CN114606276A

CN114606276A - Method for increasing fermentation yield of L-threonine

Info

Publication number: CN114606276A
Application number: CN202011438488.8A
Authority: CN
Inventors: 宫卫波; 王晓平; 吴强; 李国辉; 袁二影
Original assignee: Langfang Meihua Bio Technology Development Co Ltd
Current assignee: Langfang Meihua Bio Technology Development Co Ltd
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2022-06-10
Anticipated expiration: 2040-12-07
Also published as: CN114606276B

Abstract

The invention provides a method for improving fermentation yield of L-threonine, which takes Escherichia coli (Escherichia coli) as a fermentation strain, and improves the fermentation yield of L-threonine by adding PQQ into an initial fermentation medium or adding PQQ into the fermentation medium in the middle or later period of the fermentation process. The PQQ is added into the fermentation medium, so that the later metabolic activity of the fermentation strain is enhanced, the OD value of the strain is improved, and the yield of the L-threonine is correspondingly improved.

Description

Method for increasing fermentation yield of L-threonine

Technical Field

The invention relates to the field of microbial fermentation, in particular to a method for improving the fermentation yield of L-threonine.

Background

L-threonine is an important nutrition required by human beings and animals, has important significance on health, is one of 8 amino acids essential to human bodies, increases global demand and yield year by year, reaches 70 ten thousand tons in the world in 2019, and is widely applied to the fields of food, feed, medicines and chemical industry.

L-threonine is mainly produced by fermentation of escherichia coli, and CN106867952A replaces a promoter Ppppc of a phosphoenolpyruvate carboxylase gene (ppc) of an original strain with a promoter Pzwf of a 6-phosphoglucose dehydrogenase gene (zwf), so that the aim of regulating the L-threonine production capacity of the L-threonine by betaine is fulfilled. In the fermentation process, the yield of the L-threonine by shake flask fermentation can reach 50-55g/L by adding betaine; the yield of the 5L fermentation tank reaches 120-150g/L, and the saccharic acid conversion rate reaches 59-61%.

The stable fermentation of threonine by Escherichia coli, BIOPROCESS AND BIOSYSTEMS ENGINEERING, 2018, discloses a method for increasing the concentration of L-threonine to 127.3g/L by using a batch fermentation method, AND the conversion rate reaches 58.12%.

The yield and the conversion rate of the L-threonine are improved by improving the Escherichia coli genes and combining the mode of batch fermentation, and meanwhile, a large number of researches show that similar effects can be achieved by improving the fermentation conditions. Chenning and the like (based on the optimization of the L-threonine fermentation process of pathway analysis, the 3 rd phase of the university of Tianjin science and technology, 2008) add sodium gluconate and control dissolved oxygen by 20% through metabolic flux analysis, thereby improving the yield of L-threonine to 107 g/L. Gold and the like (L-threonine fermentation temperature change control and yeast extract fed-batch process research, modern chemical engineering S2, 2007) controls the temperature to be 37 ℃ 12h before fermentation, and increases the temperature to 39 ℃ 12h after fermentation, so that the maximum cell biomass and L-threonine yield can be obtained simultaneously; on the basis, 2.0g/L of yeast extract is continuously fed in the late stage of fermentation, so that the generation of byproduct amino acid can be effectively reduced, the acid production level of L-threonine fermentation is stabilized, the maximum acid production mass concentration reaches 105.8g/L, and the sugar acid conversion rate reaches 47.5%. Xuqingyang and the like (dissolved oxygen influences L-threonine fermentation, microbiological report 2 nd stage 2007) controls 20% dissolved oxygen in lag phase, maintains 50% dissolved oxygen after log phase, and 20% dissolved oxygen in stationary phase, and through fed-batch fermentation for 36h in a 10L tank, acid production can reach 118.9g/L, and the conversion rate of saccharic acid is 47.6%. CN107760734A, a mixed fermentation accelerator consisting of betaine hydrochloride, VB4 and VB3 is added in the L-threonine fermentation process, so that the L-threonine yield can be increased by 10-12% and reaches 133.6 g/L.

Therefore, by enhancing or weakening gene expression, fermentation conditions such as temperature, pH, dissolved oxygen and the like are improved, and the yield and the conversion rate of L-threonine can also be improved by improving the fermentation conditions at the later stage, such as fed-batch yeast extract.

Disclosure of Invention

The invention aims to provide a method for improving the fermentation yield of L-threonine.

