CN117987485A

CN117987485A - Method for producing L-threonine by fermentation, culture medium and application thereof

Info

Publication number: CN117987485A
Application number: CN202211338498.3A
Authority: CN
Inventors: 宫卫波; 袁二影; 王晓平; 赵津津; 李岩
Original assignee: Langfang Meihua Bio Technology Development Co Ltd
Current assignee: Langfang Meihua Bio Technology Development Co Ltd
Priority date: 2022-10-28
Filing date: 2022-10-28
Publication date: 2024-05-07

Abstract

The invention relates to the technical field of microbial fermentation culture, and particularly discloses a method for producing L-threonine by fermentation, a culture medium and application thereof. The method for producing L-threonine comprises the steps of directly adding beta-chloro-L-alanine into an initial fermentation medium before fermentation culture starts, or using the initial fermentation medium for fermentation culture, and adding beta-chloro-L-alanine into the fermentation medium when the fermentation culture is carried out for 55-60% of the total fermentation time. The invention provides a novel threonine fermentation production method, which can not destroy cell DNA, cell walls and the like, does not affect other functions of cells, and can realize the improvement of threonine yield.

Description

Method for producing L-threonine by fermentation, culture medium and application thereof

Technical Field

The invention relates to the technical field of microbial fermentation culture, in particular to a method for producing L-threonine by fermentation, a culture medium and application thereof.

Background

L-threonine (L-Threonine), chemical name beta-hydroxy-alpha-aminobutyric acid, molecular formula C ₄H₉NO₃, relative molecular weight 119.12. L-threonine is an essential amino acid and is mainly used in various aspects such as medicines, chemical reagents, food enhancers, feed additives and the like.

L-threonine is one of essential amino acids for human body, and has effects of relieving fatigue and promoting growth and development. At present, the microbial fermentation process for producing L-threonine mainly comprises two parts of L-threonine fermentation and extraction, wherein the L-threonine fermentation is mainly produced by adopting a nitrogen source fermentation method which takes corn starch hydrolysis sugar or molasses as a carbon source and takes corn steep liquor, hydrolysis liquid, yeast extract, peptone, ammonium salt, nitrate, urea, ammonia water, liquid ammonia and the like as nitrogen sources. The process optimization generally comprises dissolved oxygen, control of carbon source and nitrogen source, betaine dosage, other vitamins and the like.

However, fermentation still has the problem of conflict between bacterial growth and production, in order to better solve the problem, the bacteria are mainly cultured in the early stage, and after the bacteria grow sufficiently, transformation is forced, and the transformation mode adopted is a method for limiting nutrition, raising temperature and the like, which is unfavorable for the bacteria, so as to limit the bacterial growth and avoid the bacteria from competing with production of nutrient substances such as carbon sources, nitrogen sources and the like. For example, the initial culture temperature is controlled to be 32 ℃ according to the Chinese patent No. CN 112322673A; when the relative growth Δod=0.3 (this is the OD value at 620nm of the fermentation broth diluted 20 times), the temperature was switched to 37 ℃, and when Δod=0.6, the temperature was again increased to 38 ℃ to continue the fermentation until the end. As another example, chinese patent CN111356767a enhanced lipid production by limiting at least two limiting nutrient sources, including nitrogen sources, phosphate sources, vitamin sources (e.g., vitamin B2 sources, pantothenic acid sources, thiamine sources), trace metal sources (e.g., zinc sources, copper sources, cobalt sources, nickel sources, iron sources, manganese sources, molybdenum sources) and primary metal sources (e.g., magnesium sources, calcium sources, sodium sources, potassium sources), silica sources, and mixtures thereof, can achieve the production of large amounts of PUFAs (polyunsaturated fatty acids). However, these approaches have problems of limiting nutrition, increasing temperature, and side effects such as effects on bacterial DNA, RNA, other protease activities, and effects on bacterial structure.

