWTELKARLPSEVAEKLVTGPSLEKSIAPLRSFVVEPMQHGRLFLAGDAAHIVPPAGAKGLNLAASDVSTLYRLLLKAYREGRGELLERYSAICLRRIWKAERFSWWMTSVLHRFPDTDAFSQRIQQTELEYYLGSEAGLATIAENFVGLPYEEIE.

[0041] P2 nucleotide sequence (SEQ ID NO:2): TTATCTCTGGCGGTGTTGACAAGAGATAACAACGTTGATATAATTGAGCCCGTATTGTTAGCATGTACGTTTAAACCAGGAAACAGCT.

[0042] P4 nucleotide sequence (SEQ ID NO: 3): TTATCTCTGGCGGTGTTGACAAGAGATAACAACGTTGATATAATTGAGCCTGAGGTGGCTTATTATTCGTTTAAACCAGGAAACAGCT.

[0043] The method for constructing a recombinant *Escherichia coli* strain that produces gallic acid provided by this invention integrates and optimizes the endogenous shikimic acid pathway and the exogenous gallic acid synthesis pathway through metabolic engineering, achieving efficient de novo synthesis of gallic acid using glucose as a carbon source. The catalytic enzyme in the exogenous gallic acid synthesis pathway is 4-hydroxybenzoic acid hydroxylase.

[0044] The conventional experimental methods used in this invention are as follows:

[0045] 1. Preparation of competent cells by chemical transformation of Escherichia coli

[0046] A single colony of *Escherichia coli* DH5α (Beijing Qingke Biotechnology Co., Ltd.) was picked and inoculated into 3 mL of liquid LB medium and cultured at 37℃ and 250 rpm for 8–12 h to obtain a seed culture. 100 μL of the seed culture was then inoculated into 50 mL of liquid LB medium and cultured at 37℃ and 250 rpm until OD (Organic Depth) reached. 600 Approximately 0.3-0.4; incubate on ice for 15 min, then transfer the bacterial culture to a pre-chilled 50 mL centrifuge tube; centrifuge at 2000 g for 5 min, discard the supernatant, and resuspend the bacterial cells in 15 mL of pre-chilled 100 mM CaCl2 solution. Repeat this step once; centrifuge at 2000 g for 5 min, discard the supernatant, and resuspend the bacterial cells in 2 mL of pre-chilled 10% (v / v) glycerol-100 mM CaCl2 solution to obtain chemically transformed competent cells.

[0047] The LB medium contains 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl.

[0048] 2. PCR amplification reaction system and conditions

[0049] (1) When amplifying the pTargetF fragment, the high-fidelity DNA polymerase PrimeSTAR HSDNA Polymerase from TaKaRa was used. The universal amplification system was: 5× PrimeSTAR Buffer (Mg... 2+ The amplification mixture consisted of 10 μl of the following: 10 μl of the plus primer, 4 μl of dNTP Mixture (2.5 mM each), 1 μl of the upstream primer (10 μM), 1 μl of the downstream primer (10 μM), 1 μl of the template, 0.5 μl of PrimeSTAR HS DNA Polymerase (2.5 U / μl), and 32.5 μl of sterile water, for a total volume of 50 μl. The general amplification program was as follows: ① 95℃ for 3 min; ② 98℃ for 10 sec, 55℃ for 15 sec, 72℃ for 1 kb / min, repeating step ② 30 times; ③ 72℃ for 5 min.

[0050] (2) When preparing homologous recombination fragments by ligating two homologous arms, promoters, and target genes using fusion PCR, the high-fidelity DNA polymerase PrimeSTAR HS DNA Polymerase from TaKaRa was used for PCR amplification. The general preparation steps are as follows: First, amplify the two homologous arms, promoters, and target gene fragments separately using their respective specific primers. Second, using the left homologous arm and promoter as templates, amplify using the F-terminal primer of the left homologous arm and the R-terminal primer of the promoter, and ligate the two fragments to form the left homologous arm-promoter fragment; using the target gene and right homologous arm as templates, amplify using the F-terminal primer of the target gene and the R-terminal primer of the right homologous arm, and ligate the two fragments to form the target gene-right homologous arm fragment. Third, using the left homologous arm-promoter fragment and the target gene-right homologous arm fragment as templates, amplify using the F-terminal primer of the left homologous arm and the R-terminal primer of the right homologous arm, and ligate the two fragments to form the full-length fragment. Its universal amplification system is: 5×PrimeSTAR Buffer (Mg 2+ The amplification program consists of: 10 μl of (Plus) dNTP Mixture (2.5 mM each), 4 μl of F-terminal primer (10 μM), 1 μl of R-terminal primer (10 μM), 1 μl of template 1, 1 μl of template 2, 0.5 μl of PrimeSTAR HS DNA Polymerase (2.5 U / μl), and 31.5 μl of sterile water, for a total volume of 50 μl. The general amplification program is as follows: ① 95℃ for 3 min; ② 98℃ for 10 sec, 55℃ for 15 sec, 72℃ for 1 kb / min, repeat step ② 30 times; ③ 72℃ for 5 min.

