Method for improving sugar-acid conversion rate in shikimic acid fermentation production process

Abstract

The invention provides a method for improving the sugar-acid conversion rate in a shikimic acid fermentation production process. Comprising the following steps: S1, respectively preparing a seed culture medium and a fermentation culture medium, wherein the fermentation culture medium contains 0.1-0.4 g / L of methyl-alpha-D-glucopyranoside; s2, a seed culture stage: sequentially inoculating the escherichia coli bottle-combining seed solution into a primary seed tank and a secondary seed tank, and respectively culturing for a period of time; s3, fermentation culture stage: supplementing the culture solution after seed culture into a three-stage fermentation tank, and controlling the pH value of the fermentation solution to be 6.7-6.9 within 0-12 hours; the pH value of the fermentation liquor is 6.4-6.6 when the fermentation liquor is discharged from 12h to the tank, and co-culturing for 42-54h to obtain the fermentation liquor containing shikimic acid. Through double means of culture medium formula design, seed culture and fermentation culture process design, the conversion rate of synthesizing shikimic acid from glucose is synergistically increased, the overall operation is simple, the dosage of used methyl-alpha-D-glucopyranoside is small, the cost is low, more shikimic acid can be converted, and for medicine production enterprises, the method has a very good application prospect. The obvious cost-reducing and effect-increasing effects are realized.

Description

Technical Field

[0001] The invention belongs to the technical field of shikimic acid production, and particularly relates to a method for improving the sugar-acid conversion rate in the shikimic acid fermentation production process. Background Art

[0002] Shikimic acid (SA) is a natural compound with great application value in nature. The unique six-membered carbon ring and polyhydroxyl functional groups in its molecular structure give it rich biological activity. In the pharmaceutical field, shikimic acid is the core precursor for the synthesis of the anti-influenza drug oseltamivir. At the same time, its inherent antiviral, antithrombotic, analgesic and antibacterial properties make it an important raw material for innovative drug research and development. In the cosmetics and food industries, shikimic acid's antioxidant and bioactivity regulation functions are widely used in the development of functional products. In the agricultural field, shikimic acid can be used as a natural plant growth regulator and antibacterial agent to promote the development of green agriculture. Due to its important applications in many fields, shikimic acid has become an indispensable strategic biomolecule in the modern bioeconomy.

[0003] Currently, the industrial production of shikimic acid primarily relies on extraction from plants such as star anise. This traditional method has significant limitations: First, star anise has a long growth cycle and limited growing areas, making raw material supply susceptible to climate, geography, and market fluctuations. Second, the plant extraction process involves complex solvent extraction, separation, and purification steps, resulting in low spatial and temporal yields and high production costs, making it difficult to meet the growing global market demand.

[0004] With the rise of genetic and metabolic engineering, the use of microorganisms to synthesize shikimic acid has become a viable method. This method offers advantages such as a short production cycle, low cost, and minimal environmental pollution. Bioproduction of shikimic acid holds the potential to alleviate the modern pharmaceutical industry and society's reliance on the dwindling and fragile supply of plant-derived shikimic acid. Fermentation-based production processes have proven to be a successful and cost-effective method for meeting the continuous demand for high yields year-round.

[0005] Currently, the technology for producing shikimic acid by fermenting glucose and other raw materials with genetically engineered Escherichia coli has been industrialized. However, culture conditions have a significant impact on microbial fermentation production. Factors such as medium composition, temperature, pH, aeration, and agitation rate can significantly affect bacterial growth and target product synthesis.