Summarizing the major problems with the prior art are:

1. Most of fermentation is not transformed, bacteria continuously grow and threonine is produced to rob carbon sources, nitrogen sources and nutrients;

2. The existing fermentation transformation comprises the transformation of improving the temperature to inhibit the growth of thalli and producing acid, which has adverse effect on bacteria;

3. The degradation problem cannot be solved by inducing overexpression with IPTG or the like;

4. The induction using amount of arabinose and lactose is large;

5. isoleucine produced after threonine degradation is not resolvable for aspartokinase inhibition.

Therefore, further studies on the fermentative production of threonine are necessary.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a method which can not influence other functions of cells of a bacterium body, but also can effectively improve the conversion rate of threonine fermentation production.

In order to achieve the object, the technical scheme of the invention is as follows:

a method for producing L-threonine by fermentation, which comprises the steps of directly adding beta-chloro-L-alanine into an initial fermentation medium before fermentation culture starts, or using the initial fermentation medium to perform fermentation culture, and adding beta-chloro-L-alanine into the fermentation medium when the fermentation culture is performed for 55-60% of the total fermentation time.

Beta-chloro-L-alanine (beta-Chloro-L-alanine) is an antibacterial amino acid analogue for inhibiting various enzymes, can inhibit the activities of the various enzymes, and has the characteristics of no damage to cell DNA, cell walls and the like. The research of the invention finds that when the threonine is added into the fermentation culture of threonine, the degradation of the product can be effectively inhibited, and the threonine yield can be improved. In particular, when the timing of adding beta-chloro-L-alanine is controlled, the effect of the inhibitor on the growth of the fermentation tubes can be further reduced, thereby further improving the yield.

In the method, the concentration of the beta-chloro-L-alanine in the initial fermentation medium or the fermentation medium is 10-20 mug/L, preferably 10 mug/L, so that the cost can be controlled, the influence on the OD value of zymophyte is reduced, and the yield is further effectively improved.

In the method of the present invention, the initial fermentation medium comprises: corn steep liquor 5-25g/L, hydrolysate 5-10g/L, glucose 38-42g/L,MgSO₄·7H₂O 0.4-0.6g/L,KH₂PO₄ 1.8-2.2g/L,FeSO₄ 18-22mg/L,MnSO₄ 18-22mg/L,ZnSO₄ 0.04-0.06mg/L,(NH₄)₂SO₄ 4-6g/L;

Preferably comprises: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L.

The components of the culture medium are reasonably matched, so that the effect of improving threonine yield can be fully exerted.

In the method of the present invention, the initial fermentation medium further comprises: leucine, valine, isoleucine, glutamic acid, aspartic acid and citric acid.

Preferably, the mass ratio of leucine, valine and isoleucine is 1: (1.3-1.7): (1.3-1.7), more preferably 1: (1.4-1.6): (1.4 to 1.6), further preferably 1:1.5:1.5.

Preferably, the mass ratio of the glutamic acid to the aspartic acid to the citric acid is 1: (0.8-1.2): (0.8-1.2), more preferably 1: (0.9-1.1): (0.9 to 1.1), further preferably 1:1:1.

Further studies of the present invention have found that, although β -chloro-L-alanine can enhance threonine production by inhibiting product decomposition, since the inhibition of the enzyme by β -chloro-L-alanine is not specific, it affects the activities of various enzymes at the same time, and thus can also prevent further enhancement of threonine production in some aspects. Therefore, through repeated fumbling research, the invention finally discovers that the specific amino acid combination is added into the culture medium, so that certain unexpected influences on threonine yield improvement after beta-chloro-L-alanine addition can be compensated, and further the threonine yield can be further improved.

The invention discovers that the effect of beta-chloro-L-alanine on the growth of zymophyte can be further avoided when leucine, valine and isoleucine are added at the same time in a specific ratio.

When glutamic acid, aspartic acid and citric acid are added in a specific proportion, threonine production can be further promoted by matching, and the yield is further greatly improved.