[0051] (3) When performing colony PCR verification, 2×Es TaqMasterMix (Dye) from Kangwei Century Biotechnology Co., Ltd. was used for amplification. The universal amplification system is as follows: 12.5 μl of 2×Es Taq MasterMix (Dye), 1 μl of upstream primer (10 μM), 1 μl of downstream primer (10 μM), 0.5 μl of template, 10 μl of sterile water, and a total volume of 25 μl. The universal amplification program is as follows: ① 94℃ for 3 min; ② 94℃ for 30 sec, (Tm-5)℃ for 30 sec, 72℃ for 2 kb / min, repeat step ② 30 times; ③ 72℃ for 5 min.

[0052] 3. Construction of N20-sgRNA expression vector

[0053] (1) Enzyme digestion: The pTargetF fragment was amplified using PrimeSTAR HS DNA Polymerase, a high-fidelity DNA polymerase from TaKaRa, under the conditions shown in Experimental Method 2; restriction endonucleases from New England Biolabs (NEB) were used. Spe I. Single digestion of the pTargetF fragment; digestion reaction system and conditions: 3 μL of 10×CutSmartBuffer, 500-1000 ng of pTargetF fragment. Spe Add 0.5 μL of I (20 U / μL) to a final volume of sterile water to a final volume of 30 μL, and digest at 37°C for 1 h.

[0054] (2) Enzyme ligation: Using Thermo Scientific's T4 DNA ligase to ligate DNA... Spe The pTargetF fragment digested by enzyme I was circularized and self-ligated; the enzyme ligation reaction system and conditions were as follows: 10× T4 DNA Ligase Buffer 2 μL, pTargetF fragment 20-100 ng, T4 DNA Ligase (5 U / μL) 0.5 μL, and sterile water was added to bring the total volume to 20 μL. The enzyme ligation was carried out at 22℃ for 0.5 h.

[0055] (3) Transformation: Add 5-10 μL of the enzyme ligation product to 50 μL of Escherichia coli DH5α competent cells, mix gently, and incubate on ice for 30 min; heat shock at 42℃ for 45 s, incubate on ice for 2 min, add 500 μL of LB liquid medium, and incubate at 37℃ and 250 rpm for 40-60 min; centrifuge at 8000 rpm for 2 min, remove most of the supernatant, spread the remaining bacterial culture on solid LB medium containing 50 μg / mL spectinomycin, and incubate at 37℃ overnight until single colonies grow.

[0056] (4) Verification: Select 2-5 single clones and use 2×Es TaqMasterMix (Dye) of Kangwei Century Biotechnology Co., Ltd. to perform colony PCR verification. The PCR amplification conditions are as shown in Experimental Method 2. After the PCR products are detected by 1% agarose gel electrophoresis, the samples with the correct band size are sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The clones with the correct sequencing are selected and the pTargetF plasmid is extracted using the high purity plasmid small-volume rapid extraction kit of Beijing Bomed Gene Technology Co., Ltd.

[0057] 4. Preparation and transformation of competent cells by electroporation transformation of Escherichia coli

[0058] (1) Preparation of competent cells: A single colony of Escherichia coli carrying the pCas plasmid was picked and inoculated into 3 mL of liquid LB medium containing 50 μg / mL kanamycin, and cultured at 30℃ and 250 rpm for 12-16 h as seed culture; 200 μL of seed culture was inoculated into 30 mL of liquid LB medium containing 3% L-arabinose (w / v) and 50 μg / mL kanamycin, and cultured at 30℃ and 250 rpm until OD. 600 Approximately 0.5-0.7; incubate on ice for 15 min, transfer the bacterial culture to a pre-chilled 50 mL centrifuge tube, centrifuge at 2000 g for 5 min, and discard the supernatant; resuspend the bacterial pellet in 10 mL of pre-chilled 10% glycerol (v / v), centrifuge at 5000 g for 5 min, discard the supernatant, and repeat this step twice; resuspend the bacterial pellet in 300 μL of pre-chilled 10% glycerol (v / v) to obtain electrotransformation competent cells.

