CN116355972A

CN116355972A - Fermentation process for producing L-homoserine by recombinant escherichia coli

Info

Publication number: CN116355972A
Application number: CN202310208931.XA
Authority: CN
Inventors: 柳志强; 高峰; 黄良刚; 张博; 周俊平; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2023-03-07
Filing date: 2023-03-07
Publication date: 2023-06-30

Abstract

The invention relates to the technical field of biological fermentation, and discloses a fermentation process for producing L-homoserine by recombinant escherichia coli. The fermentation process for producing L-homoserine by using the recombinant escherichia coli mainly aims at solving the problems that in the existing fermentation process for producing L-homoserine by using the recombinant escherichia coli, the growth of thalli is difficult to control and the high yield is difficult to reproduce. The invention is realized by the pH, temperature, glucose content and OD in the fermentation process ₆₀₀ The value and DO value are controlled in a certain range, so that the optimal production state can be kept at each stage of the bacterial growth finally, the bacterial growth control in the fermentation process is realized, the yield of L-homoserine is finally improved, and the high yield reproducibility is good.

Description

Fermentation process for producing L-homoserine by recombinant escherichia coli

Technical Field

The invention relates to the technical field of biological fermentation, in particular to a fermentation process for producing L-homoserine by recombinant escherichia coli.

Background

L-Homoserine (HS), an unnatural amino acid belonging to the L-aspartic acid family, is a precursor for synthesizing the essential amino acids L-threonine, L-methionine and L-isoleucine. L-homoserine is also used as an important platform compound for the synthesis of high value-added compounds such as O-acetylhomoserine, homoserine lactone, isobutanol and gamma-butyrolactone. L-homoserine also has the functions of improving plant stress resistance and promoting poultry growth. Thus, L-homoserine is expected to be widely used in the fields of foods, medical treatment, agriculture and the like.

At present, the main production mode of L-homoserine is chemical synthesis, including methionine method and butyrolactone method, but the development of the L-homoserine is limited by the factors of high price of reagents used in the reaction process, long reaction time or complicated purification steps, etc. The biological method has the advantages of green and high efficiency, low cost and easy obtainment of the substrate and the like, and is concerned by people.

In 2016, university of Jiangnan Li Hua and the like, lysA, thrB, thrC and metA genes on a competitive metabolic branch and a degradation path of escherichia coli W3110L-homoserine are knocked out, and the metL genes in the metabolic path are overexpressed through a plasmid pBR322, and meanwhile, a transportation system of the L-homoserine is further modified, and finally, the L-homoserine is fermented in a 15L fermentation tank to obtain 39.54 g/L-homoserine; in 2019, the Wei Toshiba team at the university of Huadong would also obtain homoserine high-yield strain IJL by knocking out the competition and degradation pathways and combining with the modification of the central metabolic pathway, and the fed-batch fermentation yield reaches 35.8g/L; in 2021, beijing institute of microorganisms adopted a manner of controlling the balance of reducing power to realize 84.1g/L homoserine production in the production strain with released competing and degrading pathways; the applicant's prior patent CN109055290B also provides a recombinant escherichia coli with high L-homoserine production. The recombinant E.coli producing L-homoserine as described above all suffer from the common problem that Chaetomium always suffers from the essential amino acid deficiency. The defect of essential amino acid of the strain can cause the problems of complex control process control and poor production reproducibility in the tank.

Although the transformation of the homoserine chassis realizes the high yield of homoserine to a certain extent, the industrial application standard is not yet met, and the problem of the fermentation process needs to be further solved. Therefore, aiming at the problem of knocking out the essential amino acid biosynthesis pathway of the homoserine chassis bacteria, developing a fermentation process capable of shortening the fermentation period, optimizing the growth performance and improving the fermentation yield is a problem which needs to be solved by the person skilled in the art.

Disclosure of Invention

The homoserine chassis cell knocks out the essential amino acid biosynthesis gene, and the problem that the production and growth of thalli are difficult to control in the fermentation process exists. Meanwhile, the influence caused by knocking out the essential amino acid biosynthesis genes is solved by adding defective amino acids into the culture medium, but the problem of insufficient amino acid supply in the defect of the growth process still exists, and the bacterial growth in the fermentation process is influenced, so that the yield is influenced.

In view of the above, the invention provides a fermentation process for producing L-homoserine by recombinant escherichia coli. The invention aims to improve the homoserine production fermentation method of the amino acid defective strain through fermentation process optimization, improve biomass and yield, and shorten the fermentation regulation process of the fermentation period.

The specific technical scheme of the invention is as follows:

the invention provides a fermentation process for producing L-homoserine by using recombinant escherichia coli, wherein the recombinant escherichia coli for producing the L-homoserine is used as a production strain for fermentation culture, and the fermentation process is controlled by the following parameters:

(1) pH and temperature: the pH is maintained between 6.6 and 7.0, and the temperature is maintained between 30 and 32 ℃.

(2) Glucose content: the content of glucose is 0.1-5 g/L.

(3)OD ₆₀₀ Value: the bacterial cells enter a stagnation period, pulse type quantitative amino acid feeding is carried out, and OD is carried out ₆₀₀ The value is in the range of 30 to 50.

(4) DO value: the DO value of the system is reduced to 9-12% for the first time, and the DO value is maintained at 9-12% after that.

Unlike available fermentation process for producing L-homoserine with recombinant colibacillus, the present invention provides one fermentation process for producing L-homoserine with recombinant colibacillus, which aims at solving the problems of hard control of thallus growth and high yield. The invention is realized by the pH, temperature, glucose content and OD in the fermentation process ₆₀₀ The value and DO value are controlled in a certain range, so that the optimal production state can be kept at each stage of the bacterial growth finally, the bacterial growth control in the fermentation process is realized, and the yield of L-homoserine is finally improved. The fermentation process provided by the invention can obtain L-homoserine with the yield of 75g/L in the fermentation process within 70h, and the high yield has good reproducibility.

In the fermentation process provided by the invention, the pH and the temperature are controlled (1), the glucose content is controlled (2), and the OD is controlled (3) ₆₀₀ The four aspects of (4) controlling DO value can keep the best production state of each stage of the bacterial growth, thereby achieving the reproduction of high yield. If either aspect is not met, the yield may be compromised. Specifically:

in the aspect (1), the change of the pH is the comprehensive reflection of the metabolic activity of the thalli in the fermentation process, when the pH exceeds a certain range, the thalli can lose the adjusting capability, the pH of the fermentation liquor can continuously fluctuate after losing self-adjustment, and the fluctuation of the pH of the fermentation liquor can adversely affect the thalli and the pH adjusting capability thereof. The cause of the pH fluctuation depends on the metabolism of the cells themselves, but also on the surrounding environment such as the medium. In the fermentation system for producing L-homoserine by recombinant escherichia coli, the thallus and the culture environment are integrated, and the pH is maintained at 6.6-7.0, so that the thallus is in the best production state. The temperature of 30-32 ℃ can optimize the bacterial production under the environment with pH of 6.6-7.0.