The invention has the following conception: the activity of the microorganism is reduced in the later period, the later period activity can be improved by supplementing various nutrient factors such as amino acid, vitamins and the like, and the later period activity of the zymocyte is improved by supplementing pyrroloquinoline quinone (PQQ). PQQ cannot be synthesized in Escherichia coli, but can be used by enzyme on respiratory chain of Escherichia coli, and can improve respiratory efficiency and activity of late stage bacteria after PQQ is added.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a method for increasing fermentation yield of L-threonine, comprising: escherichia coli (Escherichia coli) is used as a fermentation strain, and PQQ is added to an initial fermentation medium or to a fermentation medium at the middle or later stage of the fermentation process, so as to improve the fermentation yield of L-threonine.

The concentration of PQQ in the fermentation medium is 5 mug/L-5 mg/L.

Preferably, the fermentation strain is escherichia coli, and the fermentation medium comprises the following components: 30g/L glucose, 10g/L ammonium sulfate, 6g/L corn steep liquor and KH₂PO₄ 0.5g/L，MgSO₄·7H₂O 0.5g/L，FeSO₄·7H₂O 0.01g/L，MnSO₄·H₂O 0.01g/L，pH7.0。

The method comprises the following steps:

1) activating strains and preparing a first-level seed solution;

2) preparing a secondary seed solution;

3) and inoculating the secondary seed liquid into a shake flask or a fermentation tank for fermentation culture.

The conditions for fermentation culture in the step 3) are as follows: at 37 ℃, dissolved oxygen is 20-50%, and the pH value of a fermentation system is controlled to be 7.0. Taking a fermentation tank with a volume of 50L as an example, the liquid loading amount can be 20-30L.

Further, step 1) comprises: inoculating the activated strain into a 500ml triangular shake flask filled with 100ml LB culture medium, and carrying out shake culture on a shaking table at 37 ℃ and 220rpm for 5-6 h until seed liquid OD ₆₀₀10 as first seed liquid.

Further, step 2) comprises: inoculating the primary seed solution into a 10L fermentation tank containing 4L seed culture medium, and culturing at 37 deg.C with dissolved oxygen of 20% -50% and pH of 7.0 to OD ₆₀₀20 as secondary seed liquid.

Wherein the seed culture medium comprises the following components: 25g/L glucose, 10g/L ammonium sulfate, 20g/L corn steep liquor and KH₂PO₄ 1.5g/L，MgSO₄·7H₂O 0.5g/L，FeSO₄·7H₂O 0.01g/L，MnSO₄·H₂O 0.01g/L，pH7.0。

Preferably, step 3) comprises: inoculating the secondary seed liquid into a fermentation culture medium according to the volume ratio of 1:10-1: 7.

Further, after fermentation for 13 to 17 hours (preferably 15 hours), PQQ may be added to the fermentation medium at once or at a constant flow rate. In order to prevent the interference of PQQ in the growth stage and the loss in the fermentation process and ensure the PQQ to play the maximum role, the PQQ is added in the middle and later stages of the growth of bacteria, at the moment, the strain activity in the fermentation liquor is high, and the PQQ can be efficiently utilized.

The method further comprises the following steps: after fermentation for 13-17h (preferably 15h), the inorganic salt solution is added to the fermentation medium until the end of the fermentation. At the moment, elements such as iron ions are added in the electron transfer process, so that the action of PQQ is promoted, and the activity of later fermentation of the strain is ensured; in addition, the iron ions play a role in promoting the growth of the thalli and producing the L-threonine, if the growth vigor of the thalli is too high early, excessive sugar and nutrition are consumed, the generation of amino acid is not facilitated, and the effects of promoting the growth of the thalli and producing the L-threonine are played by selecting the middle and later growth log stages.

Wherein the inorganic salt solution is prepared from phosphoric acid and ferrous sulfate heptahydrate.

The concentration of ferrous sulfate heptahydrate in the fermentation medium is controlled to be 0.01-0.1g/L, and the concentration of phosphoric acid is controlled to be 0.1-1 g/L.

In the present invention, the Escherichia coli refers to a bacterium having L-threonine producing ability, and preferably, the Escherichia coli (Escherichia coli) MHZ-0215-2. The strain MHZ-0215-2 is currently preserved in the China general microbiological culture Collection center, No. 3 of West Lu No.1 of the morning and the evening, Beijing, China academy of sciences, the postal code 100101, the preservation number CGMCC No.13403 and the preservation date 2016, 11 and 30 days.