[0059] (2) Electroporation transformation: Mix 100 ng pTargetF plasmid and 400 ng homologous recombination fragment with 50 μL competent cells and add to a 2 mm electroporation cuvette. Incubate on ice for 5 min. After drying the cuvette, place it in an electroporator for transformation at 2.5 kV. Immediately add 1 mL of pre-cooled liquid LB medium to the cuvette and incubate on ice for 5 min. Transfer the bacterial culture to a 2 mL sterile centrifuge tube and incubate at 30℃ and 250 rpm for 2 h. Centrifuge at 8000 rpm for 1 min, discard most of the supernatant, and spread the remaining bacterial culture on solid LB medium containing 50 μg / mL kanamycin and 50 μg / mL spectinomycin. Incubate at 30℃ until colonies grow, then perform colony PCR verification and sequencing.

[0060] 5. Sample detection and analysis

[0061] (1) When detecting the concentrations of protocatechuic acid, gallic acid, and glucose in the fermentation broth, the fermentation broth sample was diluted with distilled water to an appropriate ratio, centrifuged at 12,000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm aqueous microporous membrane. The analysis was performed using an Agilent 1200 high-performance liquid chromatograph equipped with a VWD ultraviolet detector and a RID differential refractive index detector. The chromatographic column was a Phenomenex Rezex RFQ-Fast Acid H+ (8%) (LC Column 100 × 7.8 mm). The chromatographic conditions were: mobile phase 5 mM H2SO4 aqueous solution, sample loading 5 μL, flow rate 0.6 mL / min, column temperature 55℃, and detection wavelength 210 nm. Gallic acid, protocatechuic acid, and glucose standards were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0062] (2) When detecting the cell concentration (OD) in the fermentation broth600 When diluting the fermentation broth sample with distilled water to an appropriate ratio, the absorbance value of the sample at a wavelength of 600 nm is measured using a 723 visible spectrophotometer from Shanghai Spectrum Instruments Co., Ltd., which is the cell concentration OD. 600 .

[0063] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be described in detail below with reference to embodiments. It should be noted that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.

[0064] Example 1: Construction of a random mutant library of 4-hydroxybenzoic acid hydroxylase

[0065] Using the coding gene of PobA derived from Pseudomonas aeruginosa as a template, we randomly mutated it using error-prone PCR.

[0066] Forward primer: GTTTAAACCAGGAAACAGCTATGAAGACCCAGGTGGCAAT

[0067] Reverse primer: TGCCGTATGAAGAAATTGAATAACATATGGAATCCTAGATCTT

[0068] The components, content, and amplification conditions of the PCR system and PCR amplification program mentioned in this invention can be adjusted as needed.

[0069] The error-prone PCR reaction system is 50 μL, which includes 1 μL template, 2.5 μL 2.5 mM MgCl2, 4 μL 1 mM MnCl2, 2 μL each of 10 μM forward and reverse primers, 4 μL dNTPs, 0.5 μL TaKaRa Taq enzyme, 5 μL 10×PCR buffer, and ddH2O to make up to 50 μL.

[0070] PCR amplification program: 94℃ for 3 min pre-denaturation; (94℃ for 30 s, 56℃ for 30 s, 72℃ for 90 s) × 35 cycles; extension at 72℃ for 5 min.

[0071] After purifying 50 μL of the error-prone PCR product using a nucleic acid gel, the target mutant gene fragment was obtained. This fragment was then ligated into the vector pACYC177 containing the constitutive promoter P2 via homologous recombination. Finally, the ligation product was transformed into *E. coli* COF04 using the chemical transformation method described in Experimental Method 1, plated on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 37°C until single colonies appeared on the plates, yielding an *E. coli* mutant library containing the pACYC177-P2-PobA plasmid.

[0072] Example 2: High-throughput screening of highly active 4-hydroxybenzoic acid hydroxylase mutants

[0073] 1. Initial screening: Single colonies of mutant libraries were selected and incubated in 96-well plates containing 500 μL of LB liquid medium with 50 μg / mL kanamycin at 37°C and 800 rpm for 12 h. Subsequently, 1% (v / v) inoculum was inoculated into 96-well plates containing 500 μL of M9 medium containing 50 μg / mL kanamycin, 1 g / L protocatechuic acid, and 4 g / L glucose, and incubated at 37°C and 800 rpm for 48 h.