In the aspect (2), the concentration of glucose is controlled to be 0.1-5 g/L, so that the concentration of glucose is uniform in the whole fermentation process, the glucose is favorable for improving the yield of the growth metabolism of thalli, and the concentration of glucose depends on the pH and the temperature range in the aspect (1). If a large amount of glucose is fed at one time, the cells grow at a larger growth speed in the initial stage of production, and the final cell density is too high, so that the growth and metabolism of the cells are not facilitated, and meanwhile, the glucose content in the later stage is low, the requirement of the growth and metabolism of the cells cannot be met, and the yield is low.

In the aspect (3), the OD of the system is controlled by pulse-type quantitative amino acid feeding ₆₀₀ The value is in the range of 30-50, so that the carbon flux can flow to the cell metabolism more, the high-yield L-homoserine is fermented, and meanwhile, in the range, the fermentation rheological property is good. If the amino acid concentration is too high, carbon flux flows to cell growth, carbon flux flowing to cell metabolism is reduced, and L-homoserine yield is reduced; in addition to the reduced flux of carbon to cellular metabolism, and the resulting excessive cell density, further impedes cellular metabolism. If the amino acid concentration is too low, the amino acid deficiency is unfavorable for the growth and metabolism of the bacterial strain because the bacterial strain is deficient in amino acid.

In the case of the (4) th aspect, the DO value of the fermentation system is reduced to 9 to 12% for the first time, and thereafter the DO value is maintained at 9 to 12%. The L-homoserine biological components are AspA and AspC paths, and the DO value of the system is 9-12% by dissolved oxygen feedback, so that the cell metabolism can be driven to synthesize homoserine by a more efficient AspC path, and the fermentation yield is improved. And combines the cell density control function of the (2) and (3) to ensure that each stage of the bacterial growth can keep the optimal production state.

As a preferable mode of the above technical scheme of the invention, the recombinant escherichia coli is escherichia coli HS33.

Coli HS33 is disclosed in the documents Multiplex design ofmetabolic network forproduction of L-homoserine in Escherichia coli [ J ]. Applied and environmental microbiology 2020,86 (20) e01477-20, provided by the university of Zhejiang Industrial microorganisms strain collection. It is noted that the above technical scheme of the invention is preferably but not limited to E.coli HS33 genetically engineered bacteria. The recombinant E.coli of the present invention may also be a methionine, lysine and/or threonine deficient strain.

Preferably, the pH of the solution of the present invention is maintained with aqueous ammonia. Preferably, the concentration of ammonia is 45 to 55wt%.

As a preferable mode of the above technical scheme of the invention, the quantitative fed-batch amino acid includes fed-batch threonine, methionine and lysine.

As a preferable mode of the above technical scheme of the invention, the quantitative feeding amount of the amino acid is 10mL.

As a preferable mode of the technical scheme, the DO value is maintained through dissolved oxygen feedback feeding. When the DO value is greater than 10%, feed medium is fed.

Dissolved oxygen feedback control under the condition of hypoxia can drive cell metabolism to synthesize homoserine through a more efficient AspC approach, thereby improving fermentation yield.

As a preferable mode of the above technical scheme of the invention, the composition of the feed medium is: glucose 500g/L, KH ₂ PO ₄ 12.5g/L，(NH ₄ ) ₂ SO ₄ 10 g/L, threonine 4g/L, L-methionine 0.5g/L, L-lysine 1g/L, metal ion 2mL/L, betaine 2g/L, VB ₁₂ 2 mg/L，VB ₁ 5mg/L, water as solvent and natural pH.

As a preferable mode of the technical scheme, the fermentation medium comprises the following components: glucose 15g/L, (NH) ₄ ) ₂ SO ₄ 17 g/L,Yeast Extract 4g/L,KH ₂ PO ₄ 1 g/L，MgSO ₄ 0.5 g/L，FeSO ₄ ·7H ₂ O 0.005g/L，MnSO ₄ ·7H ₂ O 0.005g/L，ZnSO ₄ 0.005 g/L, threonine 0.4g/L, methionine 0.125g/L, lysine 0.25g/L, VB ₁₂ 2 mg/L，VB ₁ 5mg/L, betaine 2g/L, defoamer 1mL/L, water as solvent and natural pH.

Compared with the prior art, the invention has the following technical effects:

(1) The fermentation process of L-homoserine provided by the invention can keep the optimal production state at each stage of thallus growth by controlling the pH value, the temperature, the glucose content, the OD600 value and the DO value in a certain range in the fermentation process, and realizes the control of thallus growth in the fermentation process.

(2) The fermentation process of L-homoserine provided by the invention has high yield and good reproducibility.

(3) The fermentation process of L-homoserine provided by the invention can effectively solve the problem of byproduct accumulation in the production process and improve the yield of L-homoserine through low dissolved oxygen, residual sugar and pH control.

Drawings

FIG. 1 is a diagram of the non-optimized fermentation process of E.coli HS33 in comparative example 1 of the present invention;

FIG. 2 is a graph of a 5L tank fermentation process for pulsed feed of amino acids in example 1 of the present invention;

FIG. 3 is a graph of a 5L tank fermentation process under optimal fermentation parameters in example 8 of the present invention.

Detailed Description

The invention is further described below with reference to examples.

The genetically engineered bacteria used in the following examples and comparative examples were HS33 strains constructed and deposited at the university of Zhejiang industry (Multiplex design of metabolic network for production of L-homoserine in Escherichia coli [ J ]. Applied and Environmental microbiology.2020,86 (20) e 01477-20.).

The genotype of the strain is as follows:

ΔmetJΔmetIΔmetBΔthrBΔmetAΔiclRΔptsGΔgalRΔlacI::Trc-rhtATrc-rhtATrc-eamATrc-thrATrc-me tLTrc-glkTrc-gltB。

the fermentation process for producing L-homoserine by using the recombinant escherichia coli mainly aims at solving the problems that in the existing fermentation process for producing L-homoserine by using the recombinant escherichia coli, the growth of thalli is difficult to control and the high yield is difficult to reproduce. For the purpose of understanding the technical scheme of the present invention, comparative example 1 below is compared with examples 1 to 8 of the present invention as a comparative example.