[0006] In summary, although existing technologies have achieved industrial production of shikimic acid, there is still considerable room for improvement in terms of increasing fermentation efficiency and reducing production costs. Further improving the economic benefits of shikimic acid biomanufacturing processes is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0007] The object of the present invention is to provide a method for improving the sugar-acid conversion rate in the fermentation production process of shikimic acid, comprising the following steps:

[0008] Prepare seed culture medium and fermentation medium separately:

[0009] The seed culture medium is: 5 g / L yeast powder, 10 g / L peptone, 10 g / L NaCl, 50 μg / mL chloramphenicol, 40 μg / mL kanamycin, pH 7.0;

[0010] The fermentation medium comprises: 15 g / L glucose, 13 g / L Na2HPO4·H2O, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 1.2 g / L MgSO4, 0.7 g / L L-phenylalanine, 0.35 g / L L-tryptophan, 0.7 g / L L-tyrosine, 0.3 g / L ammonium ferric citrate, 2.1 g / L citric acid monohydrate, 10 g / L yeast extract, 50 μg / mL chloramphenicol, 40 μg / mL kanamycin, and 0.1-0.4 g / L methyl-α-D-pyranoside.

[0011] Seed culture stage: The E. coli seed solution in the bottle is placed in the first-level seed tank and cultured under certain conditions for a period of time; then it is added to the second-level seed tank under sterile operation and cultured under certain conditions for a period of time;

[0012] Fermentation and culture stage: The culture solution after seed culture is aseptically added into the third-stage fermentation tank, and fermented and cultured at 33±1°C for 42-54 hours to obtain the fermentation solution containing shikimic acid.

[0013] In a preferred embodiment, the method for preparing the seed culture medium comprises the following steps:

[0014] (1) Accurately weigh the raw materials according to the formula;

[0015] (2) Dissolve yeast powder, peptone, and NaCl in 800 mL of distilled water and stir until dissolved; adjust the pH to 7.0 and dilute to 1 L;

[0016] (3) Sterilize with high-pressure steam at 121°C for 20 minutes, cool to 50°C, and add chloramphenicol and kanamycin solutions sterilized by filtration through a 0.22 μm filter membrane under a sterile environment to obtain the product.

[0017] In a preferred embodiment, the method for preparing the fermentation medium comprises the following steps:

[0018] (1) Accurately weigh the raw materials according to the formula;

[0019] (2) First, add all ingredients except chloramphenicol and kanamycin to approximately 800 mL of distilled water and stir until dissolved; adjust the pH to 7.0 and dilute to 1 L;

[0020] (3) Sterilize with high-pressure steam at 121°C for 20 minutes, cool to 50°C, and add chloramphenicol and kanamycin solutions sterilized by filtration through a 0.22 μm filter membrane under a sterile environment to obtain the product.

[0021] In a preferred embodiment, the seed culture medium is used in a primary seed tank and a secondary seed tank; and the fermentation culture medium is used in a tertiary fermentation tank.

[0022] Methyl-α-D-glucopyranoside is a glucose derivative with CAS number: 97-30-3 and molecular formula: C7H 14 O6. Through extensive experiments, the present invention discovered that adding a specific amount of methyl-α-D-pyranoglucoside to the fermentation medium in a tertiary fermenter, combined with fermentation process design, can effectively improve the sugar-acid conversion rate. Therefore, the aforementioned fermentation medium formula, dosage, and adapted fermentation process were designed.

[0023] In the present invention, lowering the fermentation tank temperature to 33±1°C slows bacterial growth, inhibiting excessive carbon source consumption and inducing the expression of enzymes involved in shikimic acid synthesis, thereby increasing sugar-acid conversion. Excessively high temperatures can accelerate bacterial growth, causing carbon sources to be used for cell maintenance rather than product synthesis; while too low a temperature can inhibit enzyme activity and prolong the fermentation cycle. Regarding the incubation time, 42-54 hours covers the main stages of shikimic acid synthesis, preventing prolonged incubation from leading to bacterial autolysis or product degradation.

[0024] In a preferred embodiment, during the seed culture stage, the inoculation amount of the E. coli bottled seed solution is 2-5% v / v.

[0025] In a preferred embodiment, during the seed culture stage, the E. coli seed culture time is 13-15 hours, and the seed solution OD 600 3-5.