[0074] The contents of each component in the M9 culture medium are as follows: Na2PO4×12H2O 15g / L, KH2PO4 3g / L, NaCl 0.5g / L, NH4Cl 1 g / L, MgSO4×7H2O 0.5g / L, CaCl2×2H2O 0.015g / L.

[0075] 2. Secondary Screening: After the 96-well plate fermentation system completed its cultivation, based on the growth of the mutant strains and the color of the fermentation broth, the fermentation broths of mutants with darker colors were selected for high-performance liquid chromatography (HPLC) to detect the content of gallic acid and protocatechuic acid. The gallic acid yield of the control strain was set as 1, and the relative gallic acid yield was used as the screening index. From the 10,000 mutant strains selected in the initial screening, mutants with relatively high gallic acid yields were identified as D37E, D37N, H72L, T356A, and A376T.

[0076] 3. Screening of saturated mutant libraries: In order to determine the best mutants for the corresponding amino acid sites, this invention designs saturated mutant primers for each amino acid mutation site obtained in step 2, constructs a saturated mutant library for the amino acid site, and screens out the best mutant amino acids.

[0077] Taking the construction of a 37-site saturated mutant library as an example, the specific method is as follows: pobA Using the gene fragment as a template, 37-site NNK-based degenerate primers were designed to construct a saturated mutant fragment, which was then ligated into the vector pACYC177 containing the constitutive promoter P2. The construction and screening methods are as described in Example 2. The saturated mutant primers used are shown in Table 1.

[0078] After screening the saturated mutant libraries at four amino acid sites, the optimal amino acids for each mutant site were determined to be D37E, H72I, T356A, and A376T.

[0079] 4. Combination Mutation: In order to obtain mutants with enhanced enzyme activity, this invention integrates four optimal PobA mutants and their combination mutations into the genome of protocatechuic acid producing bacteria, and tests the biosynthesis of gallic acid by different mutants using glucose as a substrate. This invention constructed a total of 14 bacterial strains. Strains containing four single-point mutants were named W1, W2, W3, and W4, respectively. Ten strains were constructed using combined mutants, with the following amino acid mutation combinations: D37E+H72I, D37E+T356A, D37E+A376T, H72I+T356A, H72I+A376T, T356A+A376T, D37E+H72I+T356A, D37E+H72I+A376T, D37E+T356A+A376T, and H72I+T356A+A376T. These strains were named W5, W6, W7, W8, W9, W10, W11, W12, W13, and W14, respectively. The gallic acid content of the optimal single-point mutants and the optimal combined mutants is shown in Table 2. The present invention obtained the optimal single-site mutant as D37E, the optimal two-site combination mutant as D37E+T356A, and the optimal three-site combination mutant as D37E+T356A+A376T (with a relative gallic acid yield of 3.28) through fermentation tests of single-site mutant and combined mutant strains. The amino acid sequence of the three-site combination mutant was named PobA-M1.

[0080] Taking strain W1 as an example, the specific steps are as follows:

[0081] (1) Construction ldhA N20-sgRNA expression plasmid of the gene

[0082] Using pTargetF-cadA plasmid as a template, the N20-sgRNA vector fragment pTargetF-ldhA was amplified using primers ldhA-N20-F / pTargetF-R and TaKaRa's high-fidelity DNA polymerase PrimeSTAR HS DNA Polymerase. The PCR amplification system and procedure are shown in Experimental Method 2, and the primers used are shown in Table 1.

[0083] Using restriction endonucleases from NEB Spe I For the vector fragment pTargetF- ldhA Enzyme digestion was performed, and the enzyme digestion reaction system and conditions are shown in Experimental Method 3.

[0084] The vector fragment pTargetF- was digested using T4 DNA ligase from Thermo Scientific. ldhA The cyclization self-ligation reaction system and conditions are shown in Experimental Method 3.

[0085] Escherichia coli DH5α competent cells were prepared according to Experimental Method 1. Then, 5 μL of enzyme ligation product was used to transform 50 μL of competent cells, and the transformation method was as shown in Experimental Method 3.

[0086] Three single clones were selected and colony PCR was performed using pTargetF-seq-F / pTargetF-seq-R primers via 2×Es Taq MasterMix (Dye) from Kangwei Century Biotechnology Co., Ltd. PCR amplification conditions were as shown in Experimental Method 2. After PCR product detection by 1% agarose gel electrophoresis, samples with correct band sizes were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Clones with correct sequencing were then extracted using a high-purity plasmid small-scale rapid extraction kit from Beijing Bomed Gene Technology Co., Ltd. ldhA The N20-sgRNA expression plasmid pTargetF- of the gene ldhA .