In a second aspect, the present invention provides the use of PQQ in the fermentative production of L-threonine or to increase the fermentative production of L-threonine.

In the above application, Escherichia coli is used as a fermentation strain.

The PQQ is added into the fermentation medium, so that the later metabolic activity of the fermentation strain is enhanced, the OD value of the strain is improved, and the yield of the L-threonine is correspondingly improved.

Drawings

FIGS. 1 to 4 show the growth of bacterial cells in the experimental group and the control group in the preferred embodiment of the present invention. Wherein, 1: OD value of experimental group, 2: OD value of control group.

FIG. 5 shows the growth of bacterial cells in the experimental group and the control group according to the preferred embodiment of the present invention. Wherein, 1-4 respectively represent the initial fermentation medium added with PQQ of 5. mu.g/L, 50. mu.g/L, 500. mu.g/L and 5mg/L, and 5 is the control group OD.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

Escherichia coli MHZ-0215-2 used in the following examples was purchased from Touchfany plum biotechnological development, Inc.

The content of L-threonine in the fermentation liquor is determined by adopting a High Performance Liquid Chromatography (HPLC) method, and the derivatization method is a PITC-trifluoroacetic acid method (see the ceramic champion et al, the HPLC quantitative analysis of glutamine in protein and peptide, 20 vol. 4 of Zheng Zhou food academy, 1999), and an Agilen C18 chromatographic column is adopted. The OD value was measured using a model 721 spectrophotometer.

Example 1 method for increasing fermentation yield of L-threonine

1. Preparing culture medium

(1) Culture medium for preparing first-stage seed liquid

LB medium (g/L): yeast powder 5, peptone 10 and sodium chloride 5.

(2) Culture medium for preparing secondary seed liquid

Seed medium (g/L): 25 parts of glucose, 10 parts of ammonium sulfate, 20 parts of corn steep liquor and KH₂PO₄ 1.5，MgSO₄·7H₂O0.5，FeSO₄·7H₂O 0.01，MnSO₄·H₂O0.01; sterilizing at 121 deg.C for 20min at pH 7.0.

(3) Fermentation medium (g/L): 30 parts of glucose, 10 parts of ammonium sulfate, 6 parts of corn steep liquor and KH₂PO₄ 0.5，MgSO₄·7H₂O，0.5，FeSO₄·7H₂O 0.01，MnSO₄·H₂O0.01; sterilizing at 121 deg.C for 20min at pH 7.0.

2. Preparing feeding nutrient solution

The PQQ nutrient solution was prepared in 1000. mu.g/L ultrapure water.

3. Experimental methods

Preparing frozen glycerol tube, preparing 50% glycerol with ultrapure water, sterilizing at 121 deg.C for 20min, sucking 500 μ l, packaging into 2ml glycerol tube, and adding OD₆₀₀1ml of seed solution of 10 deg.C, and freezing at-70 deg.C. Taking out the frozen glycerol tube, thawing, sucking 100 μ l of seed liquid, coating LB slant for activation, and culturing at 37 deg.C for 12 h; transferring the inclined plane into a triangular shake flask with the volume of 500ml (100 ml LB culture medium is filled in the shake flask), placing the shake flask in a 37 ℃ shaking table after inoculation, and carrying out shaking culture on the shake table at 220rpm for 5-6 h. Shake flask seed OD₆₀₀Growing to 10, namely, obtaining a first-grade seed solution; inoculating the first-stage seed solution into a seed tank (4L seed culture medium in the seed tank) with a volume of 10L, and culturing under the conditions: controlling the pH value to be 7.0 by 25-28% ammonia water at 37 ℃, and dissolving 20-50% of oxygen; seed solution OD₆₀₀Growing to 20, namely obtaining a secondary seed solution; inoculating 2.5L of the secondary seed solution into a fermentation tank with the volume of 50L (17.5L of fermentation medium is filled in the fermentation tank) for fermentation culture, wherein the culture conditions are as follows: controlling the pH value to be 7.0 at 37 ℃ by 25-28% ammonia water, dissolving 20% -50% of oxygen, and adding 500mL of PQQ nutrient solution into a fermentation tank when fermenting for 15 h. The total fermentation time is 36 h.

