A Corynebacterium glutamicum with high L-serine productivity and a method for improving L-serine production

Abstract

The present invention relates to a Corynebacterium glutamicum strain with high L-serine productivity and a method for improving L-serine production. This strain is named Corynebacterium glutamicum A36-pDser, and was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 11, 2018. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 15172. The yield of L-serine produced by the Corynebacterium glutamicum obtained in the present invention reaches 33.7 g / L, which is 10.86% higher than that of the starting strain. Moreover, after adding α-ketoglutaric acid, the biomass changes little, and the L-serine yield is increased by 36.2%, greatly improving the efficiency of producing L-serine using Corynebacterium glutamicum.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a Corynebacterium glutamicum with high L-serine yield and a method for improving L-serine production. Background Art

[0002] L-serine is currently one of the thirty most promising skeletal compounds in the chemical and materials fields, and has wide applications in the fields of cosmetics, food, and medicine. However, there has always been a lack of relatively economical and effective industrial production methods. More than 80% of L-serine in China is extracted by acid hydrolysis of hair. Some international companies use enzymatic semi-synthesis technology, but there are still problems such as low conversion rate and expensive precursor glycine, resulting in high prices of L-serine. Compared with enzymatic and acid hydrolysis methods, the production of L-serine by microbial fermentation has advantages such as less pollution and low cost, but its development has been relatively lagging. Realizing the production of L-serine by microbial fermentation is of great significance for improving the variety of amino acids in China. However, due to the limitation of strains, this production method has not achieved further breakthroughs, which also makes L-serine a bottleneck amino acid.

[0003] Corynebacterium glutamicum is an important amino acid-producing strain, which is widely used in the fermentation production of glutamic acid, lysine, valine, etc. In recent years, the main research on the production of L-serine by microbial fermentation at home and abroad includes: Tian Dandan et al. reported that the methylotrophic strain Pseudomonas WGP35 produced 25 g / L of L-serine using methanol; Yang Hui et al. reported that Brevibacterium flavum C-11 produced 22.65 g / L of L-serine using sugar; Chinese Patent CN201510570110.6 discloses a method for promoting the growth of Corynebacterium glutamicum and producing L-serine. Protocatechuic acid was added during the cultivation of Corynebacterium glutamicum SYPS-062-33aΔSSA, and the final yield reached 19.7511; Chinese Patent CN201310389798.9 discloses a method for constructing a Corynebacterium glutamicum SYPS-062 resistant to L-serine feedback inhibition, which was obtained by genetically modifying the 3-phosphoglycerate dehydrogenase-encoding gene serA at the genomic level of Corynebacterium glutamicum SYPS-062. In shake flask fermentation, the yield of the recombinant strain was 21 - 26 g / L, which was nearly 2.4 times higher than that of the original strain; Chinese Patent CN202011001880.6 discloses a Corynebacterium glutamicum containing an aminodeoxychorismate synthase mutant, and the yield of L-serine in fermentation production reached 30.4 g / L. However, this cannot meet the actual production of L-serine, and the yield of L-serine still needs to be further improved. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a Corynebacterium glutamicum with high yield of L-serine through laboratory adaptive evolution. The yield reaches 33.7 g / L after fermentation. Subsequently, in order to further improve the yield of L-serine, α-ketoglutaric acid was added to the synthetic medium of Corynebacterium glutamicum A36-pDser for shake-flask fermentation culture. It was found that after adding 12 mM α-ketoglutaric acid, the biomass of Corynebacterium glutamicum A36-pDser changed little, and the yield of L-serine increased by 36.2%, improving the efficiency of producing L-serine using Corynebacterium glutamicum. Whether α-ketoglutaric acid was added had no significant effect on the starting strain A36.

[0005] The first object of the present invention is to provide a Corynebacterium glutamicum with high yield of L-serine, named Corynebacterium glutamicum A36-pDser, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 11, 2018. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 15172.

[0006] The second object of the present invention is to provide the application of the above-mentioned Corynebacterium glutamicum with high yield of L-serine in the production of L-serine.

[0007] Furthermore, fermentation production is carried out using sucrose as a substrate.

[0008] Furthermore, the fermentation production is to inoculate the Corynebacterium glutamicum with high yield of L-serine into a seed medium for cultivation to obtain a seed solution, and then transfer the seed solution to a fermentation medium for cultivation.

