Production strain screening and production method of L-serine

Abstract

The invention belongs to the technical field of microorganisms, and discloses a production strain screening and production method of L-serine, which comprises the following steps: adding quantitative ectoine and L-serine into a screening culture medium to screen out a strain capable of tolerating and producing L-serine at high yield; and then adding ectoine while adding an inducer L-arabinose during fermentation, so that the content of L-serine in the fermentation liquor is remarkably increased. The method is simple, the screened high-yield strain is high in stability, the yield of L-serine produced through fermentation is high, and the method is suitable for large-scale industrial production.

Description

Technical Field

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

[0002] As an important skeleton compound, L-serine has wide application value in many fields, especially in cosmetics, food and medicine. However, its industrial production method has always faced challenges. More than 80% of L-serine in China is extracted by hair acid hydrolysis, which is not only limited in source but also has environmental pollution problems. Although the enzymatic semi-synthesis technology has made some progress, it is still subject to the problems of low conversion rate and high cost of the precursor glycine. Therefore, it is particularly important to develop an economical, efficient and environmentally friendly method for producing L-serine. As a production technology with great potential, microbial fermentation has attracted attention due to its advantages such as low pollution and low cost, but its development is relatively lagging, especially in terms of strain screening and fermentation efficiency. There are obvious shortcomings, low fermentation level, poor production capacity, and poor stability of strains with higher yields, which are not suitable for large-scale industrial production. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a method for screening and producing L-serine producing strains, by adding a certain amount of ectoine and L-serine to the screening culture medium, successfully screening strains that can tolerate and produce high L-serine, and achieving high production of L-serine through fermentation.

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

[0005] A method for screening and producing L-serine production strains comprises the following steps:

[0006] a. The recombinant L-serine-producing Escherichia coli (the strain previously purchased by the applicant from the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 20947 and a deposit date of October 23, 2020) was gradiently diluted with sterilized water to obtain a dilution;

[0007] 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, 2g / L L-serine, 1mmol / L ectoine, 50mg / L streptomycin sulfate, and the rest is deionized water, the pH value is natural; and culture at 37°C for 10 to 18h;

[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 with a formula of 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 2 g / L L-serine, and the rest is deionized water with a natural pH value; cultured at 37°C for 10 to 16 hours;

[0009] d. A portion of the colony obtained by culture in step c was used for preservation, and a portion was inoculated into a 50 ml liquid culture medium shake flask, the liquid culture medium formula was 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, the pH value was natural; the culture was kept constant at 37 ° C and 220 ~ 260 rpm until the bacterial solution OD600 value was 1.8 ~ 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 5% for fermentation culture. The culture was cultured at 37°C and 220 rpm until the OD600 value of the bacterial solution was 2.5. The inducer L-arabinose and 1 mmol / L ectoine were added at a final concentration of 2 g / 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 best bacteria were selected.

[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 colonies are white in morphology, have raised colonies, and have a colony diameter of 2 to 4 mm; and are cultured 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 is 2 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, and pH 7.0.

[0016] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0017] The present invention: 1. By adding a certain amount of ectoine and L-serine to the screening culture medium, a strain that can tolerate the high L-serine environment is successfully screened out, and ectoine is added while adding an inducer L-arabinose during the fermentation process to exert the antioxidant and protective functions of ectoine, so that the strain can continuously produce high L-serine; this screening and production method not only improves the tolerance and acid production efficiency of the strain, but also lays a solid foundation for subsequent industrial production; 2. By screening out high-yield and highly tolerant strains, the production cost is effectively reduced and the production efficiency is improved; experimental data show that the yield of L-serine is increased by 30% compared with the traditional method by using the strain screened by the present invention for fermentation; 3. Compared with the acid hydrolysis method and the enzyme method, the production by using the microbial fermentation method has less pollution and is more in line with the requirements of green production.

[0018] In conclusion, the method of the present invention is simple, the screened high-yield strain has strong stability, the yield of L-serine produced by fermentation is high, and it is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 HPLC chart of the fermentation product of strain S109 in Example 1 of the present invention;

[0020] Figure 2 This is the HPLC chart of L-serine standard. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below in conjunction with embodiments:

[0022] Embodiment 1:

[0023] a. The recombinant L-serine production E. coli was diluted with sterile water, the gradient dilution was from 10 -1 to 10 -8 Obtaining a dilution;

[0024] b. Inoculate the dilution in step a onto the selection medium and culture at 37°C for 18h; the selection medium formula is: 5g / L yeast powder, 10g / L peptone, 10g / L sodium chloride, 20g / L agar powder, 2g / L L-serine, 1mmol / L ectoine, and the rest is deionized water, with a natural pH value; after sterilization at 120°C, add streptomycin sulfate at a final concentration of 50mg / L when the temperature drops to 40-50°C;

[0025] c. Select the colonies cultured on the culture medium in step b, which are white, have raised colonies, and have a colony diameter of 2 to 4 mm, and inoculate them on a plate culture medium, and culture them at 37°C for 16 hours; the plate culture medium formula is 5g / L yeast powder, 10g / L peptone, 10g / L sodium chloride, 20g / L agar powder, 2g / L L-serine, and the rest is deionized water, and the pH value is natural;