After the end of fermentation, the L-threonine treatment in the fermentation broth of the experimental group (with PQQ added) was increased by 1.5g/L and the glucose conversion by 1% compared to the control group (without PQQ added) (Table 1). The growth of the bacteria in the experimental group and the control group is shown in FIG. 1.

TABLE 1 increase in L-threonine production following PQQ addition

Example 2 method for increasing fermentation yield of L-threonine

1. Preparing inorganic salt nutrient solution

A phosphoric acid solution having a concentration of 10g/L was prepared, and ferrous sulfate heptahydrate was dissolved to give a final concentration of 0.1g/L as an inorganic salt solution (inorganic salt nutrient solution). 1L of inorganic salt solution is prepared. Sterilizing at 121 deg.C for 20 min.

2. An LB culture medium, a seed culture medium, a fermentation culture medium and a feeding nutrient solution are respectively prepared, and specifically, refer to example 1.

3. Experimental method

Preparing frozen glycerol tube, preparing 50% glycerol with ultrapure water, sterilizing at 121 deg.C for 20min, sucking 500 μ l, packaging into 2ml glycerol tube, and freezing seed with OD of 10 at-70 deg.C to obtain the final product. Taking out the frozen glycerol tube, thawing, sucking 100 μ l of seed liquid, coating LB slant for activation, and culturing at 37 deg.C for 12 h; transferring the inclined plane into a triangular shake flask with the volume of 500ml (100 ml LB culture medium is filled in the shake flask), placing the shake flask in a 37 ℃ shaking table after inoculation, and carrying out shaking culture on the shake table at 220rpm for 5-6 h. Shake flask seed OD₆₀₀Growing to 10, namely, obtaining a first-grade seed solution; inoculating the primary seed liquid into a seed tank with a volume of 10L (Seed tank is filled with 4L seed culture medium), the culture conditions are as follows: controlling the pH value to be 7.0 by 25-28% ammonia water at 37 ℃, and dissolving 20-50% of oxygen; seed solution OD₆₀₀Growing to 20, namely secondary seed liquid; inoculating 2.5L of the secondary seed solution into a fermentation tank with the volume of 50L (17.5L of fermentation medium is filled in the fermentation tank) for fermentation culture, wherein the culture conditions are as follows: controlling pH to 7.0 by 25-28% ammonia water at 37 ℃, dissolving 20% -50% oxygen, and adding 500mL of PQQ nutrient solution and 500mL of inorganic salt nutrient solution into a fermentation tank when fermenting for 15 h. The total fermentation time is 36 h.

After the fermentation was completed, the L-threonine content in the fermentation broth was increased by 3g/L and the glucose conversion rate by 2% as compared to the control (without PQQ and inorganic salt nutrient solution) (Table 2). The growth of the cells in the experimental group and the control group is shown in FIG. 2.

TABLE 2L-threonine production improvement after PQQ and inorganic salt addition

Example 3 method for increasing fermentation yield of L-threonine

1. Inorganic salt nutrient solutions were prepared, see in particular example 2.

2. LB medium, seed medium, fermentation medium and PQQ nutrient solution were prepared separately, see example 1.

3. Experimental methods

Preparing frozen glycerol tube, preparing 50% glycerol with ultrapure water, sterilizing at 121 deg.C for 20min, sucking 500 μ l, packaging into 2ml glycerol tube, and freezing seed with OD of 10 at-70 deg.C to obtain the final product. Taking out the frozen glycerol tube, thawing, sucking 100 μ l of seed liquid, coating LB slant for activation, and culturing at 37 deg.C for 12 h; transferring the inclined plane into a triangular shake flask with the volume of 500ml (100 ml LB culture medium is filled in the shake flask), placing the shake flask in a 37 ℃ shaking table after inoculation, and carrying out shaking culture on the shake table at 220rpm for 5-6 h. Shake flask seed OD₆₀₀Growing to 10, namely, obtaining a first-grade seed solution; inoculating the first-stage seed liquid into a seeding tank with the volume of 10L (4L of seed culture medium is filled in the seeding tank), and culturing under the following conditions: controlling the pH value to 7.0 by 25-28% ammonia water at 37 ℃, and dissolving 20-50% of oxygen; seed treating liquid OD₆₀₀Growing to 20, namely obtaining a secondary seed solution; inoculating 2.5L of the secondary seed solution into a fermentation tank with the volume of 50L (17.5L of fermentation medium is filled in the fermentation tank) for fermentation culture, wherein the culture conditions are as follows: controlling the pH value to be 7.0 by 25-28% ammonia water at 37 ℃, dissolving 20-50% oxygen, adding 500mL of inorganic salt nutrient solution into a fermentation tank once when fermenting for 15h, wherein the total fermentation time is 36 h.