[0026] In a preferred embodiment, in the seed culture stage, the culture conditions in the first-level seed tank include: culture temperature 37±2°C, sterile air flow rate (0.8-1.2):1vvm (referring to the ratio of the volume of air introduced per minute to the volume of the fermentation liquid), stirring speed 200-400rpm, tank pressure 0.03-0.05Mpa, and DO (referring to the oxygen concentration in the fermentation liquid) is controlled to be 25-40% by adjusting the stirring speed during the culture process; preferably, the culture conditions in the first-level seed tank include: culture temperature 37°C, sterile air flow rate 1:1vvm, stirring speed 300rpm, tank pressure 0.03-0.05Mpa, and DO is controlled to be 25-40% by adjusting the stirring speed during the culture process.

[0027] Around 37°C is the optimal growth temperature for Escherichia coli. At this temperature, the bacterial metabolic activity is strong and the division rate is fast, which is conducive to obtaining a high-concentration seed liquid in a short period of time. If the temperature is too high (>39°C), it may cause the bacterial protein to denature or enter the stable period prematurely; if it is too low (<35°C), the culture time will be prolonged. The sterile air flow rate (0.8-1.2): 1vvm and the stirring speed of 200-400rpm can meet the aerobic growth needs of Escherichia coli and avoid metabolic abnormalities due to insufficient dissolved oxygen. If the ventilation volume is too high or the stirring is too fast, the shear force will increase and damage the bacteria; if it is too low, it will lead to insufficient dissolved oxygen and affect the vitality of the seeds. The tank pressure of 0.03-0.05MPa can prevent the invasion of external bacteria and increase the solubility of oxygen in the fermentation liquid. By adjusting the stirring speed to maintain DO in the appropriate range, it is ensured that the bacteria reproduce rapidly during the logarithmic growth period. Therefore, the present invention limits the aforementioned seed culture conditions. In a preferred embodiment, during the seed culture stage, the culture time in the primary seed tank is 2-6 hours; preferably, the culture time in the primary seed tank is 2-3 hours.

[0028] In a preferred embodiment, in the seed culture stage, after the primary seed tank culture is completed, the OD 600 is 2-4.

[0029] In a preferred embodiment, in the seed culture stage, the culture conditions in the secondary seed tank include: culture temperature 37±2°C, sterile air flow (0.8-1.2):1vvm, stirring speed 200-400rpm, tank pressure 0.03-0.05Mpa, and DO is controlled to 25-40% by adjusting the stirring speed during the culture process; preferably, the culture conditions in the secondary seed tank include: culture temperature 37°C, sterile air flow 1:1vvm, stirring speed 300rpm, tank pressure 0.03-0.05Mpa, and DO is controlled to 25-40% by adjusting the stirring speed during the culture process; more preferably, the culture conditions of the first-level seed tank and the second-level seed tank are the same.

[0030] In a preferred embodiment, during the seed cultivation stage, the cultivation time in the secondary seed tank is 2-6 hours; preferably, the cultivation time in the secondary seed tank is 2-3 hours.

[0031] During the seed culture stage, the OD 600 is 2-4.

[0032] In a preferred embodiment, during the fermentation culture stage, the culture conditions in the three-stage fermentation tank include: a stirring speed of 150-250 rpm, a tank pressure of 0.03-0.05 MPa, and the DO is controlled to be 25-40% by adjusting the stirring speed during the culture process; preferably, the culture conditions in the three-stage fermentation tank include: a stirring speed of 200 rpm, a tank pressure of 0.03-0.05 MPa, and the DO is controlled to be 25-40% by adjusting the stirring speed during the culture process.

[0033] In a preferred embodiment, during the fermentation stage, the fermentation temperature of the tertiary fermentation tank is 33° C., and the fermentation time is 48 hours.

[0034] In a preferred embodiment, during the fermentation and culturing stage, the pH of the fermentation liquid in the three-stage fermentation tank is 6.7-6.9 during the fermentation and culturing period from 0 to 12 hours; and the pH of the fermentation liquid from 12 hours to the time of tank discharge is 6.4-6.6.

[0035] In a preferred embodiment, during the fermentation culture stage, the reagents for adjusting the pH in the tertiary fermentation tank include: glucose (to lower the pH of the fermentation liquid) and / or alkaline reagents (to increase the pH of the fermentation liquid); preferably, the alkaline reagents include NaOH, Na2CO3, NaHCO3, ammonia water, etc.