Comparative example 1

The formula of the culture medium is as follows:

the first seed culture medium consists of: 10g/L peptone, 5g/L yeast powder and 10g/L NaCl;

The composition of the secondary seed culture medium is as follows: 10g/L peptone, 5g/L yeast powder and 10g/L NaCl;

the composition of the fermentation medium is: glucose 40g/L, (NH) ₄ ) ₂ SO ₄ 17 g/L,Yeast Extract 4g/L,KH ₂ PO ₄ 1 g/L，MgSO ₄ 0.5 g/L，FeSO ₄ ·7H ₂ O 0.005g/L，MnSO ₄ ·7H ₂ O 0.005g/L，ZnSO ₄ 0.005 g/L,0.4g/L threonine, 0.125g/L methionine, 0.25g/L lysine, 1mL/L defoamer, water as solvent, and natural pH.

The feed medium composition is: 500g/L glucose, 12.5g/L KH ₂ PO ₄ ，10g/L(NH ₄ ) ₂ SO ₄ 4g/L threonine, 0.5g/L L-methionine, 1g/L L-lysine, 2mL/L metal ion, water as solvent, and natural pH.

The L-homoserine fermentation production method comprises the following steps:

seed liquid culture:

the genetic engineering bacteria are activated by inclined plane, bacterial colony is picked up and inoculated into a test tube filled with a primary seed culture medium for culture to be used as primary seed liquid, and the culture conditions of the primary seed liquid are as follows: single colonies were picked into 10mL test tubes and placed in a constant temperature shaking incubator at 37℃for 10 hours at 180 rpm. Inoculating the primary seed liquid into a shake flask filled with a secondary seed culture medium to culture the primary seed liquid as a secondary seed liquid, wherein the culture conditions of the secondary seed liquid are as follows: the primary seed solution was inoculated into a 500mL shake flask having a liquid loading amount of 200mL at an inoculum size of 1%, and placed in a constant temperature shaking incubator at 37℃for shaking culture at 180rpm for 10 hours.

Culturing in a fermentation tank:

inoculating the second-level seed liquid with the inoculation amount of 10% into a 5L fermentation tank with the liquid loading amount of 2L, wherein the culture conditions are as follows: the temperature was 28℃and the aeration ratio was 2vvm, the rotational speed was 300rpm, the pH was 6.80 and the dissolved oxygen was 20%. The pH was adjusted to 6.60 with 50% (v/v) of sterilized water and 50% (v/v) of aqueous ammonia in advance (the pH was raised to 6.80 after inoculation, and the pH set point was changed to 6.80). When the fermentation time is about 4 hours, sampling and OD detection are started ₆₀₀ And residual sugar content, and preserving the fermentation broth for subsequent detection of L-homoserine content, wherein samples are taken at intervals of 2h in the early stage and 4h in the middle and later stagesAnd (5) sampling once. DO naturally drops to 20% after inoculation, and DO-Stat feedback feed is started. Namely, when DO is more than 20%, the feeding is started, the residual sugar is controlled to be 0.1g/L-5g/L, and the feeding period is adjusted according to the residual sugar residual quantity. Fermentation was ended when DO reached 100%. As shown in FIG. 1, the cells stopped growing and OD after fermentation for 12 hours ₆₀₀ The sugar consumption speed is slow and the yield is 15.4g/L at the maximum by fluctuation around 13.

Offline parameter determination involved in the above method:

biomass measurement: after a fermentation broth sample was obtained through the sampling port, 2m L was diluted appropriately and placed in a cuvette, and the absorbance (OD) was measured at 600nm using an ultraviolet spectrophotometer ₆₀₀ )。

Residual sugar detection, principle: the glucose concentration was measured by the 3, 5-dinitrosalicylic acid method (reducing sugar by DNS method). The DNS, namely dinitrosalicylic acid method, utilizes the principle that dinitrosalicylic acid and reducing sugar undergo oxidation-reduction reaction under alkaline condition to generate 3-amino-5-nitrosalicylic acid, the product shows brownish red color under boiling condition, and the color depth and the reducing sugar content are in proportional relation in a certain concentration range, and the reducing sugar content is measured by a colorimetric method. Reagent: 1g/L glucose solution. 3, 5-dinitrosalicylic acid 6.3g of 3, 5-dinitrosalicylic acid was dissolved in 400mL of distilled water, 21g of sodium hydroxide was gradually added, 182g of potassium sodium tartrate tetrahydrate, 5.0g of phenol, 5.0g of anhydrous sodium sulfite were added, and the solution was stirred continuously in a warm water bath until the solution was clear and transparent. Distilled water was used to volume to 1000mL and stored in a brown bottle. Isolated from carbon dioxide. Standing for 5-7 days. Taking five centrifuge tubes with 1.5mL, sequentially adding 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1mL of glucose standard solution into each centrifuge tube, adding water to supplement 1.0mL, and shaking and mixing uniformly to obtain glucose solutions with the concentration of 0.2 g/L, 0.4 g/L, 0.6 g/L, 0.8 g/L and 1.0 g/L. Five 1.5mL centrifuge tubes were each aspirated with 500. Mu.L glucose solution and 500. Mu.L of DNS reagent was added. The mixture was heated in a water bath for 5 minutes, and then 600. Mu.L of the reaction mixture was taken and 2.4mL of distilled water was added. Detection was performed at 540nm in a spectrophotometer. The standard curve is plotted with the measured value at 540nm as ordinate and the glucose concentration as abscissa.

The liquid phase detection method comprises the following steps: the fermentation broth detection time on the tank was chosen every 4 hours. 1mL of the fermentation broth was centrifuged at 12000rpm for 1min at room temperature, and the supernatant was diluted 20-fold and then examined for L-homoserine amino acid production by HPLC. Mobile phase a: pure acetonitrile is filtered by a filter membrane of 0.22 mu m, is subjected to ultrasonic deaeration and is prepared in an on-line way.

Mobile phase B:50mM HAc-NaAc buffer, pure water, acetonitrile, triethylamine (82.8:17:0.2) acetic acid to adjust pH to 4.9. Filtering with 0.22 μm filter membrane, ultrasonic degassing, and preparing. Derivatizing agent I:0.2700g CNBF (3, 5-dinitro-4-chloro-trifluoromethylbenzene) per 10ml acetonitrile; buffer II: pH 9.0H ₃ BO ₃ -Na ₂ B ₄ O ₇ Buffer solution 0.2mol/L boric acid (H) ₃ BO ₃ ) With 0.05mol/L borax (Na) ₂ B ₄ O ₇ ) A solution. After being prepared, the materials need to be stored in a dark place. Derivatization method: the sample was diluted to 0.1-2 g/L, 100. Mu.L sample, 300. Mu.L derivatization reagent I, 500. Mu.L buffer II. Placing in a mixing instrument, and reacting at 60 ℃ and 400rpm in a dark place for 1h. After the reaction is finished, the mixture is filtered by a 0.22 mu m syringe type organic filter membrane and is reserved for detection. Detection conditions: chromatographic column: j (J)&K C18-H Column (4.6X250 mm,5 μm); flow rate: 0.80mL/min; column temperature: 30 ℃; sample injection amount: 10.0. Mu.L; detection wavelength: 260nm.