After the fermentation was completed, the L-threonine treatment in the fermentation broth of the experimental group (with inorganic salt nutrient solution added) was improved by 0.5g/L and the glucose conversion rate was improved by 0.5% as compared with the control group (without inorganic salt nutrient solution added) (Table 3). The growth of the cells in the experimental group and the control group is shown in FIG. 3.

TABLE 3 increase in L-threonine production after addition of inorganic salts

Example 4 method for increasing fermentation yield of L-threonine

2. LB medium, seed medium, fermentation medium and PQQ nutrient solution were prepared separately, as described in example 1.

3. Experimental methods

Preparing frozen glycerol tube, preparing 50% glycerol with ultrapure water, sterilizing at 121 deg.C for 20min, sucking 500 μ l, packaging into 2ml glycerol tube, and freezing seed with OD of 10 at-70 deg.C to obtain the final product. Taking out the frozen glycerol tube, thawing, sucking 100 mu l of seed liquid, coating an LB inclined plane for activation, and culturing for 12h at 37 ℃; transferring the inclined plane into a triangular shake flask with the volume of 500ml (100 ml LB culture medium is filled in the shake flask), placing the shake flask in a 37 ℃ shaking table after inoculation, and carrying out shaking culture on the shake table at 220rpm for 5-6 h. Shake flask seed OD₆₀₀Growing to 10, namely, obtaining a first-grade seed solution; inoculating the first-stage seed liquid into a seeding tank with the volume of 10L (4L of seed culture medium is filled in the seeding tank), and culturing under the following conditions: controlling the pH value to be 7.0 by 25-28% ammonia water at 37 ℃, and dissolving 20-50% of oxygen; seed solution OD₆₀₀Growing to 20, namely obtaining a secondary seed solution; inoculating 2.5L of the second-stage seed liquid into a fermentation tank with a volume of 50L(17.5L fermentation medium is filled in a fermentation tank) for fermentation culture, and the culture conditions are as follows: controlling pH to 7.0 by 25-28% ammonia water at 37 ℃, dissolving 20% -50% of oxygen, feeding PQQ nutrient solution into the fermentation tank at a constant speed of 50mL/h when fermenting for 15h, and feeding inorganic salt nutrient solution into the fermentation tank at a constant speed of 50mL/h until the feeding is finished. The total fermentation time is 36 h.

After the fermentation was completed, the L-threonine content in the fermentation broth was increased by 2g/L and the glucose conversion rate was increased by 1.5% as compared with the control (no PQQ and no inorganic salt nutrient solution added). The growth of the cells in the experimental group and the control group is shown in FIG. 4.

TABLE 4L-threonine production improvement by PQQ addition and inorganic salt addition

Example 5 method for increasing fermentation yield of L-threonine

1. LB medium, seed medium, fermentation medium were prepared separately, and specifically referring to example 1, PQQ was added to the initial fermentation medium at 5. mu.g/L, 50. mu.g/L, 500. mu.g/L, and 5mg/L, respectively.

2. Experimental methods

Preparing frozen glycerol tube, preparing 50% glycerol with ultrapure water, sterilizing at 121 deg.C for 20min, sucking 500 μ l, packaging into 2ml glycerol tube, and freezing seed with OD of 10 at-70 deg.C to obtain the final product. Taking out the frozen glycerol tube, thawing, sucking 100 μ l of seed liquid, coating LB slant for activation, and culturing at 37 deg.C for 12 h; transferring the inclined plane into a triangular shake flask with the volume of 500ml (100 ml LB culture medium is filled in the shake flask), placing the shake flask in a 37 ℃ shaking table after inoculation, and carrying out shaking culture on the shake table at 220rpm for 5-6 h. Shake flask seed OD₆₀₀Growing to 10, namely, obtaining a first-grade seed solution; inoculating the first-stage seed liquid into a seeding tank (6L of seed culture medium is filled in the seeding tank) with the volume of 10L, and culturing under the following conditions: controlling the pH value to be 7.0 by 25-28% ammonia water at 37 ℃, and dissolving 20-50% of oxygen; when the OD600 of the seed liquid grows to 20, the secondary seed liquid is obtained; inoculating 2.5L of the secondary seed solution into two fermentation tanks with 50L volume (17.5L of the secondary seed solution contained 5 μ g/L and 17.5L of the secondary seed solution contained in the fermentation tanks respectively)5mg/L PQQ fermentation medium), and the culture conditions are as follows: controlling the pH value to 7.0 at 37 ℃ by 25-28% ammonia water, dissolving oxygen by 20% -50% and fermenting for 36 h.