[0009] Furthermore, by mass percentage, the composition of the seed medium is: sucrose 1-10%, brain heart infusion 1-10%, ammonium sulfate 1-5%, magnesium sulfate 0.01-0.1%, dihydrogen phosphate 0.01-0.1%, hydrogen phosphate 0.01-0.1%.

[0010] Furthermore, by mass percentage, the composition of the fermentation medium is: sucrose 5-15%, ammonium sulfate 1-5%, calcium carbonate 1-5%, magnesium sulfate 0.01-0.1%, ferric sulfate 0.001-0.01%, manganese sulfate 0.001-0.01%, hydrogen phosphate 0.1-0.5%, biotin 50-70 μg / L, vitamin B1 450-550 μg / L.

[0011] The third object of the present invention is to provide a method for improving the production of L-serine by Corynebacterium glutamicum, including the step of adding α-ketoglutaric acid to the Corynebacterium glutamicum medium.

[0012] Furthermore, the Corynebacterium glutamicum is the above-mentioned Corynebacterium glutamicum with high yield of L-serine.

[0013] Furthermore, the concentration of α-ketoglutaric acid in the culture medium is 6-15 mM, and the most preferred is 12 mM.

[0014] By the above solution, the present invention has at least the following advantages:

[0015] (1) Using A36 as the starting strain, a Corynebacterium glutamicum A36-pDser capable of directly fermenting sucrose to produce L-serine was obtained through laboratory adaptive evolution. Through shake-flask fermentation, the yield of L-serine reached 33.7 g / L.

[0016] (2) The present invention also provides a method for adding α-ketoglutaric acid (α-KG) to improve the production of L-serine by Corynebacterium glutamicum. The results show that when an appropriate amount of α-ketoglutaric acid is added, the highest yield of L-serine reaches 45.90 g / L, which is 36.2% higher than that before addition, greatly improving the efficiency of producing L-serine using Corynebacterium glutamicum A36-pDser.

[0017] The above description is only an overview of the technical solution of the present invention. In order to be able to understand the technical means of the present invention more clearly and implement it according to the content of the specification, the following describes the preferred embodiments of the present invention as follows.

[0018] Biological material preservation

[0019] Corynebacterium glutamicum A36-pDser. The Corynebacterium glutamicum A36-pDser was deposited with the China General Microbiological Culture Collection Center on January 11, 2018, with the deposit number CGMCC No. 15172 and the deposit address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Detailed implementation manners

[0020] The present invention will be further described below in conjunction with specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments cited do not limit the present invention.

[0021] Example 1 Obtaining of Corynebacterium glutamicum A36-pDser

[0022] Using Corynebacterium glutamicum A36 (accession number: CGMCC No. 15171) as the starting strain, the strain was subjected to laboratory adaptive evolution (ALE) by adding different concentrations of sucrose, and then strain A36-pDser was obtained through fluorescence screening and shake flask screening.

[0023] Growth and L-serine production ability of Corynebacterium glutamicum A36-pDser without adding α-ketoglutaric acid in Example 2

[0024] 1. Strains: Fermentative production of L-serine was carried out using Corynebacterium glutamicum A36-pDser and the starting strain A36.

[0025] 2. Seed medium: Sucrose 2%; BHI 3%; Ammonium sulfate 1.5%; MgSO4·7H2O 0.06%; NaH2PO4 0.02%; K2HPO4 0.02%;

[0026] Fermentation medium: Sucrose 10%; (NH4)2SO4 2.0%; CaCO3 2.0%; K2HPO4 0.3%; MgSO4·7H2O 0.05%; FeSO4·7H2O 0.002%; MnSO4·H2O 0.002%; Biotin 60 μg / L; Thiamine·HCl 500 μg / L; pH 7.0;

[0027] 3. Fermentation culture of Corynebacterium glutamicum: Use an inoculation loop to streak the strain on a solid plate of the seed medium, place the plate in an incubator at 30 °C until single colonies grow, inoculate into 20 mL of the seed medium, culture at 30 °C and 120 rpm until the mid-logarithmic growth phase, and then inoculate into 25 mL of the fermentation medium at an inoculation amount of 5% and carry out fermentation culture at 30 °C and 120 rpm. Each group was repeated 3 times.