Gradient elution and mobile phase gradient elution times are shown in table 1, for example, as compared to flow.

TABLE 1

Example 1 amino acid pulsed fed-batch fermentation

The formula of the culture medium is as follows:

fermentationThe composition of the culture medium is as follows: glucose 40g/L, (NH) ₄ ) ₂ SO ₄ 17 g/L，Yeast Extract 4g/L，KH ₂ PO ₄ 1 g/L，MgSO ₄ 0.5 g/L，FeSO ₄ ·7H ₂ O 0.005g/L，MnSO ₄ ·7H ₂ O 0.005g/L，ZnSO ₄ 0.005 g/L,0.4g/L threonine, 0.125g/L methionine, 0.25g/L lysine, 1mL/L defoamer, water as solvent, and natural pH.

The amino acid feeding bottle is additionally arranged, and the composition of the amino acid feeding bottle is as follows: 80g/L threonine, 40g/L methionine and 20g/L lysine.

The L-homoserine fermentation production method comprises the following steps:

seed liquid culture:

Culturing in a fermentation tank:

inoculating the second-level seed liquid with the inoculation amount of 10% into a 5L fermentation tank with the liquid loading amount of 2L, wherein the culture conditions are as follows: the temperature was 28℃and the aeration ratio was 2vvm, the rotational speed was 300rpm, the pH was 6.80 and the dissolved oxygen was 20%. The pH was adjusted to 6.60 with 50% (v/v) of sterilized water and 50% (v/v) of aqueous ammonia in advance (the pH was raised to 6.80 after inoculation, and the pH set point was changed to 6.80). When the fermentation time is about 4 hours, sampling and OD detection are started ₆₀₀ And residual sugar content, and preserving the fermentation broth for subsequent useAnd detecting the content of L-homoserine, wherein samples are taken at intervals of 2 hours in the early stage and at intervals of 4 hours in the middle and later stages. DO naturally drops to 20% after inoculation, and DO-Stat feedback feed is started. Namely, when DO is more than 20%, the feeding is started, the residual sugar is controlled to be 0.1g/L-5g/L, and the feeding period is adjusted according to the residual sugar residual quantity.

Amino acid addition time to OD 1 hr before and after fermentation process ₆₀₀ The pulse type quantitative feeding amino acid is fed by 10mL each time without obvious change. And OD is taken to ₆₀₀ Controlled between 30-50. When the DO reaches 100%, the fermentation is ended. As a result, as shown in FIG. 2, the yield reached a maximum of 29.8g/L, OD at 96h ₆₀₀ A maximum of 42.15 is reached at 84 h. The yield was improved by 96.1% compared to comparative example 1.

Example 2 fermentation process initial glucose concentration optimization

When the nutrient concentration such as carbon source in the culture system exceeds a certain critical value, the microorganism can form ion gradient to resist high osmotic pressure and maintain the intracellular environment, which is energy consuming, and the result can obviously inhibit the growth of the thallus, while if the initial glucose concentration is too low, the thallus can be forced to stop growing due to the exhaustion of the carbon source when the thallus is not in the optimal state, so that the selection of the initial carbon source with proper concentration is necessary, and four gradients are set for the initial sugar concentration in the embodiment: 10g/L,15g/L,20g/L and 30g/L.

The formula of the culture medium is as follows:

the composition of the fermentation medium is: glucose was set to 4 concentration gradients (10 g/L,15g/L,20g/L,30 g/L), (NH 4) 2SO417g/L, yeast Extract 4g/L, KH2PO41 g/L, mgSO40.5 g/L, feSO4.7H2O 0.005g/L, mnSO4.7H2O 0.005g/L, znSO40.005 g/L,0.4g/L threonine, 0.125g/L methionine, 0.25g/L lysine, defoamer 1mL/L, solvent water, pH was natural.

The feed medium composition is: 500g/L glucose, 12.5g/L KH2PO4, 10g/L (NH) ₄ ) ₂ SO ₄ 4g/L threonine, 0.5g/L methionine, 1g/L lysine, and 2mL/L metal ion.

Amino acid bottle: 80g/L threonine, 40g/L methionine and 20g/L lysine.

The L-homoserine fermentation production method comprises the following steps:

seed liquid culture:

Culturing in a fermentation tank:

inoculating the second-level seed liquid with the inoculation amount of 10% into a 5L fermentation tank with the liquid loading amount of 2L, wherein the culture conditions are as follows: the temperature was 28℃and the aeration ratio was 2vvm, the rotational speed was 300rpm, the pH was 6.80 and the dissolved oxygen was 20%. The pH was adjusted to 6.60 with 50% (v/v) of sterilized water and 50% (v/v) of aqueous ammonia in advance (the pH was raised to 6.80 after inoculation, and the pH set point was changed to 6.80). When the fermentation time is about 4 hours, sampling and OD detection are started ₆₀₀ And residual sugar content, and preserving the fermentation broth for later detection of L-homoserine content, wherein samples are taken at intervals of 2 hours in the early stage and at intervals of 4 hours in the middle and later stages. DO naturally drops to 20% after inoculation, and DO-Stat feedback feed is started. Namely, when DO is more than 20%, the feeding is started, the residual sugar is controlled to be 0.1g/L-5g/L, and the feeding period is adjusted according to the residual sugar residual quantity. Amino acid addition time to OD 1 hr before and after fermentation process ₆₀₀ The pulse type quantitative feeding amino acid is fed by 10mL each time without obvious change. And OD is taken to ₆₀₀ Controlled between 30-50. When the DO reaches 100%, the fermentation is ended. The final optimal initial sugar concentration is 15g/L, the maximum yield after fermentation to 96h is 40.14g/L, and the yield is improved by 2.6 times compared with the comparative example。

Example 3 fermentation process temperature optimization

The temperature in the fermentation tank has great influence on the activity of microbial cellular enzyme, and the intracellular enzyme activity determines the physiological metabolism activity of cells so as to influence the growth of thalli and the synthesis of products. In this embodiment, the fermentation temperature is gradient optimized, and four temperature gradients are set: 28 ℃,31 ℃,34 ℃ and 37 ℃.