After the fermentation is finished, compared with a control group (without PQQ), the L-threonine in the fermentation liquor added with 5 mu g/L is improved by 0.5g/L, and the conversion rate of the glucose is improved by 0.2%; the addition of L-threonine in 50. mu.g/L of the fermentation broth increased 1.5g/L, the conversion to glucose increased 1%, the addition of L-threonine in 500. mu.g/L of the fermentation broth increased 1.6g/L, the conversion to glucose increased 1.1%, the addition of L-threonine in 5mg/L of the fermentation broth increased 0.4g/L, and the conversion to glucose increased 0.15% (Table 5). The growth of the cells in the experimental group and the control group is shown in FIG. 5.

TABLE 5 increase in L-threonine production following PQQ addition to initial fermentation Medium

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method for increasing the fermentation yield of L-threonine, comprising: escherichia coli (Escherichia coli) is used as a fermentation strain, and PQQ is added to an initial fermentation medium or to a fermentation medium at the middle or later stage of the fermentation process, so as to improve the fermentation yield of L-threonine.

2. The method according to claim 1, wherein the concentration of PQQ in the fermentation medium is 5 μ g/L to 5 mg/L.

3. The method according to claim 1 or 2, wherein the fermentative strain is large intestineBacillus, the composition of the fermentation medium is as follows: 30g/L glucose, 10g/L ammonium sulfate, 6g/L corn steep liquor and KH₂PO₄ 0.5g/L，MgSO₄·7H₂O 0.5g/L，FeSO₄·7H₂O 0.01g/L，MnSO₄·H₂O 0.01g/L，pH7.0。

4. A method according to claim 3, characterized in that the method comprises the steps of:

1) activating strains and preparing a first-level seed solution;

2) preparing a secondary seed solution;

3) inoculating the secondary seed liquid into a fermentation tank for fermentation culture;

the conditions for fermentation culture in the step 3) are as follows: at 37 ℃, dissolved oxygen is 20-50%, and the pH value of a fermentation system is controlled to be 7.0.

5. The method of claim 4, wherein step 1) comprises: inoculating the activated strain into a 500ml triangular shake flask filled with 100ml LB culture medium, and carrying out shake culture on a shaking table at 37 ℃ and 220rpm for 5-6 h until seed liquid OD₆₀₀10 as first seed liquid.

6. The method of claim 5, wherein step 2) comprises: inoculating the primary seed solution into a 10L fermentation tank containing 4L seed culture medium, and culturing at 37 deg.C with dissolved oxygen of 20% -50% and pH of 7.0 to OD₆₀₀20 as secondary seed liquid;

wherein the seed culture medium comprises the following components: 25g/L glucose, 10g/L ammonium sulfate, 20g/L corn steep liquor and KH₂PO₄1.5g/L，MgSO₄·7H₂O 0.5g/L，FeSO₄·7H₂O 0.01g/L，MnSO₄·H₂O 0.01g/L，pH7.0。

7. The method of claim 6, wherein step 3) comprises: inoculating the secondary seed liquid into a fermentation culture medium according to the volume ratio of 1:10-1: 7.

8. The method of claim 7, wherein PQQ is added to the fermentation medium after 13-17 hours of fermentation.

9. The method according to any one of claims 3-7, further comprising: after fermenting for 13-17h, adding inorganic salt solution into the fermentation medium until the fermentation is finished;

wherein the inorganic salt solution is prepared from phosphoric acid and ferrous sulfate heptahydrate; the concentration of ferrous sulfate heptahydrate in the fermentation medium is controlled to be 0.01-0.1g/L, and the concentration of phosphoric acid is controlled to be 0.1-1 g/L.

10. The method according to any one of claims 3 to 7, wherein the Escherichia coli is Escherichia coli (Escherichia coli) MHZ-0215-2, having a accession number of CGMCC No. 13403.