[0087] (2) Preparation of homologous recombination fragments at the ldhA site

[0088] Amplification was performed using PrimeSTAR HS DNA Polymerase, a high-fidelity DNA polymerase from TaKaRa. ldhA The homologous recombination fragments at specific sites, the PCR amplification system and procedure are as shown in Experimental Method 2, and the preparation steps for the homologous recombination fragments are as follows:

[0089] The first step involved using E. coli bacterial culture as a template and amplifying the culture using ldhA-1F / ldhA-1R primers. ldhA Left homologous arm fragment of gene locus ldhA- 1; Amplification was obtained using ldhA-2F / ldhA-2R primers. ldhA Right homologous arm fragment of gene locus ldhA- 2; Using DNA containing the artificially synthesized regulatory element P4 (as shown in SEQ ID NO:3) as a template, the promoter P4 was amplified using PF / PR primers; pobA D37E Using the pAYCYC177 recombinant plasmid as a template, the 4-hydroxybenzoic acid hydroxylase mutant gene fragment was amplified using the pobA-F1 / pobA-R1 primers. pobA D37E .

[0090] The second step is to ldhA- Using 1 and P4 as templates, the ldhA-1-P4 fragment was amplified using ldhA-1F / PR primers; pobA D37E and ldhA- 2 is the template, using pobA-Amplification with F1 / ldhA-2R primers yielded... pobA D37E -ldhA-2 fragment.

[0091] The third step is to ldhA- 1-P4 and pobA D37E - ldhA- Using 2 as a template, amplification was performed using primers ldhA-1F / ldhA-2R to obtain... ldhA- P 4- pobA D37E Full-length clip.

[0092] (3) Construction pobA D37E Genome-integrating strain W1

[0093] Using the protocatechuic acid producing bacterium (patent application number 202410963207.2) as the starting strain, electrocompetent cells were prepared according to the method shown in Experimental Method 4.

[0094] 100ng pTargetF- ldhA plasmid and 400ng ldhA- P4- pobA D37E Homologous recombination fragments were transformed into 50 μL of competent cells using the electroporation transformation method shown in Experimental Method 4, and single colonies were obtained after overnight culture.

[0095] Five single clones were selected and colony PCR was performed using ldhA-1F / ldhA-2R primers via 2×Es Taq MasterMix (Dye) from Kangwei Century Biotechnology Co., Ltd. The PCR amplification conditions were as shown in Experimental Method 2. After the PCR products were detected by 1% agarose gel electrophoresis, samples with the correct band size were selected and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Finally, the 4-hydroxybenzoic acid hydroxylase expression strain, namely the genetically engineered strain W1, was obtained.

[0096] (4) Fermentation test of strain W1

[0097] The culture medium used for the fermentation test of recombinant Escherichia coli strain W1 was a de novo synthesis of gallic acid using glucose as a substrate.

[0098] The fermentation medium contains 40 g / L glucose, 10 g / L K2HPO4, 4 g / L MgSO4, 1.6 g / L (NH4)2SO4, 0.1 g / L FeSO4×7H2O, 4 g / L citric acid, 4.5 mg / L MnSO4×H2O, 20 mg / L Na2SO4, 6.4 mg / L ZnSO4×7H2O, 4 mg / L CoCl2×6H2O, and 0.6 mg / L CuSO4×5H2O.

[0099] The fermentation test of strain W1 was conducted in the same manner as the initial screening procedure for the mutant library, except for the different culture medium composition. The fermentation methods for strains W1-W14 were consistent.