The formula of the culture medium is as follows:

the composition of the fermentation medium is: glucose 15g/L, (NH) ₄ ) ₂ SO ₄ 17 g/L,Yeast Extract 4g/L,KH ₂ PO ₄ 1 g/L，MgSO ₄ 0.5 g/L，FeSO ₄ ·7H ₂ O 0.005g/L，MnSO ₄ ·7H ₂ O 0.005g/L，ZnSO ₄ 0.005 g/L,0.4g/L threonine, 0.125g/L methionine, 0.25g/L lysine, 1mL/L defoamer, water as solvent, and natural pH.

The feed medium composition is: 500g/L glucose, 12.5g/LKH ₂ PO ₄ ，10g/L(NH ₄ ) ₂ SO ₄ 4g/L threonine, 0.5g/L L-methionine, 1g/L L-lysine, 2mL/L metal ion, water as solvent, and natural pH.

Amino acid bottle: 80g/L threonine, 40g/L methionine and 20g/L lysine.

The L-homoserine fermentation production method comprises the following steps:

seed liquid culture:

Culturing in a fermentation tank:

inoculating the second-level seed liquid with the inoculation amount of 10% into a 5L fermentation tank with the liquid loading amount of 2L, wherein the culture conditions are as follows: the temperature was set at 4 gradients (28 ℃,31 ℃,34 ℃,37 ℃) and the aeration ratio was 2vvm, the rotational speed 300rpm, the pH 6.80, and the dissolved oxygen 20%. The pH was adjusted to 6.60 with 50% (v/v) of sterilized water and 50% (v/v) of aqueous ammonia in advance (the pH was raised to 6.80 after inoculation, and the pH set point was changed to 6.80). When the fermentation time is about 4 hours, sampling and OD detection are started ₆₀₀ And residual sugar content, and preserving the fermentation broth for later detection of L-homoserine content, wherein samples are taken at intervals of 2 hours in the early stage and at intervals of 4 hours in the middle and later stages. DO naturally drops to 20% after inoculation, and DO-STAT feedback feed is started. Namely, when DO is more than 20%, the feeding is started, the residual sugar is controlled to be 0.1g/L-5g/L, and the feeding period is adjusted according to the residual sugar residual quantity. Amino acid addition time to OD 1 hr before and after fermentation process ₆₀₀ Pulse type quantitative feeding of amino acid 10mL each time without obvious change, OD ₆₀₀ Controlled between 30-50. When the DO reaches 100%, the fermentation is ended. Finally, the optimal fermentation temperature is 31 ℃, the yield reaches the highest value in 72 hours and is 42.56g/L, and the yield is improved by 2.76 times compared with the comparative example; it is notable that the fermentation period is greatly shortened.

Example 4 dissolved oxygen feedback threshold optimization

Coli is a facultative anaerobe that metabolizes and grows in both aerobic and anaerobic environments, but the metabolic pathways are altered, homoserine biosynthesis can also occur through both aerobic and anaerobic pathways, where anaerobic pathways have higher theoretical conversion rates, and it should be noted that too high dissolved oxygen may be detrimental to cell growth, as some thiol-bearing enzymes can be very sensitive to high concentrations of oxygen; at the same time, disturbances in cellular metabolism caused by oxygen starvation may lead to the accumulation of large amounts of byproducts. How to coordinate the two synthetic pathways can be achieved from control of the oxygen concentration of the fermentation process. In order to balance the homoserine biosynthetic pathway, the critical oxygen concentration and the optimal oxygen concentration of the fermentation product need to be examined, so that the fermentation process is kept at the optimal oxygen concentration. In this embodiment, four parameters are set for the dissolved oxygen feedback threshold: 10%,20%,30% and 40%.

The hair medium formulation is as follows:

the composition of the fermentation medium is: glucose 15g/L, (NH) ₄ ) ₂ SO ₄ 17 g/L，Yeast Extract 4g/L，KH ₂ PO ₄ 1 g/L，MgSO ₄ 0.5 g/L，FeSO ₄ ·7H ₂ O 0.005g/L，MnSO ₄ ·7H ₂ O 0.005g/L，ZnSO ₄ 0.005 g/L,0.4g/L threonine, 0.125g/L methionine, 0.25g/L lysine, 1mL/L defoamer.

The feed medium composition is: 500g/L glucose, 12.5g/L KH ₂ PO ₄ ，10g/L(NH ₄ ) ₂ SO ₄ 4g/L threonine, 0.5g/L methionine, 1g/L lysine, and 2mL/L metal ion.

Amino acid bottle: 80g/L threonine, 40g/L methionine and 20g/L lysine.

The L-homoserine fermentation production method comprises the following steps:

seed liquid culture:

Culturing in a fermentation tank:

inoculating the second-level seed liquid with the inoculation amount of 10% into a 5L fermentation tank with the liquid loading amount of 2L, wherein the culture conditions are as follows: the temperature was 28℃and the aeration ratio was 2vvm, the rotational speed was 300rpm, the pH was 6.80, and the dissolved oxygen was 4 gradients (10%. 20%,30%, 40%). The pH was adjusted to 6.60 with 50% (v/v) of sterilized water and 50% (v/v) of aqueous ammonia in advance (the pH was raised to 6.80 after inoculation, and the pH set point was changed to 6.80). When the fermentation time is about 4 hours, sampling and OD detection are started ₆₀₀ And residual sugar content, and preserving the fermentation broth for later detection of L-homoserine content, wherein samples are taken at intervals of 2 hours in the early stage and at intervals of 4 hours in the middle and later stages. DO naturally drops to dissolved oxygen feedback threshold (10%, 20%,30%, 40%) after inoculation, DO-STAT feedback feed is started. Namely, when DO is larger than a set threshold, feeding is started, residual sugar is controlled to be 0.1g/L-5g/L, and the feeding period is adjusted according to the residual sugar residual quantity. Amino acid addition time to OD 1 hr before and after fermentation process ₆₀₀ The pulse type quantitative feeding amino acid is fed by 10mL each time without obvious change. And OD is taken to ₆₀₀ Controlled between 30-50. Fermentation was ended when DO reached 100%. The optimal dissolved oxygen concentration is 10% after screening, the yield is highest after fermentation for 72 hours and reaches 43.43g/L, which is 2.8 times higher than that of the comparative example.

Example 5 betaine concentration optimization

Betaine (N, N-trimethylglycine) is a major compatible solute and also an osmoprotectant, which, due to its specific methylation-related structure, in escherichia coli, is an indispensable role in the growth and production of bacteria. Especially in the field of fermentation, betaine can improve the growth rate and biomass of bacteria in the fermentation process, so that the content of target metabolites is improved, and the osmotic pressure in a liquid culture medium can be increased in the process of producing biological products such as threonine by escherichia coli fermentation, and then the betaine is added into the liquid culture medium by escherichia coli. Meanwhile, betaine has the effect of enhancing the expression of zwf genes related to reducing power and providing reducing power for the synthesis of L-homoserine. In the embodiment, the concentration gradient is set by carrying out gradient optimization on the betaine concentration: 0g/L,1g/L,2g/L and 3g/L.