In the method of the present invention, the initial fermentation medium further comprises: leucine in an amount of 0.1-0.3g/L, valine in an amount of 0.2-0.4g/L, isoleucine in an amount of 0.2-0.4g/L, glutamic acid in an amount of 0.8-1.2g/L, aspartic acid in an amount of 0.8-1.2g/L, and citric acid in an amount of 0.8-1.2 g/L;

preferably, the method further comprises: leucine at 0.2g/L, valine at 0.3g/L, isoleucine at 0.3g/L, glutamic acid at 1g/L, aspartic acid at 1g/L and citric acid at 1 g/L.

In the method of the invention, the conditions of fermentation culture are as follows: aeration rate of 0.7-0.9vvm, rotation speed of 300-600rpm, culture pH of 7.0-7.2, temperature of 36-38deg.C and dissolved oxygen of 28-32%;

Preferably, the aeration rate is 0.8vvm, the rotating speed is 300rpm-600rpm, the culture pH is 7.0, the temperature is 37 ℃, and the dissolved oxygen is 30%;

when the residual sugar content in the fermentation liquid is 0.1-0.2g/L, glucose is fed in, so that the concentration of glucose is controlled to be 0-1g/L, and nitrogen source ammonia water is used as a pH regulator;

and/or the fermentation bacteria are Escherichia coli capable of producing threonine.

As a particularly preferred embodiment, the method of the present invention comprises:

1. the initial medium was supplemented with beta-chloro-L-alanine:

(1) Preparing a seed culture medium and culturing seeds;

(2) A fermentation medium was prepared, and 10. Mu.g/L of beta-chloro-L-alanine was added thereto for fermentation culture.

(3) Controlling fermentation conditions; the reduction in OD of fermentation was greater but the final yield was increased compared to the absence of beta-chloro-L-alanine.

2. Beta-chloro-L-alanine is added in the fermentation culture process;

(1) Preparing a seed culture medium and culturing seeds;

(2) Preparing a fermentation medium, and fermenting and culturing;

(3) Controlling fermentation conditions, and adding 10 mug/L beta-chloro-L-alanine when the total fermentation time reaches 57%; the OD is hardly reduced, and the yield and the conversion rate are greatly improved.

3. Adding beta-chlorine-L-alanine, a specific organic nitrogen source and amino acid into an initial culture medium;

(1) Preparing a seed culture medium and culturing seeds;

(2) Preparing a fermentation medium, adding 10 mug/L beta-chloro-L-alanine, leucine, valine, isoleucine, glutamic acid, aspartic acid and citric acid, and fermenting and culturing;

(3) Controlling fermentation conditions; there was little reduction in OD and further improvement in yield and conversion.

The present invention also provides a medium for fermentatively producing L-threonine, comprising: corn steep liquor 5-25g/L, hydrolysate 5-10g/L, glucose 38-42g/L,MgSO₄·7H₂O 0.4-0.6g/L,KH₂PO₄ 1.8-2.2g/L,FeSO₄ 18-22mg/L,MnSO₄ 18-22mg/L,ZnSO₄ 0.04-0.06mg/L,(NH₄)₂SO₄ 4-6g/L,β- chloro-L-alanine 10-20 μg/L; preferably further comprising: leucine in an amount of 0.1-0.3g/L, valine in an amount of 0.2-0.4g/L, isoleucine in an amount of 0.2-0.4g/L, glutamic acid in an amount of 0.8-1.2g/L, aspartic acid in an amount of 0.8-1.2g/L, and citric acid in an amount of 0.8-1.2 g/L;

More preferably, the method comprises: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L,β- chloro-L-alanine 10 μg/L, leucine 0.2g/L, valine 0.3g/L, isoleucine 0.3g/L, glutamic acid 1g/L, aspartic acid 1g/L and citric acid 1 g/L.

The threonine yield can be effectively improved and the conversion rate can be improved through the culture medium.

The invention also provides an application of the method or the culture medium in improving the conversion rate of threonine production by fermentation.

The invention has the advantages that:

The invention provides a novel threonine fermentation production method and a culture medium, which can not destroy cell DNA, cell walls and the like, not affect other functions of cells and realize the improvement of threonine yield when being used for producing threonine by fermentation. Especially, the time for adding the beta-chloro-L-alanine is controlled, and the specific organic acid and the amino acid are preferably matched, so that the negative influence of the beta-chloro-L-alanine on the growth of zymophyte can be avoided, and the fermentation effect is further improved.