[0036] In a preferred embodiment, at the end of the fermentation culture, the sugar-acid conversion rate of glucose and shikimic acid is 47-49%.

[0037] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0038] 1. The present invention utilizes a dual approach of culture medium formulation design, seed culture, and fermentation process design to synergistically increase the conversion rate of glucose to shikimic acid. The overall operation is simple and cost-effective. In particular, methyl-α-D-glucopyranoside, while requiring minimal use and at low cost, can convert more shikimic acid. For pharmaceutical manufacturers, the present process offers significant cost-reduction and efficiency-enhancing benefits.

[0039] 2. In the present invention, one to three levels of tanks are designed for seed culture and fermentation culture. The purpose is: the first-level seed tank is used for long-term culture through high ventilation volume and suitable temperature for bacterial amplification; the second-level seed tank is used to further expand the number of bacteria while stabilizing the state of the bacteria, so that the same fermentation conditions as the first-level seed tank can be maintained; the third-level fermentation tank is used for product synthesis, and by lowering the fermentation temperature (33°C) and stirring speed (200rpm) to inhibit excessive bacterial proliferation, reduce energy consumption and activate the expression of key enzymes in the shikimic acid synthesis pathway to increase the yield.

[0040] 3. In the fermentation process of the present invention, by limiting fermentation conditions such as fermentation temperature, stirring speed, dissolved oxygen content, and incubation time, bacterial growth is promoted and the bacterial population is expanded during the seeding stage; during the fermentation stage, growth is inhibited and the process shifts to product synthesis, achieving a metabolic pathway switch between the "growth-synthesis" stage. Simultaneously, this prevents rapid glucose consumption, which leads to the accumulation of byproducts such as acetic acid. This maximizes sugar-acid conversion, reduces byproducts, and reduces energy consumption.

[0041] 4. In the three-stage fermentation tank, the pH is designed to be 6.7-6.9 in the initial fermentation period of 0-12 hours, which can enable bacterial growth and basal metabolism, and accumulate enough biomass for subsequent acid production. At the same time, a higher pH value can suppress the accumulation of organic acids, avoiding the growth inhibition effect caused by excessive acidic substances in the early stage. And the pH is designed to be 6.4-6.6 in the middle and late fermentation period from 12 hours to the tank release, which can activate the shikimic acid synthesis pathway and inhibit bacterial growth in an acidic environment, and use energy and metabolic precursors for shikimic acid synthesis rather than cell proliferation. At the same time, the acidic environment can also suppress the synthesis pathway of by-products, reduce carbon source waste, and improve sugar acid conversion rate. Therefore, the present invention specifically limits the staged pH regulation, accurately guides the carbon source to flow to the shikimic acid synthesis pathway, reduces by-product consumption, and maximizes the efficiency of glucose conversion to shikimic acid. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in detail below in conjunction with specific embodiments. However, it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0043] The embodiments of the present invention provide a method for improving the sugar-acid conversion rate in the fermentation production process of shikimic acid, thereby solving the problems of low fermentation efficiency and low economic benefits in the industrial production of shikimic acid by traditional Chinese medicine enterprises in the prior art.

[0044] The technical solution of this application is described in detail below through specific embodiments:

[0045] Unless otherwise specified, the technical means used in the present invention are conventional means well known to those skilled in the art. The various raw materials, reagents, instruments, and equipment used in the present invention can be purchased from the market or prepared by existing methods. Unless otherwise specified, the reagents used in the present invention are of analytical grade.

[0046] Example 1

[0047] (1) preparing a seed culture medium and a fermentation culture medium respectively, using the seed culture medium in a first-stage seed tank and a second-stage seed tank; and using the fermentation culture medium in a third-stage fermentation tank;

[0048] The seed culture medium comprises the following ingredients: 5 g / L yeast extract, 10 g / L peptone, 10 g / L NaCl, 50 μg / mL chloramphenicol, and 40 μg / mL kanamycin. The preparation method comprises the following steps:

[0049] Accurately weigh the raw materials according to the formula; dissolve yeast powder, peptone, and NaCl in 800 mL of distilled water and stir until dissolved; adjust the pH to 7.0 and make the volume to 1 L; sterilize with high-pressure steam at 121°C for 20 minutes, cool to 50°C, and add chloramphenicol and kanamycin solutions sterilized by filtration through a 0.22 μm filter membrane under a sterile environment to obtain the product.