The formula of the culture medium is as follows:

the composition of the fermentation medium is: glucose 15g/L, (NH) ₄ ) ₂ SO ₄ 17 g/L，Yeast Extract 4g/L,KH2PO41 g/L，MgSO ₄ 0.5 g/L，FeSO ₄ ·7H ₂ O 0.005g/L，MnSO ₄ ·7H ₂ O 0.005g/L，ZnSO ₄ 0.005 g/L,0.4g/L threonine, 0.125g/L methionine, 0.25g/L lysine, 1mL/L defoamer, water as solvent, and natural pH.

The feed medium composition is: 500g/L glucose, 12.5g/L KH ₂ PO ₄ ，10g/L(NH ₄ ) ₂ SO ₄ 4g/L threonine, 0.5g/L L-methionine, 1g/L L-lysine, 2mL/L metal ion, betaine set concentration gradient (0 g/L,1g/L,2g/L,3 g/L), water as solvent, and natural pH.

Amino acid bottle: 80g/L threonine, 40g/L methionine and 20g/L lysine.

The L-homoserine fermentation production method comprises the following steps:

seed liquid culture:

Culturing in a fermentation tank:

inoculating the second-level seed liquid with the inoculation amount of 10% into a 5L fermentation tank with the liquid loading amount of 2L, wherein the culture conditions are as follows: the temperature is 31 ℃, the aeration ratio is 2vvm, the rotating speed is 300rpm, the pH is 6.80, and the dissolved oxygen is10%. The pH was adjusted to 6.60 with 50% (v/v) of sterilized water and 50% (v/v) of aqueous ammonia in advance (the pH was raised to 6.80 after inoculation, and the pH set point was changed to 6.80). When the fermentation time is about 4 hours, sampling and OD detection are started ₆₀₀ And residual sugar content, and preserving the fermentation broth for later detection of L-homoserine content, wherein samples are taken at intervals of 2 hours in the early stage and at intervals of 4 hours in the middle and later stages. DO naturally drops to 10% after inoculation, and DO-STAT feedback feed is started. Namely, when DO is more than 10%, the feeding is started, the residual sugar is controlled to be 0.1g/L-5g/L, and the feeding period is adjusted according to the residual sugar residual quantity. Amino acid addition time to OD 1 hr before and after fermentation process ₆₀₀ The pulse type quantitative feeding amino acid is fed by 10mL each time without obvious change. And OD is taken to ₆₀₀ Controlled between 30-50. Fermentation was ended when DO reached 100%. The optimal addition amount of the screened betaine is 2g/L, and the yield reaches the highest 41.39g/L when the fermentation is carried out for 60 hours.

EXAMPLE 6VB ₁₂ Concentration optimization

VB ₁₂ Can transfer methyl and transfer one carbon under the action of enzyme, and can provide universal one carbon unit for bioconversion so as to be favorable for synthesizing various cell substances. Further VB ₁₂ The molecules contain cobalt and corrines, and contain Co ²⁺ Under the condition of high concentration, the activity of methylmalonyl CoA isomerase can be effectively improved, and the tricarboxylic acid cycle efficiency and the oxaloacetic acid yield can be effectively improved by isomerizing the methylmalonyl CoA into succinyl CoA and entering a tricarboxylic acid cycle path. The embodiment is directed to VB ₁₂ Gradient optimization is carried out on the concentration, and the concentration gradient is set: 1mg/L,2mg/L,4mg/L,8mg/L.

The formula of the culture medium is as follows:

the composition of the fermentation medium is: glucose 15g/L, (NH) ₄ ) ₂ SO ₄ 17 g/L,Yeast Extract 4g/L,KH ₂ PO ₄ 1 g/L，MgSO ₄ 0.5 g/L，FeSO ₄ ·7H ₂ O 0.005g/L，MnSO ₄ ·7H ₂ O 0.005g/L，ZnSO ₄ 0.005 g/L,0.4g/L threonine, 0.125g/L methionine, 0.25g/L lysine, 2g/L betaine and 1mL/L defoamer.

The composition of the feed medium was: 500g/L glucose, 12.5g/LKH ₂ PO ₄ ，10g/L(NH ₄ ) ₂ SO ₄ 4g/L threonine, 0.5g/L L-methionine, 1g/L L-lysine, metal ion 2mL/L betaine 2g/L VB ₁₂ The concentration gradient (1 mg/L,2mg/L,4mg/L,8 mg/L) is set, the solvent is water, and the pH is natural.

Amino acid bottle: 80g/L threonine, 40g/L methionine and 20g/L lysine.

The L-homoserine fermentation production method comprises the following steps:

Seed liquid culture:

Culturing in a fermentation tank:

inoculating the second-level seed liquid with the inoculation amount of 10% into a 5L fermentation tank with the liquid loading amount of 2L, wherein the culture conditions are as follows: the temperature was 31℃and the aeration ratio was 2vvm, the rotational speed was 300rpm, the pH was 6.80 and the dissolved oxygen was 10%. The pH was adjusted to 6.60 with 50% (v/v) of sterilized water and 50% (v/v) of aqueous ammonia in advance (the pH was raised to 6.80 after inoculation, and the pH set point was changed to 6.80). When the fermentation time is about 4 hours, sampling and OD detection are started ₆₀₀ And residual sugar content, and preserving the fermentation broth for later detection of L-homoserine content, wherein samples are taken at intervals of 2 hours in the early stage and at intervals of 4 hours in the middle and later stages. DO naturally drops to 10% after inoculation, and DO-STAT feedback feed is started. Namely, when DO is more than 10%, the feeding is started and the residual sugar is controlled The feeding period is regulated according to the residual sugar content and is controlled to be 0.1g/L-5 g/L. Amino acid addition time to OD 1 hr before and after fermentation process ₆₀₀ The pulse type quantitative feeding amino acid is fed by 10mL each time without obvious change. And OD is taken to ₆₀₀ Controlled between 30-50. Fermentation was ended when DO reached 100%. The optimal adding amount of VB12 is 2mg/L, and the yield reaches the highest 40.64g/L when the fermentation is performed for 60 hours.