Drawings

FIG. 1 shows the OD ₆₀₀ statistics of the fermentation of the medium of example 1 containing various concentrations of beta-Chloro-L-alanine (beta-chloro-L-alanine). The ordinate is OD value, the abscissa is incubation time, in h.

FIG. 2 shows the statistical results of OD ₆₀₀ at fermentation after addition of β -Chloro-L-alanine (β -chloro-L-alanine) at various time points during fermentation in example 2. The ordinate is OD value, the abscissa is incubation time, in h.

FIG. 3 shows the results of OD ₆₀₀ statistics of the different media in example 3 during fermentation. The ordinate is OD value, the abscissa is incubation time, in h.

FIG. 4 is a graph showing the statistical results of OD ₆₀₀ at the time of fermentation for the different media of comparative example 1. The ordinate is OD value, the abscissa is incubation time, in h.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used in the examples below, unless otherwise indicated, are all those available commercially or may be prepared by methods conventional in the art.

The escherichia coli used in the specific embodiment part of the invention is escherichia coli MHZ-0216-5 for producing threonine, and the construction method of the escherichia coli MHZ-0216-5 is disclosed in Chinese patent application CN113846132A.

Example 1

The embodiment provides a culture medium and a method for producing threonine by fermentation, and the culture medium and the method are specifically as follows:

(1) L-threonine seed medium: 15g/L corn steep liquor, 5g/L hydrolysate and glucose 20g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L.

(2) Seed culture: the strain adopts escherichia coli for production, the glycerol pipe frozen strain is firstly coated on an LB culture medium for activation culture overnight, a certain amount of bacterial colonies are selected and inoculated in a 5L triangular flask filled with 1000mL of seed culture medium, the bacterial colonies are put into a constant temperature reciprocating shaking table for shaking culture at the rotation speed of 120rpm and the temperature of 37 ℃ for 8-12 hours until the concentration of the strain in the seed liquid is OD ₆₀₀ = 10, and then the culture is finished.

(3) L-threonine fermentation medium: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L,, and beta-chloro-L-alanine 0, 1, 10, 20 μg/L.

(4) Fermentation culture: the cultured seed liquid was introduced into a 50L fermenter containing 20L of fermentation medium, the aeration rate was 0.8vvm, and the rotation speed was gradually increased from 300rpm to 600rpm to ensure 30% dissolved oxygen, and the culture was conducted at pH7.0 and 37 ℃. Culturing for 28 hours.

And starting feeding a carbon source when the residual sugar content in the fermentation liquid is 0.1g/L, controlling the glucose concentration to be 0-1g/L, controlling pH to 7.0 by taking nitrogen source ammonia water as a pH regulator, preparing a 50% glucose solution as the fed-batch carbon source, and taking 25% ammonia water as the fed-batch nitrogen source.

(5) Glucose content was measured using an SBA biosensor, and L-threonine and L-isoleucine content were measured by HPLC.

The fermentation results are shown in Table 1, and the OD value statistics during fermentation are shown in FIG. 1.

TABLE 1 fermentation results of initial Medium addition of beta-chloro-L-alanine

Example 2

This example provides a method for producing threonine by fermentation, specifically as follows:

(3) L-threonine fermentation medium: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L.

(4) Fermentation culture: the cultured seed liquid is inoculated into a 50L fermentation tank filled with 20L fermentation medium, the aeration rate is 0.8vvm, the rotating speed is 300-600rpm, the pH value is 7.0, the temperature is 37 ℃, and the dissolved oxygen is 30%. Culturing for 28 hours.

Beta-chloro-L-alanine was added at a final concentration of 10. Mu.g/L at 12, 16, 20h of fermentation, respectively.

The results of the fermentation are shown in Table 2, and the OD statistics at the time of fermentation are shown in FIG. 2 (the medium without beta-chloro-L-alanine is used as a control, and the medium without beta-chloro-L-alanine is used as a group).