[0028] 4. Detection of biomass: Samples were taken every 12 h, and the biomass (OD 562 ) was measured using a spectrophotometer.

[0029] 5. Detection of amino acid concentration: The amino acid concentration in the fermentation broth was determined by HPLC method.

[0030] The experimental results are shown in Table 1. When α-ketoglutaric acid was not added, the growth OD 562 of Corynebacterium glutamicum A36-pDser was 57.4, the L-serine yield was 33.7 g / L, and the yield of the starting strain A36 was 30.4 g / L (fermentation for 120 h).

[0031] Table 1 Growth and acid production ability of Corynebacterium glutamicum A36-pDser without adding α-ketoglutaric acid

[0032]

[0033] Example 3 Effects of Different Concentrations of α-Ketoglutaric Acid on the Growth of Corynebacterium glutamicum and the Yield of L-Serine

[0034] 1. The strain, culture medium, and seed medium were the same as those in Example 1. The concentrations of α-ketoglutaric acid added to the fermentation medium were 3 mM, 6 mM, 9 mM, 12 mM, and 15 mM;

[0035] 2. Fermentation culture of Corynebacterium glutamicum, detection of biomass, and detection of amino acid concentration: Refer to Example 1;

[0036] The relevant experimental results of the effects of adding different concentrations of α-ketoglutaric acid on the growth of Corynebacterium glutamicum A36-pDser and the yield of L-serine are shown in Table 2-6. The results show that the effect is best when 12 mM α-ketoglutaric acid is added. The growth OD of Corynebacterium glutamicum A36-pDser 562 reached 57.78, showing little change compared with that without adding α-ketoglutaric acid; the yield of L-serine reached 45.90 g / L, an increase of 36.2% compared with that before adding. Thus, adding α-ketoglutaric acid during the fermentation process of A36-pDser can increase the yield of L-serine. Whether to add α-ketoglutaric acid has no obvious effect on the original strain A36.

[0037] Table 2 Effects of 3 mM α-Ketoglutaric Acid on the Growth and Acid Production of Corynebacterium glutamicum A36-pDser

[0038]

[0039] Table 3 Effects of 6 mM α-Ketoglutaric Acid on the Growth and Acid Production of Corynebacterium glutamicum A36-pDser

[0040]

[0041] Table 4 Effects of 9 mM α-Ketoglutaric Acid on the Growth and Acid Production of Corynebacterium glutamicum A36-pDser

[0042]

[0043] Table 5 Effects of 12 mM α-Ketoglutaric Acid on the Growth and Acid Production of Corynebacterium glutamicum A36-pDser

[0044]

[0045] Table 6 Effects of 15 mM α-Ketoglutaric Acid on the Growth and Acid Production of Corynebacterium glutamicum A36-pDser

[0046]

[0047] Obviously, the above embodiments are merely examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or alterations derived therefrom still fall within the protection scope of the present invention.

Claims

1. A method for producing L-serine, characterized in that: Inoculate Corynebacterium glutamicum into a seed medium for cultivation to obtain a seed solution, then transfer the seed solution to a fermentation medium for cultivation, and add α-ketoglutaric acid to the fermentation medium. The concentration of α-ketoglutaric acid in the fermentation medium is 6-15 mM. The Corynebacterium glutamicum is named Corynebacterium glutamicum ( Corynebacterium glutamicum ) A36-pDser, and the deposit number is CGMCC No. 15172.

2. The method according to claim 1, wherein: Perform fermentation production using sucrose as a substrate.

3. The method according to claim 1, characterized in that: By mass percentage, the composition of the seed medium is: sucrose 1-10%, brain heart infusion 1-10%, ammonium sulfate 1-5%, magnesium sulfate 0.01-0.1%, dihydrogen phosphate 0.01-0.1%, hydrogen phosphate 0.01-0.1%.

4. The method according to claim 1, wherein: By mass percentage, the composition of the fermentation medium is: sucrose 5-15%, ammonium sulfate 1-5%, calcium carbonate 1-5%, magnesium sulfate 0.01-0.1%, ferric sulfate 0.001-0.01%, manganese sulfate 0.001-0.01%, hydrogen phosphate 0.1-0.5%, biotin 50-70 μg / L, vitamin B1 450-550 μg / L.

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