EXAMPLE 7VB ₁ Concentration optimization

The bioactive form of vitamin B1 is thiamine Jiao Linzhi (thiamine pyrophosphate, TPP). High amounts of NADPH are required during homoserine synthesis, and TPP is required as a cofactor for the enzymatic reaction of its biosynthesis; furthermore, TPP is an important cofactor in the pyruvate dehydrogenase complex (PDHC), alpha-ketoglutarate dehydrogenase complex (KGDHC) and Transketolase (TK) reactions of the pentose phosphate pathway. PDHC and KGDHC are important components of the ATP pathway that cells use glucose to produce; TK is a key enzyme for gluconeogenesis. Thiamine has an important role in glucose metabolism as a coenzyme for two key catalytic enzymes in glycolysis. Thus, the concentration of VB1 in this example was gradient optimized, and the concentration gradient was set: 1mg/L;3mg/L;5mg/L;7mg/L.

The formula of the culture medium is as follows:

the composition of the fermentation medium is: glucose 15g/L, (NH) ₄ ) ₂ SO417 g/L,Yeast Extract 4g/L,KH ₂ PO ₄ 1 g/L，MgSO ₄ 0.5 g/L，FeSO ₄ ·7H ₂ O 0.005g/L，MnSO ₄ ·7H ₂ O 0.005g/L，ZnSO ₄ 0.005 g/L,0.4g/L threonine, 0.125g/L methionine, 0.25g/L lysine, betaine 2g/L, VB ₁₂ 2. 2 mg/L, defoamer 1mL/L.

The composition of the feed medium was: 500g/L glucose, 12.5g/L KH ₂ PO ₄ ，10g/L(NH ₄ ) ₂ SO ₄ ，4gthreonine/L, 0.5g/L L-methionine, 1g/L L-lysine, metal ion 2mL/L, betaine 2g/L, VB ₁₂ 2 mg/L，VB ₁ The concentration gradient (1 mg/L;3mg/L;5mg/L;7 mg/L) is set, the solvent is water, and the pH is natural.

Amino acid bottle: 80g/L threonine, 40g/L methionine and 20g/L lysine.

The L-homoserine fermentation production method comprises the following steps:

seed liquid culture:

Culturing in a fermentation tank:

inoculating the second-level seed liquid with the inoculation amount of 10% into a 5L fermentation tank with the liquid loading amount of 2L, wherein the culture conditions are as follows: the temperature was 31℃and the aeration ratio was 2vvm, the rotational speed was 300rpm, the pH was 6.80 and the dissolved oxygen was 10%. The pH was adjusted to 6.60 with 50% (v/v) of sterilized water and 50% (v/v) of aqueous ammonia in advance (the pH was raised to 6.80 after inoculation, and the pH set point was changed to 6.80). When the fermentation time is about 4 hours, sampling and OD detection are started ₆₀₀ And residual sugar content, and preserving the fermentation broth for later detection of L-homoserine content, wherein samples are taken at intervals of 2 hours in the early stage and at intervals of 4 hours in the middle and later stages. DO naturally drops to 10% after inoculation, and DO-STAT feedback feed is started. Namely, when DO is more than 10%, the feeding is started, the residual sugar is controlled to be 0.1g/L-5g/L, and the feeding period is adjusted according to the residual sugar residual quantity. Amino acid addition time to OD 1 hr before and after fermentation process ₆₀₀ The pulse type quantitative feeding amino acid is fed by 10mL each time without obvious change. And OD is taken to ₆₀₀ Controlled between 30-50. Fermentation was ended when DO reached 100%. VB (VB) ₁ The optimal addition amount is 5mg/L, and the yield reaches the highest 45.32g/L when fermentation is carried out for 60 hours.

Example 8 Combined fermentation of optimal factors

Based on the optimal influence factors screened in examples 1 to 7, the present example performs a combination test on the above optimal factors. The formula of the culture medium is as follows:

the composition of the fermentation medium is: 15g/L of glucose, (NH 4) 2SO417 g/L, yeast Extract 4g/L, KH2PO41 g/L, mgSO40.5 g/L, feSO4.7H2O 0.005g/L, mnSO4.7H2O 0.005g/L, znSO40.005 g/L,0.4g/L threonine, 0.125g/L methionine, 0.25g/L lysine, betaine 2g/L, VB122 mg/L, VB15mg/L and defoamer 1mL/L.

The composition of the feed medium was: 500g/L glucose, 12.5g/LKH ₂ PO ₄ ，10g/L(NH ₄ ) ₂ SO ₄ 4g/L threonine, 0.5g/L L-methionine, 1g/L L-lysine, 2mL/L metal ion, 2g/L betaine, 2mg/LVB12,5mg/LVB1, water as solvent, and natural pH.

Amino acid supplementing bottle: 80g/L threonine, 40g/L methionine and 20g/L lysine.

The L-homoserine fermentation production method comprises the following steps:

the genetic engineering bacteria are activated by inclined plane, bacterial colony is picked up and inoculated into a test tube filled with a primary seed culture medium for culture to be used as primary seed liquid, and the culture conditions of the primary seed liquid are as follows: single colonies were picked into test tubes of 10ml in liquid loading amount, placed in a constant temperature shaking incubator at 37℃and cultured for 10 hours at 180 rpm.

Inoculating the primary seed liquid into a shake flask filled with a secondary seed culture medium to culture the primary seed liquid as a secondary seed liquid, wherein the culture conditions of the secondary seed liquid are as follows: the primary seed solution was inoculated into a 500mL shake flask having a liquid loading amount of 200mL at an inoculum size of 1%, and placed in a constant temperature shaking incubator at 37℃for shaking culture at 180rpm for 10 hours.

Culturing in a fermentation tank:

inoculating the second-level seed liquid with the inoculation amount of 10% into a 5L fermentation tank with the liquid loading amount of 2L, wherein the culture conditions are as follows: the temperature was 31℃and the aeration ratio was 2vvm, the rotational speed was 300rpm, the pH was 6.80 and the dissolved oxygen was 10%. The pH was adjusted to 6.60 with 50% (v/v) of sterilized water and 50% (v/v) of aqueous ammonia in advance (the pH was raised to 6.80 after inoculation, and the pH set point was changed to 6.80). When the fermentation time is about 4 hours, sampling and OD detection are started ₆₀₀ And residual sugar content, and preserving the fermentation broth for later detection of L-homoserine content, wherein samples are taken at intervals of 2 hours in the early stage and at intervals of 4 hours in the middle and later stages. DO naturally drops to 10% after inoculation, and DO-Stat feedback feed is started. Namely, when DO is more than 10%, the feeding is started, the residual sugar is controlled to be 0.1g/L-5g/L, and the feeding period is adjusted according to the residual sugar residual quantity. Amino acid addition time to OD 1 hr before and after fermentation process ₆₀₀ The pulse type quantitative feeding amino acid is fed by 10mL each time without obvious change. And OD is taken to ₆₀₀ Controlled between 30-50. Fermentation was ended when DO reached 100%. As a result of fermentation under the optimal conditions of all examples, 75.28g/L of the highest yield was detected at 64h of fermentation, which is 3.82 times that of the comparative example, and the sugar acid conversion rate was 0.45g/g, as shown in FIG. 3.

