L-serine production method

Abstract

The invention belongs to the technical field of microbial fermentation, and discloses an L-serine production method, which comprises the following steps: screening recombinant escherichia coli for producing L-serine by selecting a culture medium, and regularly and quantitatively adding scyllo-inositol into the obtained stable strain to control the fermentation conditions to produce L-serine. According to the method, the strain with high production stability can be obtained, and the yield of the L-serine is remarkably increased by controlling fermentation conditions.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and specifically relates to a method for producing L-serine. Background Art

[0002] As an important backbone compound, L-serine has wide application value in many fields, especially in cosmetics, food and medicine. The existing production of L-serine is mainly achieved by fermentation of recombinant Escherichia coli. However, in the process of continuous fermentation of multiple batches, as the number of fermentation batches increases, the growth environment and cell health of Escherichia coli are often affected, resulting in a gradual decrease in the production of L-serine and large fluctuations in production. This technical problem limits the industrial production efficiency and stability of L-serine. Summary of the invention

[0003] The first technical problem to be solved by the present invention is to provide a method for producing L-serine. The method obtains strains with high production stability by directional screening of production strains, and significantly improves the yield of L-serine by controlling fermentation conditions.

[0004] In order to solve the above technical problems, the technical solution of the present invention is:

[0005] A method for producing L-serine comprises the following steps:

[0006] a. The recombinant Escherichia coli of L-serine was gradiently diluted with sterile water to obtain a dilution;

[0007] b. Inoculate the dilution in step a onto a selective medium, wherein the selective medium formula is 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 0.1 mmol / L scyllo-inositol, 1 g / L L-serine, 50 mg / L streptomycin sulfate, and the rest is deionized water, with a natural pH value; and culture at 37°C for 10 to 18 hours until the colony diameter is 2 to 4 mm;

[0008] c. In step b, the colonies cultured on the culture medium with uniform single color, raised colonies and a colony diameter of 2 to 4 mm are selected and inoculated on a plate culture medium, wherein the culture medium formula is 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, and the rest is deionized water, and the pH value is natural; culture at a constant temperature of 37°C for 10 to 16 hours until the colony diameter is 2 to 4 mm;

[0009] d. A portion of the colony obtained by culturing in step c was used for preservation, and a portion was inoculated into a 50 ml liquid culture medium shake flask, wherein the liquid culture medium formula was 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and the rest was deionized water, and the pH value was natural; the culture was cultured at 37 ° C and 220 to 260 rpm until the OD600 value of the bacterial solution was 1.8 to 2.5;

[0010] e. The single colony cultured in the shake flask in step d was inoculated into 500 ml of fermentation medium at an inoculum size of 10% for fermentation culture, and cultured at a constant temperature of 37°C and 220 rpm. 0.1 mmol / L scyllo-inositol was added at 6h, 12h, and 18h ​​of fermentation. When the OD600 value of the bacterial solution was 2.5, the inducer L-arabinose was added at a final concentration of 2g / L. The fermentation was completed after constant temperature induction at 37°C for 24 hours; the content of L-serine in the fermentation broth was detected by HPLC, and the bacterial cells were selected for stability experiments, and the above-mentioned fermentation method was used to produce L-serine.

[0011] Preferably, the gradient dilution in step a is from 10 -1 to 10 -8 .

[0012] Preferably, the step b is cultured at 37° C. for 18 h.

[0013] Preferably, in step c, the colony morphology is white and the culture is carried out at a constant temperature of 37° C. for 16 hours.

[0014] Preferably, in step d, the colonies are white in morphology, have raised colonies, and have a colony diameter of 2 to 4 mm; and the culture is carried out at a constant temperature of 240 rpm until the OD600 value of the bacterial solution is 2.0.

[0015] Preferably, in step e, the fermentation medium comprises 1-5 g / L citric acid, 10 g / L potassium dihydrogen phosphate, 30 g / L glucose, 1 g / L magnesium sulfate, 10 mg / L vitamin B1, 0.5 g / L lysine, 0.5 g / L methionine, 0.5 g / L isoleucine, 0.5 g / L threonine, 0.01 g / L scyllo-inositol, and pH 7.0.

[0016] Furthermore, in the step e, the amount of citric acid added to the fermentation medium is 3 g / L.