TABLE 2 post-fermentation results of beta-chloro-L-alanine addition during fermentation

Example 3

(3) L-threonine fermentation medium A: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L,10μg/Lβ- chloro-L-alanine.

(4) L-threonine fermentation medium B: corn steep liquor 25g/L, hydrolysate 10g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L,10μg/Lβ- chloro-L-alanine.

(5) L-threonine fermentation medium C: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L, added with 10 mu g/L beta-chloro-L-alanine, leucine 0.2g/L, valine 0.3g/L, isoleucine 0.3g/L, glutamic acid 1g/L, aspartic acid 1g/L, citric acid 1g/L.

(6) Fermentation culture: the cultured seed solutions are respectively inoculated into a 50L fermentation tank filled with 20L of the fermentation medium, the aeration rate is 0.8vvm, the rotating speed is 300-600rpm, the culture pH is 7.0, the temperature is 37 ℃, and the dissolved oxygen is 30%. Culturing for 28 hours.

(7) Glucose content was measured using an SBA biosensor, L-threonine and L-isoleucine content was measured by HPLC, the fermentation results are shown in Table 3, and the OD value statistics at the time of fermentation are shown in FIG. 3. The L-threonine production was 120.3g/L with 58.6% conversion using L-threonine fermentation medium B, while the OD growth was substantially recovered with L-threonine production 121.5g/L with 59.1% conversion using L-threonine fermentation medium C (containing a specific variety of amino acid supplements).

TABLE 3 fermentation results of initial Medium addition of beta-chloro-L-alanine

Comparative example 1

This comparative example provides 9 methods for producing threonine by fermentation, which are the same as in example 1, except for the fermentation medium.

The fermentation media used for each of the 9 methods of this comparative example were as follows:

Fermentation medium 1: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L, added with 10 mu g/L beta-chloro-L-alanine, leucine 0.2g/L, valine 0.2g/L, isoleucine 0.3g/L, glutamic acid 1g/L, aspartic acid 1g/L, citric acid 1g/L.

Fermentation medium 2: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L, added with 10 mu g/L beta-chloro-L-alanine, leucine 0.2g/L, valine 0.3g/L, isoleucine 0.3g/L, glutamic acid 1g/L, aspartic acid 1g/L, citric acid 0.5g/L.

Fermentation medium 3: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L, added with 10 mu g/L beta-chloro-L-alanine, leucine 0.2g/L, isoleucine 0.3g/L, glutamic acid 1g/L, aspartic acid 1g/L, citric acid 1g/L.

Fermentation medium 4: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L, added with 10 mu g/L beta-chloro-L-alanine, leucine 0.2g/L, valine 0.3g/L, glutamic acid 1g/L, aspartic acid 1g/L, citric acid 1g/L.

Fermentation medium 5: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L, added with 10 mu g/L beta-chloro-L-alanine, leucine 0.2g/L, valine 0.3g/L, isoleucine 0.3g/L, glutamic acid 1g/L, aspartic acid 1g/L.

Fermentation medium 6: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L, added with 10 mu g/L beta-chloro-L-alanine, leucine 0.2g/L, valine 0.24g/L, isoleucine 0.24g/L, glutamic acid 1g/L, aspartic acid 1g/L, citric acid 1g/L.

Fermentation medium 7: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L, added with 10 mu g/L beta-chloro-L-alanine, leucine 0.2g/L, valine 0.36g/L, isoleucine 0.36g/L, glutamic acid 1g/L, aspartic acid 1g/L, citric acid 1g/L.

Fermentation medium 8: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L, added with 10 mu g/L beta-chloro-L-alanine, leucine 0.2g/L, valine 0.3g/L, isoleucine 0.3g/L, glutamic acid 1g/L, aspartic acid 0.7g/L, citric acid 0.7g/L.

Fermentation medium 9: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L, added with 10 mu g/L beta-chloro-L-alanine, leucine 0.2g/L, valine 0.3g/L, isoleucine 0.3g/L, glutamic acid 1g/L, aspartic acid 1.3g/L, citric acid 1.3g/L. The results of the fermentation are shown in Table 4 and FIG. 4 (the medium without addition of beta-chloro-L-alanine and leucine, valine, isoleucine, glutamic acid, aspartic acid and citric acid is used as a control, and the group without addition is shown).