Example 9 Combined fermentation of optimal factors

The main difference from example 8 is the type of fermenter used for the cultivation, and the other reference example 8.

The L-homoserine fermentation production method comprises the following steps:

Culturing in a fermentation tank:

inoculating the secondary seed liquid with the inoculation amount of 10% into a 10L fermentation tank with the liquid loading amount of 7L, wherein the culture conditions are as follows: the temperature was 31℃and the aeration ratio was 2vvm, the rotational speed was 300rpm, the pH was 6.80 and the dissolved oxygen was 10%. The pH was adjusted to 6.60 with 50% (v/v) of sterilized water and 50% (v/v) of aqueous ammonia in advance (the pH was raised to 6.80 after inoculation, and the pH set point was changed to 6.80). When the fermentation time is about 4 hours, sampling and OD detection are started ₆₀₀ And residual sugar content, and preserving the fermentation broth for later detection of L-homoserine content, wherein samples are taken at intervals of 2 hours in the early stage and at intervals of 4 hours in the middle and later stages. DO naturally drops to 10% after inoculation, and DO-Stat feedback feed is started. Namely, when DO is more than 10%, the feeding is started, the residual sugar is controlled to be 0.1g/L-5g/L, and the feeding period is adjusted according to the residual sugar residual quantity. Amino acid addition time to OD 1 hr before and after fermentation process ₆₀₀ The pulse type quantitative feeding amino acid is fed by 10mL each time without obvious change. And OD is taken to ₆₀₀ Control ofBetween 30-50. Fermentation was ended when DO reached 100%. At the time of fermentation for 66h, 75.21g/L of the highest yield is detected, and the sugar acid conversion rate reaches 0.46g/g.

Example 10 Combined fermentation of optimal factors

The L-homoserine fermentation production method comprises the following steps:

Culturing in a fermentation tank:

inoculating the secondary seed liquid with the inoculation amount of 10% into a 10L fermentation tank with the liquid loading amount of 6L, wherein the culture conditions are as follows: the temperature was 31℃and the aeration ratio was 2vvm, the rotational speed was 300rpm, the pH was 6.80 and the dissolved oxygen was 10%. The pH was adjusted to 6.60 with 50% (v/v) of sterilized water and 50% (v/v) of aqueous ammonia in advance (the pH was raised to 6.80 after inoculation, and the pH set point was changed to 6.80). When the fermentation time is about 4 hours, sampling and OD detection are started ₆₀₀ And residual sugar content, and preserving the fermentation broth for later detection of L-homoserine content, wherein samples are taken at intervals of 2 hours in the early stage and at intervals of 4 hours in the middle and later stages. DO naturally drops to 10% after inoculation, and DO-Stat feedback feed is started. Namely, when DO is more than 10%, the feeding is started, the residual sugar is controlled to be 0.1g/L-5g/L, and the feeding period is adjusted according to the residual sugar residual quantity. Amino acid addition time to OD 1 hr before and after fermentation process ₆₀₀ The pulse type quantitative feeding amino acid is fed by 10mL each time without obvious change. And OD is taken to ₆₀₀ Controlled between 30-50. Fermentation was ended when DO reached 100%. At 66h of fermentation, 75.19g/L of the highest yield is detected, and the sugar acid conversion rate reaches 0.44g/g.

The raw materials and equipment used in the invention are common raw materials and equipment in the field unless specified otherwise; the methods used in the present invention are conventional in the art unless otherwise specified.

The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent transformation of the above embodiment according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A fermentation process for producing L-homoserine by recombinant escherichia coli is characterized in that: taking recombinant escherichia coli for producing L-homoserine as a production strain for fermentation culture, wherein the fermentation process controls the following parameters:

(1) pH and temperature: the pH is maintained at 6.6-7.0, and the temperature is maintained at 30-32 ℃;

(2) Glucose content: the content of glucose is 0.1-5 g/L;

(3)OD ₆₀₀ value: the bacterial cells enter a stagnation period, pulse type quantitative amino acid feeding is carried out, and OD is carried out ₆₀₀ The value is in the range of 30-50;

2. The fermentation process for producing L-homoserine by recombinant E.coli according to claim 1, wherein: the recombinant escherichia coli is escherichia coli HS33.

3. The fermentation process for producing L-homoserine by recombinant E.coli according to claim 1, wherein: the pH was maintained with ammonia.

4. A fermentation process for producing L-homoserine by recombinant escherichia coli as claimed in claim 3, wherein: the concentration of the ammonia water is 45-55wt%.

5. The fermentation process for producing L-homoserine by recombinant E.coli according to claim 1, wherein: the quantitative fed-batch amino acid comprises fed-batch threonine, methionine and lysine.

6. The fermentation process for producing L-homoserine by recombinant E.coli according to claim 1 or 5, wherein: the quantitative feeding amount of the amino acid is 10mL in each feeding.

7. The fermentation process for producing L-homoserine by recombinant E.coli according to claim 1, wherein: the DO value is maintained by dissolved oxygen feedback feeding.

8. The fermentation process for producing L-homoserine by recombinant E.coli according to claim 7, wherein: the method for maintaining DO value comprises the following steps: when the DO value is greater than 10%, feed medium is fed.

9. The fermentation process for producing L-homoserine by recombinant E.coli according to claim 8, wherein: the feed medium comprises the following components: glucose 500g/L, KH ₂ PO ₄ 12.5g/L，(NH ₄ ) ₂ SO ₄ 10g/L threonine 4g/L, L-methionine 0.5g/L, L-lysine 1g/L, metal ion 2mL/L, betaine 2g/L, VB ₁₂ 2mg/L，VB ₁ 5mg/L, water as solvent and natural pH.

10. The fermentation process for producing L-homoserine by recombinant E.coli according to claim 1, wherein: the fermented culture medium comprises the following components: glucose 15g/L, (NH) ₄ ) ₂ SO ₄ 17g/L,Yeast Extract 4g/L,KH ₂ PO ₄ 1g/L，MgSO ₄ 0.5g/L，FeSO ₄ ·7H ₂ O 0.005g/L，MnSO ₄ ·7H ₂ O 0.005g/L，ZnSO ₄ 0.005g/L, threonine 0.4g/L, methionine 0.125g/L, lysine 0.25g/L, VB ₁₂ 2mg/L，VB ₁ 5mg/L, betaine 2g/L, defoamer 1mL/L, water as solvent and natural pH.