[0050] The fermentation medium comprises: 15 g / L glucose, 13 g / L Na2HPO4·H2O, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 1.2 g / L MgSO4, 0.7 g / L L-phenylalanine, 0.35 g / L L-tryptophan, 0.7 g / L L-tyrosine, 0.3 g / L ammonium ferric citrate, 2.1 g / L citric acid monohydrate, 10 g / L yeast extract, 50 μg / mL chloramphenicol, 40 μg / mL kanamycin, and 0.194 g / L methyl-α-D-pyranoside. The preparation method comprises the following steps:

[0051] Accurately weigh the raw materials according to the formula; first add the ingredients except chloramphenicol and kanamycin to approximately 800 mL of distilled water and stir until dissolved; adjust the pH to 7.0 and adjust the volume to 1 L; sterilize with high-pressure steam at 121°C for 20 minutes, cool to 50°C, and add chloramphenicol and kanamycin solutions sterilized by filtration through a 0.22 μm filter membrane under a sterile environment to obtain the product.

[0052] (2) The E. coli shake flask seed solution was inoculated into a first-level seed tank at a 3% inoculation rate for cultivation; wherein the preparation method of the shake flask seed solution comprises: placing 50 ml of culture medium in a 500 ml Erlenmeyer flask, culturing at 200 rpm and 37°C for 13.5 h until the OD 600The shake flask medium formula is: 5g / L yeast powder, 10g / L peptone, 10g / L NaCl, 50μg / mL chloramphenicol, and 40μg / mL kanamycin. The shake flask medium is prepared by dissolving yeast powder, peptone, and NaCl in 800mL of distilled water and stirring until dissolved; adjusting the pH to 7.0 and making the volume to 1L; sterilizing by high-pressure steam at 121°C for 20 minutes, cooling to 50°C, and adding chloramphenicol and kanamycin solutions sterilized by filtration through a 0.22μm filter membrane under a sterile environment.

[0053] During the cultivation, the temperature of the first seed tank was controlled at 37°C, the sterile air flow was controlled at 1:1 vvm, the initial stirring speed was 300 rpm, the tank pressure was controlled at 0.03-0.05 MPa, and the stirring speed was adjusted to control the DO at 35±5% during the cultivation process. The culture was continued for 12 h until the OD 600 is 2.8;

[0054] (3) Aseptically adding the fermentation liquid in the first seed tank into the second seed tank for cultivation;

[0055] During the cultivation, the temperature of the secondary seed tank was controlled at 37°C, the sterile air flow was controlled at 1 vvm, the initial stirring speed was 300 r / min, the tank pressure was controlled at 0.03-0.05 MPa, and the stirring speed was adjusted to control the DO at 35±5% during the cultivation process. The culture was continued for 3 h until the OD 600 is 2.6;

[0056] (4) Aseptically add the fermentation liquid in the secondary seed tank to the tertiary seed tank for cultivation;

[0057] During cultivation, the temperature of the three-stage seed tank is controlled at 33°C, the initial stirring speed is 200 r / min, the tank pressure is controlled at 0.03-0.05 MPa, and the DO is controlled at 30±5% by adjusting the stirring speed during the cultivation process. The cultivation cycle is 48 hours. During cultivation, the pH of the fermentation liquid is controlled at 6.7-6.9 from 0 to 12 hours; the pH of the fermentation liquid from 12 hours to the time of tank release is 6.4-6.6. The control method is: if the pH is higher than the above range, an appropriate amount of glucose is added; if the pH is lower than the above range, an appropriate amount of sodium bicarbonate is added.