TABLE 4 Table 4

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. A method for producing L-threonine by fermentation is characterized in that beta-chloro-L-alanine is directly added into an initial fermentation medium before fermentation culture starts, or the initial fermentation medium is used for fermentation culture, and when the fermentation culture is carried out for 55-60% of the total fermentation time, the beta-chloro-L-alanine is added into the fermentation medium.

2. The method according to claim 1, characterized in that the concentration of β -chloro-L-alanine in the initial fermentation medium or the fermentation medium is 10-20 μg/L, preferably 10 μg/L.

3. The method according to claim 1 or 2, wherein the initial fermentation medium comprises: corn steep liquor 5-25g/L, hydrolysate 5-10g/L, glucose 38-42g/L,MgSO₄·7H₂O 0.4-0.6g/L,KH₂PO₄ 1.8-2.2g/L,FeSO₄ 18-22mg/L,MnSO₄ 18-22mg/L,ZnSO₄ 0.04-0.06mg/L,(NH₄)₂SO₄ 4-6g/L;

Preferably comprises: corn steep liquor 5/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄0.05mg/L,(NH₄)₂SO₄ 5g/L.

4. A method according to claim 3, wherein the initial fermentation medium further comprises: leucine, valine, isoleucine, glutamic acid, aspartic acid and citric acid.

5. The method according to claim 4, wherein the mass ratio of leucine, valine and isoleucine is 1: (1.3-1.7): (1.3-1.7), preferably 1:1.5:1.5.

6. The method according to claim 4 or 5, wherein the mass ratio of glutamic acid, aspartic acid and citric acid is 1: (0.8-1.2): (0.8-1.2), preferably 1:1:1.

7. The method of any one of claims 4-6, wherein the initial fermentation medium further comprises: leucine in an amount of 0.1-0.3g/L, valine in an amount of 0.2-0.4g/L, isoleucine in an amount of 0.2-0.4g/L, glutamic acid in an amount of 0.8-1.2g/L, aspartic acid in an amount of 0.8-1.2g/L, and citric acid in an amount of 0.8-1.2 g/L;

8. The method according to any one of claims 1 to 7, wherein the conditions of the fermentation culture are: aeration rate of 0.7-0.9vvm, rotation speed of 300-600rpm, culture pH of 7.0-7.2, temperature of 36-38deg.C and dissolved oxygen of 28-32%;

Preferably, the aeration rate is 0.8vvm, the rotating speed is 300rpm-600rpm, the dissolved oxygen is 30%, the culture pH is 7.0, and the temperature is 37 ℃;

9. A medium for fermentative production of L-threonine, comprising: corn steep liquor 5-25g/L, hydrolysate 5-10g/L, glucose 38-42g/L,MgSO₄·7H₂O 0.4-0.6g/L,KH₂PO₄ 1.8-2.2g/L,FeSO₄ 18-22mg/L,MnSO₄18-22mg/L,ZnSO₄0.04-0.06mg/L,(NH₄)₂SO₄ 4-6g/L,β- chloro-L-alanine 10-20 μg/L; preferably further comprising: leucine in an amount of 0.1-0.3g/L, valine in an amount of 0.2-0.4g/L, isoleucine in an amount of 0.2-0.4g/L, glutamic acid in an amount of 0.8-1.2g/L, aspartic acid in an amount of 0.8-1.2g/L, and citric acid in an amount of 0.8-1.2 g/L;

More preferably, the method comprises: corn steep liquor 5g/L, hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L,β- chloro-L-alanine 10 μg/L, leucine 0.2g/L, valine 0.3g/L, isoleucine 0.3g/L, glutamic acid 1g/L, aspartic acid 1g/L and citric acid 1 g/L.

10. Use of the method of any one of claims 1 to 8 or the medium of claim 9 for increasing the conversion of threonine produced by fermentation.