[0017] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0018] The invention utilizes a selective culture medium for directional screening, and can effectively screen strains with high yield and strong stability, and improves the production stability and the yield of L-serine by controlling the fermentation conditions. Compared with the prior art, the yield of L-serine is increased by 5-10%. Therefore, the invention has broad industrial application prospects, provides an efficient and stable technical path for the industrial production of L-serine, and can effectively reduce production costs and improve market competitiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Attached Figure 1 HPLC chart of A110 fermentation product under the conditions of Example 1;

[0020] Attached Figure 2 HPLC chart of the product of Example 1 control experiment performed by A110 in Example 5;

[0021] Attached Figure 3 This is the HPLC chart of the A109 fermentation product in Example 4. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below in conjunction with embodiments:

[0023] Embodiment 1:

[0024] a. The recombinant Escherichia coli of L-serine (Deposit No.: CGMCC No.20947) was diluted with sterile water in a gradient from 10 -1 to 10 -8 Obtaining a dilution;

[0025] b. Inoculate the dilution in step a onto the selection medium, the selection medium formula is 5g / L yeast powder, 10g / L peptone, 10g / L sodium chloride, 20g / L agar powder, 1g / L L-serine, 0.1mmol / L scyllo-inositol, add 50mg / L streptomycin sulfate at a final concentration when the temperature drops to 40-50°C, the rest is deionized water, the pH value is natural; and culture at 37°C for 18h until the colony diameter is 2-4mm;

[0026] c. In step b, the colonies cultured on the culture medium with uniform single color, raised colonies and a colony diameter of 2 to 4 mm are selected and inoculated on a plate culture medium, wherein the culture medium formula is 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, and the rest is deionized water, and the pH value is natural; the culture is kept at 37°C for 16 hours until the colony diameter is 2 to 4 mm;

[0027] d. A portion of the colonies obtained by culturing in step c was used for preservation, and a portion was inoculated into a 50 ml liquid culture medium shake flask, wherein the liquid culture medium was formulated as 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 1 g / L L-serine, and the rest was deionized water, and the pH value was natural; the culture was cultured at 37 ° C and 240 rpm until the OD600 value of the bacterial solution was 2.0; the preserved colonies were named A101, A102, A103........A110;

[0028] e. The single colony cultured in the shake flask in step d was inoculated into 500 ml of fermentation medium at an inoculum size of 10% for fermentation culture, wherein the fermentation medium contained 3 g of citric acid, 5 g of potassium dihydrogen phosphate, 15 g of hydatidiform mole, 0.5 g of magnesium sulfate, 5 mg of vitamin B1, 0.25 g of lysine, 0.25 g of methionine, 0.25 g of isoleucine, 0.25 g of threonine, 0.005 g of scyllo-inositol, and pH 7.0; cultured at 37° C. and 220 rpm; 0.1 mmol / L scyllo-inositol was added at 6 h, 12 h, and 18 h of fermentation; when the OD600 value of the bacterial solution was 2.5, an inducer L-arabinose was added at a final concentration of 2 g / L; and the fermentation was completed after cultured at 37° C. for 24 hours; the content of L-serine in the fermentation broth was detected by HPLC, and the results are shown in Table 1.

[0029] Embodiment 2:

[0030] The 10 strains selected in Example 1 were cultured according to the method of step 1, wherein the seed solution culture method in step d was "constant temperature culture at 37°C and 260 rpm until the OD600 value of the bacterial solution was 2.5", and the citric acid content of the fermentation medium was 5 g / L; other conditions remained unchanged; the content of L-serine in the fermentation broth was detected by HPLC, and the results are shown in Table 1.

[0031] Embodiment 3:

[0032] The 10 strains selected in Example 1 were cultured according to the method of step 1, wherein the seed solution culture method in step d was "constant temperature culture at 37°C and 220 rpm until the OD600 value of the bacterial solution was 1.8", and the citric acid content of the fermentation medium was 1 g / L; other conditions remained unchanged; the content of L-serine in the fermentation broth was detected by HPLC, and the results are shown in Table 1.

[0033] Embodiment 4:

[0034] No scyllo-inositol was added to the screening medium and fermentation medium and during the fermentation process. The remaining components and operations were the same as in Example 1. A total of 10 single colonies were screened and named A201 to A210. The L-serine yields after fermentation are shown in Table 1.

[0035] Embodiment 5:

[0036] The 10 strains selected in Example 1 were cultured according to the seed culture and fermentation production methods in Examples 1-3, respectively, wherein scyllo-inositol was not added to the fermentation medium and during the culture process, as a control. The L-serine yield after fermentation is shown in Table 1.