[0058] After the incubation period, the fermentation broth was collected and assayed using high-performance liquid chromatography (HPLC) (General Method 0512). The mobile phase consisted of 0.05 mol / L sodium dihydrogen phosphate (pH 6.3) and acetonitrile in a ratio of 75:25. The column was packed with octadecylsilane-bonded silica gel (C18, 250 x 4.6 mm, 5 μm). The fermentation broth was diluted with 65% ethanol. The flow rate was 1.0 ml / min, the column temperature was 40°C, the detection wavelength was 229 nm, and the injection volume was 20 μL. The shikimic acid content was determined to be 125 g / L, and the sugar-acid conversion rate was calculated to be 47%.

[0059] Example 2

[0060] The only difference from Example 1 is that in step (1), the fermentation medium is: 15 g / L glucose, 13 g / L Na2HPO4·H2O, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 1.2 g / L MgSO4, 0.7 g / L L-phenylalanine, 0.35 g / L L-tryptophan, 0.7 g / L L-tyrosine, 0.3 g / L ammonium ferric citrate, 2.1 g / L citric acid monohydrate, 10 g / L yeast extract, 50 μg / mL chloramphenicol, 40 μg / mL kanamycin, and 0.223 g / L methyl-α-D-pyranoside; other raw materials and methods are exactly the same as those in Example 1.

[0061] The shikimic acid content in the fermentation broth was determined to be 126 g / L using the same method, and the sugar-acid conversion rate was calculated to be 48%.

[0062] Example 3

[0063] The only difference from Example 1 is that in step (1), the fermentation medium is: 15 g / L glucose, 13 g / L Na2HPO4·H2O, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 1.2 g / L MgSO4, 0.7 g / L L-phenylalanine, 0.35 g / L L-tryptophan, 0.7 g / L L-tyrosine, 0.3 g / L ammonium ferric citrate, 2.1 g / L citric acid monohydrate, 10 g / L yeast extract, 50 μg / mL chloramphenicol, 40 μg / mL kanamycin, and 0.252 g / L methyl-α-D-pyranoside; other raw materials and methods are exactly the same as those in Example 1.

[0064] The shikimic acid content in the fermentation broth was determined to be 129 g / L using the same method, and the sugar-acid conversion rate was calculated to be 49%.

[0065] Comparative Example 1

[0066] The only difference from Example 1 is that in step (1), the fermentation medium comprises: 15 g / L glucose, 13 g / L Na2HPO4·H2O, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 1.2 g / L MgSO4, 0.7 g / L L-phenylalanine, 0.35 g / L L-tryptophan, 0.7 g / L L-tyrosine, 0.3 g / L ammonium ferric citrate, 2.1 g / L citric acid monohydrate, 10 g / L yeast extract, 50 μg / mL chloramphenicol, and 40 μg / mL kanamycin; other raw materials and methods are exactly the same as those in Example 1.

[0067] The shikimic acid content in the fermentation broth was determined to be 117 g / L using the same method, and the sugar-acid conversion rate was calculated to be 38%.

[0068] Comparative Example 2

[0069] The only difference from Example 1 is that in step (4), the temperature of the third-stage seed tank is controlled at 37° C. Other raw materials and methods are exactly the same as those in Example 1.

[0070] The shikimic acid content in the fermentation broth was determined to be 116 g / L using the same method, and the sugar-acid conversion rate was calculated to be 38%.

[0071] Comparative Example 3

[0072] The only difference from Example 1 is that in step (4), the pH is kept between 6.7 and 6.9 during the 48-hour culture period. Other materials and methods are the same as those in Example 1.

[0073] The shikimic acid content in the fermentation broth was determined to be 115 g / L using the same method, and the sugar-acid conversion rate was calculated to be 40%.

[0074] Comparative Example 4

[0075] The only difference from Example 1 is that in step (4), the pH is kept between 6.4 and 6.6 during the 48-hour culture period. Other materials and methods are the same as those in Example 1.

[0076] The shikimic acid content in the fermentation broth was determined to be 105 g / L using the same method, and the sugar-acid conversion rate was calculated to be 39%.