[0100] 5. Second round of screening of the PobA mutant library: After obtaining the highly fermenting PobA-M1, in order to further improve the catalytic activity of PobA-M1 for the substrate protocatechuic acid, this invention conducted a second round of screening of the PobA mutant library. The selected libraries were used in the screening process. pob-M1 Random mutant libraries were constructed using the method described in Example 1 as templates for error-prone PCR, and the screening method was consistent with the first round of screening. The optimal mutant amino acids obtained from primary screening, secondary screening, and saturated mutant library screening of 10,000 mutant strains in this round were A10V, Q19L, M65T, A125C, T241E, and I336V. The six single-point mutants and combined mutant fragments were integrated into the genome of protocatechuic acid-producing bacteria, respectively. Among them, strains containing single-point mutants of A10V, Q19L, M65T, A125C, T241E, and I336V are named W15, W16, W17, W18, W19, and W20, respectively; strains containing combined mutants of A10V+Q19L, A10V+M65T, A10V+A125C, A10V+T241E, A10V+I336V, Q19L+M65T, Q19L+A125C, Q19L+T241E, Q19L+I336V, M65T+A125C, M65T+T241E, M65T+I336V, A125C+T241E, A125C+I336V, and T241E+I336V are named W21-W35, respectively. The culture conditions were the same as those for strain W1, etc. The relative gallic acid yields of the optimal single-point mutant and the optimal combination mutant are shown in Table 3. Among them, the combination mutant strain with the highest catalytic activity towards protocatechuic acid was W26 (Q19L+M65T), with a relative gallic acid content of 4.03. This mutant was named PobA-M2 based on its amino acid sequence. The screening results of the combination mutant strains are as follows: Figure 1 As shown, Figure 1Figure a in the figure represents the relative gallic acid content of the combined mutant strains in the second round of PobA mutant library screening results, specifically strain W26 (PobA). D37E / T356A / A376T / Q19L / M65T Gallic acid content was the highest, with a relative content of 4.03%. Figure 1 Figure b in the figure represents the relative content of protocatechuic acid in the combined mutant strains in the second round of PobA mutant library screening results. The W26 strain has the lowest relative content of protocatechuic acid, which is 0.72.

[0101] Table 1. Primers used to construct gallic acid-producing strains

[0102]

[0103] Table 2. Relative gallic acid yields in the first round of PobA mutant library screening.

[0104]

[0105] Table 3. Relative gallic acid yields in the second round of PobA mutant library screening.

[0106]

[0107] Example 3: Application of a highly active 4-hydroxybenzoic acid hydroxylase mutant in the synthesis of gallic acid

[0108] The following examples demonstrate the use of shake-flask fermentation and fed-batch fermentation to test the gallic acid synthesis capacity and protocatechuic acid accumulation level of strain W26.

[0109] (1) The shake-flask fermentation process is described below as an example.

[0110] Seed culture medium (LB): tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L

[0111] Shake-flask fermentation medium: glucose 40 g / L, K2HPO4 10 g / L, MgSO4 4 g / L, (NH4)2SO4 1.6 g / L, FeSO4×7H2O 0.1 g / L, citric acid 4 g / L, CaCO3 10 g / L, MnSO4×H2O 4.5 mg / L, Na2SO4 20 mg / L, ZnSO4×7H2O 6.4 mg / L, CoCl2×6H2O 4 mg / L, CuSO4×5H2O 0.6 mg / L.

[0112] The shake-flask fermentation steps are as follows:

[0113] Genetically engineered strain W26 was selected and inoculated into test tubes containing 3 mL of LB medium. The culture was carried out at 37°C and 250 rpm for 12 h to obtain the seed culture.

[0114] The seed culture in the test tube was transferred at an inoculation rate of 1% to a 100 mL Erlenmeyer flask containing 15 mL of shake-flask fermentation medium. The flask was incubated at 37 °C and 250 rpm. Samples were taken at the required time points during the fermentation process.

[0115] Take an appropriate amount of fermentation broth sample and determine the concentrations of protocatechuic acid and gallic acid in the fermentation broth according to the analytical method in Experiment 5.

[0116] Results: After 48 hours of shake-flask fermentation, the gallic acid content in the fermentation broth of the genetically engineered strain W26 was 3.92 g / L, and the protocatechuic acid residue was 0.07 g / L. In contrast, the control strain using the original PobA strain had a gallic acid content of only 0.20 g / L and a protocatechuic acid residue of 4.03 g / L. The gallic acid yield of strain W26 was 18.6 times higher than that of the control strain. The highly active 4-hydroxybenzoic acid hydroxylase mutant described in this example can both efficiently synthesize gallic acid and significantly reduce the residual level of protocatechuic acid.

[0117] (2) The following is an example of a fed-batch fermentation process:

[0118] Fed-batch fermentation medium: glucose 40 g / L, K2HPO4 10 g / L, MgSO4 4 g / L, (NH4)2SO4 1.6 g / L, FeSO4×7H2O 0.1 g / L, citric acid 4 g / L, MnSO4×H2O 4.5 mg / L, Na2SO4 20 mg / L, ZnSO4×7H2O 6.4 mg / L, CoCl2×6H2O 4 mg / L, CuSO4×5H2O 0.6 mg / L.