[0026] d. Ten colonies obtained by culture in step c were selected and named S101, S102, S103, ... A110; and part of these colonies were used for preservation, and part was inoculated in a shake flask containing 50 ml of liquid culture medium, the liquid culture medium formula was 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, pH value natural; constant temperature culture at 37 ° C, 220 rpm speed until the bacterial solution OD600 value was 2.0;

[0027] e. The single colonies cultured in step d were inoculated into 500 ml of fermentation medium at a 5% inoculum for fermentation culture. The fermentation medium was 2 g / L citric acid, 10 g / L potassium dihydrogen phosphate, 30 g / L glucose, 1 g / L magnesium sulfate, and vitamin B1 10 mg / L, lysine 0.5 g / L, methionine 0.5 g / L, isoleucine 0.5 g / L, threonine 0.5 g / L, pH 7.0; culture at 37°C, 220 rpm until the bacterial solution has an OD600 value of 2.5, add a final concentration of 2 g / L inducer L-arabinose and 1 mmol / L ectoine, and culture at 37°C for 24 hours before fermentation is complete; the content of L-serine in the fermentation broth is detected by HPLC (chromatographic column: Alltech amino column (4.6 mm×250 mm), mobile phase: acetonitrile: water = 75:25, flow rate: 1 mL / min, column constant temperature: 30°C, detector: differential detector, injection volume: 10 μL), the yield of each strain is shown in Table 1.

[0028] Embodiment 2:

[0029] No ectoine was added to the screening medium and the fermentation medium. The other components and operations were the same as in Example 1. A total of 10 single colonies were screened and named S201 to S210. The L-serine yields after fermentation are shown in Table 1.

[0030] Embodiment 3:

[0031] No L-serine was added to the screening medium and the fermentation medium. The remaining components and operations were the same as in Example 1. A total of 10 single colonies were screened and named S301 to S310. The L-serine yields after fermentation are shown in Table 1.

[0032] The results of L-serine of each strain in Examples 1-3 are shown in Table 1:

[0033] Table 1 L-serine fermentation yield of each strain in Examples 1-3

[0034]

[0035]

[0036] From the above results, it can be seen that the strains S105, S107, S109, S110, S207, and S209 have higher yields.

[0037] Example 4: Stability test

[0038] The genetic stability experiment of strains S105, S107, S109, S110, S207 and S209 showed that the yield of strains S105, S109 and S207 remained unchanged after 50 generations, which proved that they can be used for industrial production.

[0039] Result analysis:

[0040] The following conclusions were drawn from the analysis of the L-serine yield of each strain in Examples 1-3: L-serine and ectoine can significantly affect the screening of high-yield strains and the yield of L-serine, and the lack of any one of them will affect the reduction of the final amount of L-serine synthesized, especially ectoine.

[0041] The method for producing L-serine by microbial fermentation provided by the present invention solves many problems in traditional production methods, can provide mainstream technology for the future industrial production of L-serine, and make important contributions to promoting green development and technological innovation in related industries.

[0042] 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 screening and producing L-serine, characterized in that: The following steps are involved: a. The recombinant L-serine production Escherichia coli was gradiently diluted with sterile water to obtain a dilution; 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, 2g / L L-serine, 1mmol / L ectoine, 50mg / L streptomycin sulfate, and the rest is deionized water, the pH value is natural; and culture at 37°C for 10 to 18h; 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 with a formula of 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, 2 g / L L-serine, and the rest is deionized water with a natural pH value; cultured at 37°C for 10 to 16 hours; d. A portion of the colony obtained by culture in step c was used for preservation, and a portion was inoculated into a 50 ml liquid culture medium shake flask, the liquid culture medium formula was 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, the pH value was natural; the culture was kept constant at 37 ° C and 220 ~ 260 rpm until the bacterial solution OD600 value was 1.8 ~ 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. The culture was cultured at 37°C and 220 rpm until the OD600 value of the bacterial solution was 2.

5. The inducer L-arabinose and 1 mmol / L ectoine were added at a final concentration of 2 g / 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.

2. The method for producing L-serine by strain screening and production as claimed in claim 1, characterized in that: The gradient dilution in step a is from 10 -1 to 10 -8 .

3. The method for producing L-serine by strain screening and production as claimed in claim 1, characterized in that: In step b, the mixture is cultured at a constant temperature of 37° C. for 18 hours.

4. The method for producing L-serine by strain screening and production as claimed in claim 1, characterized in that: In the step c, the colonies are white in morphology, convex in colonies, and have a colony diameter of 2 to 4 mm; and the colonies are cultured at a constant temperature of 37° C. for 16 hours.

5. The method for screening and producing L-serine as claimed in claim 1, characterized in that: 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 screening and producing L-serine as claimed in claim 1, characterized in that: In step e, the fermentation medium comprises 2 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, and pH 7.0.

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