[0077] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for improving the sugar-acid conversion rate in the fermentation production process of shikimic acid, characterized in that: The following steps are involved: Prepare seed culture medium and fermentation medium separately: The seed culture medium is: 5 g / L yeast powder, 10 g / L peptone, 10 g / L NaCl, 50 μg / mL chloramphenicol, 40 μg / mL kanamycin, pH 7.0; The fermentation medium comprises: 15 g / L glucose, 13 g / L Na2HPO4·H2O, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 1.2 g / L MgSO4, 0.7 g / L L-phenylalanine, 0.35 g / L L-tryptophan, 0.7 g / L L-tyrosine, 0.3 g / L ammonium ferric citrate, 2.1 g / L citric acid monohydrate, 10 g / L yeast extract, 50 μg / mL chloramphenicol, 40 μg / mL kanamycin, and 0.1-0.4 g / L methyl-α-D-pyranoside. Seed culture stage: the E. coli seed solution in the bottle is placed in the first-level seed tank and cultured for a period of time under certain conditions; Sterile operation is carried out to fill the seed tank into the secondary seed tank and cultured under certain conditions for a period of time; Fermentation and culture stage: The culture solution after seed culture is aseptically added into the third-stage fermentation tank, and fermented and cultured at 33±1°C for 42-54 hours to obtain the fermentation solution containing shikimic acid.

2. The method for improving the sugar-acid conversion rate in the fermentation production process of shikimic acid according to claim 1, characterized in that: During the seed culture stage, the inoculation amount of the Escherichia coli bottled seed solution is 2-5%.

3. The method for improving the sugar-acid conversion rate in the fermentation production process of shikimic acid according to claim 1, characterized in that: During the seed culture stage, the culture conditions in the first-level seed tank include: culture temperature 37±2°C, sterile air flow rate (0.8-1.2): 1vvm, stirring speed 200-400rpm, tank pressure 0.03-0.05Mpa, and DO is controlled to 25-40% by adjusting the stirring speed during the culture process.

4. The method for improving the sugar-acid conversion rate in the fermentation production process of shikimic acid according to claim 1, characterized in that: During the seed cultivation stage, the cultivation time in the primary seed tank is 10-14 hours.

5. The method for improving the sugar-acid conversion rate in the fermentation production process of shikimic acid according to claim 1, characterized in that: During the seed culture stage, the culture conditions in the secondary seed tank include: culture temperature 37±2°C, sterile air flow rate (0.8-1.2):1vvm, stirring speed 200-400rpm, tank pressure 0.03-0.05Mpa, and DO is controlled to 25-40% by adjusting the stirring speed during the culture process.

6. The method for improving the sugar-acid conversion rate in the fermentation production process of shikimic acid according to claim 1, characterized in that: During the seed cultivation stage, the cultivation time in the secondary seed tank is 2-6 hours.

7. The method for improving the sugar-acid conversion rate in the fermentation production process of shikimic acid according to claim 1, characterized in that: During the fermentation culture stage, the culture conditions in the three-stage fermentation tank include: a stirring speed of 150-250 rpm, a tank pressure of 0.03-0.05 MPa, and DO is controlled to be 25-40% by adjusting the stirring speed during the culture process.

8. The method for improving the sugar-acid conversion rate in the fermentation production process of shikimic acid according to claim 1, characterized in that: During the fermentation and cultivation stage, the fermentation temperature of the three-stage fermentation tank is 33° C., and the fermentation and cultivation time is 48 hours.

9. The method for improving the sugar-acid conversion rate in the fermentation production process of shikimic acid according to claim 1, characterized in that: During the fermentation and cultivation stage, the pH of the fermentation liquid in the three-stage fermentation tank is 6.7-6.9 from 0 to 12 hours of fermentation and cultivation; and the pH of the fermentation liquid from 12 hours to the time of tank discharge is 6.4-6.

6.

10. The method for improving the sugar-acid conversion rate in the fermentation production process of shikimic acid according to claim 1, characterized in that: At the end of the fermentation culture, the sugar-acid conversion rate of glucose and shikimic acid was 47-49%.