[0119] The feeding medium was a 600 g / L glucose solution.

[0120] The batch feeding fermentation steps are as follows:

[0121] Primary seed culture: Single colonies of recombinant Escherichia coli strain W26 were picked and inoculated into 5 mL LB medium and cultured at 37℃ and 250 rpm for 10 h to obtain the primary seed culture.

[0122] Secondary seed culture: The primary seed culture was transferred to 200 mL LB medium at an inoculation rate of 0.1% and cultured at 37℃ and 250 rpm for 10 h to obtain the secondary seed culture.

[0123] Fed-batch fermentation: The secondary seed culture was transferred at a 10% inoculum to a 5 L fermenter (Shanghai Baoxing Bio-Equipment Engineering Co., Ltd., BIOTECH-5BG fermenter) containing 1.8 L of fed-batch fermentation medium. The fermentation temperature was 37℃, and the aeration rate was 1 vvm. During fermentation, the agitator speed was gradually increased to maintain dissolved oxygen at 30% and the pH of the fermentation broth at 6.5. The initial glucose concentration was approximately 40 g / L. As fermentation progressed, when the glucose concentration in the fermentation broth decreased to approximately 1 g / L, feeding was initiated, and the feeding rate was controlled to ensure that the glucose concentration in the fermentation broth remained below 5 g / L. Samples were taken at appropriate time points during fermentation as needed.

[0124] Sample testing: Take an appropriate amount of fermentation broth sample and test the concentrations of gallic acid, protocatechuic acid, and glucose, as well as the cell concentration OD, in the fermentation broth according to the analytical method in Experimental Method 5. 600 .

[0125] Results: After 72 h of fermentation, the gallic acid yield of Escherichia coli strain W26 was 58.65 g / L. Throughout the fermentation process, protocatechuic acid residue remained at a low level, with a residual amount of only 0.33 g / L at the end of fermentation.

[0126] The recombinant Escherichia coli strain W26 provided in this embodiment is a novel microbial strain that can be used to produce gallic acid. This strain is plasmid-free, exhibits strong genetic stability, has a simple fermentation process, a short fermentation cycle, and achieves a gallic acid yield of up to 58.65 g / L with minimal residual precursor protocatechuic acid. The gallic acid yield is 13.72% higher than the highest reported yield of gallic acid synthesized by microbial fermentation (51.57 g / L) in the literature (Guo J, Ren X, Lu L, et al. Microbialsynthesis of gallic acid and its glucoside β-glucogallin. BiotechnolBioeng.2024, 121(11):3527-3536.). The recombinant Escherichia coli strain W26 provided in this embodiment shows promising application prospects in the large-scale industrial fermentation production of gallic acid and its derivatives.

[0127] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications or equivalent substitutions made within the spirit and technical principles of the present invention are included within the protection scope of the present invention.

Claims

1. A 4-hydroxybenzoic acid hydroxylase mutant, characterized in that, The following mutation is present only in the parental amino acid sequence of 4-hydroxybenzoic acid hydroxylase, SEQ ID NO. 1: (1) D37E (2) M65T (3) T356A (4) A376T (5)D37E / T356A / A376T, (6)D37E / T356A / A376T / A10V, (7)D37E / T356A / A376T / Q19L, (8)D37E / T356A / A376T / M65T, (9)D37E / T356A / A376T / T241E, (10)D37E / T356A / A376T / A125C、 (11)D37E / T356A / A376T / I336V, or (12)D37E / T356A / A376T / Q19L / M65T.

2. A nucleic acid encoding the 4-hydroxybenzoic acid hydroxylase mutant as described in claim 1.

3. A recombinant expression vector containing the nucleic acid encoding as described in claim 2.

4. A recombinant genetically engineered bacterium, characterized in that, It contains recombinant Escherichia coli encoding nucleic acid as described in claim 3.

5. The recombinant genetically engineered bacteria as described in claim 4, characterized in that, The encoded nucleic acid is integrated into the genome.

6. The use of the 4-hydroxybenzoic acid hydroxylase mutant of claim 1, the nucleic acid encoding of claim 2, or the recombinant expression vector of claim 3, or the recombinant genetically engineered strain of claim 4 or 5 in the synthesis of gallic acid or its salts.