[0037] The L-serine yields of each strain in Examples 1 to 5 are summarized in Table 1:

[0038] Table 1 L-serine yields in Examples 1-5

[0039]

[0040]

[0041] From the above results, it can be seen that strains A101, A106, A108, A109, and A110 have higher yields.

[0042] Example 6: Stability test

[0043] The A101, A106, A108, A109, and A110 strains were subjected to genetic stability experiments, and the results showed that the yield of A108 and A110 remained unchanged after 50 generations, proving that they can be used for industrial production.

[0044] Result analysis:

[0045] The following conclusions were drawn from the analysis of the L-serine production of the strains in each example: the addition of scyllo-inositol during strain screening, fermentation medium and fermentation process can significantly affect the L-serine production of the high-yield strain, and the lack of any of these steps will reduce the final L-serine synthesis amount or affect the stability of its production.

[0046] The method for producing L-serine by fermentation using recombinant Escherichia coli provided by the present invention solves the problems of poor strain stability and gradually reduced yield in traditional production methods, and has broad market application prospects. With the growing demand for L-serine in the fields of cosmetics, food, and medicine, the production method of the present invention can meet the growing demand of the future market and make important contributions to promoting the green development and technological innovation of related industries.

[0047] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

Claims

1. A method for producing L-serine, characterized in that: The following steps are involved: a. The recombinant Escherichia coli of L-serine was gradiently diluted with sterile water to obtain a dilution; b. Inoculate the dilution in step a onto a selective medium, wherein the selective medium formula is 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 0.1 mmol / L scyllo-inositol, 1 g / L L-serine, 50 mg / L streptomycin sulfate, and the rest is deionized water, with a natural pH value; and culture at 37°C for 10 to 18 hours until the colony diameter is 2 to 4 mm; c. In step b, the colonies cultured on the culture medium with uniform single color, raised colonies and a colony diameter of 2 to 4 mm are selected and inoculated on a plate culture medium, wherein the culture medium formula is 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, and the rest is deionized water, and the pH value is natural; culture at a constant temperature of 37°C for 10 to 16 hours until the colony diameter is 2 to 4 mm; d. A portion of the colony obtained by culturing in step c was used for preservation, and a portion was inoculated into a 50 ml liquid culture medium shake flask, wherein the liquid culture medium formula was 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, and the rest was deionized water, and the pH value was natural; the culture was cultured at 37 ° C and 220 to 260 rpm until the OD600 value of the bacterial solution was 1.8 to 2.5; e. The single colony cultured in the shake flask in step d was inoculated into 500 ml of fermentation medium at an inoculum size of 10% for fermentation culture, and cultured at a constant temperature of 37°C and 220 rpm. 0.1 mmol / L scyllo-inositol was added at 6h, 12h, and 18h ​​of fermentation. When the OD600 value of the bacterial solution was 2.5, the inducer L-arabinose was added at a final concentration of 2g / L. The fermentation was completed after constant temperature induction at 37°C for 24 hours; the content of L-serine in the fermentation broth was detected by HPLC, and the bacterial cells were selected for stability experiments, and the above-mentioned fermentation method was used to produce L-serine.

2. The method for producing L-serine according to claim 1, wherein: The gradient dilution in step a is from 10 -1 to 10 -8 .

3. The method for producing L-serine according to claim 1, wherein: In step b, the mixture is cultured at a constant temperature of 37° C. for 18 hours.

4. The method for producing L-serine according to claim 1, wherein: In the step c, the colony morphology is white and the culture is carried out at a constant temperature of 37° C. for 16 hours.

5. The method for producing L-serine according to claim 1, wherein: In the step d, the colonies are white in morphology, convex in colonies, and have a colony diameter of 2 to 4 mm; the culture is carried out at a constant temperature of 240 rpm until the OD600 value of the bacterial solution is 2.

0.

6. The method for producing L-serine according to claim 1, wherein: The fermentation medium in step e is 1-5 g / L citric acid, 10 g / L potassium dihydrogen phosphate, 30 g / L glucose, 1 g / L magnesium sulfate, 10 mg / L vitamin B1, 0.5 g / L lysine, 0.5 g / L methionine, 0.5 g / L isoleucine, 0.5 g / L threonine, 0.01 g / L scyllo-inositol, and pH 7.

0.

7. The method for producing L-serine according to claim 6, wherein: In the step e, the amount of citric acid added to the fermentation medium is 3 g / L.

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