7. A method for synthesizing gallic acid or its salts, characterized in that, Using protocatechuic acid as a substrate, gallic acid or its salts are synthesized by whole-cell catalysis using recombinant genetically engineered bacteria as described in claim 4 or 5. or Using glucose as a substrate, gallic acid or its salts are synthesized by aerobic fermentation using recombinant genetically engineered bacteria as described in claim 4 or 5.

8. The method as described in claim 7, characterized in that, The whole-cell catalytic reaction uses recombinant Escherichia coli expressing the nucleic acid encoded as described in claim 2 as a catalyst and protocatechuic acid as a substrate to synthesize gallic acid or its salt under the following reaction system and conditions: protocatechuic acid concentration of 1-20 g / L, reaction pH of 5.0-8.0, reaction temperature of 25℃-40℃, and reaction time of 12 h-60 h.

9. The method as described in claim 7, characterized in that, The recombinant genetically engineered bacteria express the encoded nucleic acid as described in claim 2 in recombinant Escherichia coli that produces protocatechuic acid, and carry out aerobic fermentation in a culture medium containing glucose to achieve efficient synthesis of gallic acid or its salts under the following fermentation system and fermentation conditions: fermentation pH 5.5-7.5, fermentation temperature 25℃-40℃, and fermentation time 48 h-84 h.

10. The method as described in claim 9, characterized in that, Fermentation medium: K₂HPO₄ content 2.5-12.5 g / L; The MgSO4 content is 1-5 g / L; the (NH4)2SO4 content is 0.8-4.0 g / L; The FeSO4×7H2O content is 0.025-0.125 g / L; The content of citric acid monohydrate is 1-5 g / L; The CaCO3 content is 0-50 g / L; The MnSO4×H2O content is 1.5-7.5 mg / L; The Na2SO4 content is 10-50 mg / L; The ZnSO4×7H2O content is 0.4-6.4 mg / L; The CoCl2×6H2O content is 1-5 mg / L; The CuSO4×5H2O content is 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, or 1.0 mg / L.

11. The method as described in claim 10, characterized in that, The K2HPO4 content is 2.5 g / L, 5.0 g / L, 7.5 g / L, 10 g / L, or 12.5 g / L; The MgSO4 content is 1 g / L, 2 g / L, 3 g / L, 4 g / L, or 5 g / L; The (NH4)2SO4 content is 0.8 g / L, 1.6 g / L, 2.4 g / L, 3.2 g / L, or 4.0 g / L; The FeSO4×7H2O content is 0.025 g / L, 0.050 g / L, 0.075 g / L, 0.100 g / L, or 0.125 g / L; The content of citric acid monohydrate is 1 g / L, 2 g / L, 3 g / L, 4 g / L, or 5 g / L; The CaCO3 content is 0 g / L or 10 g / L or 20 g / L or 30 g / L or 40 g / L or 50 g / L; The MnSO4×H2O content is 1.5 mg / L, 3.0 mg / L, 4.5 mg / L, 6.0 mg / L, or 7.5 mg / L; The Na2SO4 content is 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, or 50 mg / L; The ZnSO4×7H2O content is 0.4 mg / L, 1.6 mg / L, 3.2 mg / L, 4.8 mg / L, or 6.4 mg / L; The CoCl2×6H2O content is 1 mg / L or 2 mg / L or 3 mg / L or 4 mg / L or 5 mg / L; The CuSO4×5H2O content is 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, or 1.0 mg / L.

12. The method as described in claim 9, characterized in that, In the aforementioned aerobic fermentation culture method, the initial concentration of the substrate glucose is 10-60 g / L; The aerobic fermentation culture method uses a glucose solution as the feeding medium. The aerobic fermentation culture method wherein the fermentation pH is 5.0-8.0; The aerobic fermentation culture method wherein the fermentation temperature is 25℃-42℃; The aerobic fermentation culture method has a fermentation cycle of 48h-84h.

13. The method as described in claim 12, characterized in that, The aerobic fermentation culture method wherein the initial concentration of the substrate glucose is 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, or 60 g / L. The aerobic fermentation culture method uses a glucose solution as the feeding medium, with a concentration of 100 g / L, 200 g / L, 300 g / L, 400 g / L, 500 g / L, 600 g / L, or 700 g / L. The aerobic fermentation culture method wherein the fermentation pH is 5.0 or 5.5 or 6.0 or 6.5 or 7.0 or 7.5 or 8.0; The aerobic fermentation culture method wherein the fermentation temperature is 25℃, 30℃, 37℃, 40℃, or 42℃. The aerobic fermentation culture method has a fermentation cycle of 48 h, 60 h, 72 h, or 84 h.

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