Mutagenesis breeding and application of serine high-yield strain

Abstract

According to the invention, corynebacterium glutamicum for producing serine is modified and mutagenized by combining metabolic engineering with normal-pressure and room-temperature plasma (ARTP) mutagenesis technology to breed a mutant strain with high yield of L-serine. When the mutant strain is used for producing serine, the yield, the conversion rate and the production efficiency of the serine can be greatly improved, and the production cost is effectively reduced. The invention further provides an ARTP-based mutagenesis and breeding technology, the performance of serine production strains is effectively improved and optimized, and reference and guidance are provided for efficient screening of other production strains.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a mutagenesis and screening technology for a serine high-yield strain. Background Art

[0002] L-serine is a non-essential amino acid and is widely used in the food, feed, medicine, agriculture and cosmetics industries as an important biological reagent and pharmaceutical agent. L- The main production methods of serine include protein hydrolysis, chemical synthesis, enzyme conversion and microbial fermentation. Among them, microbial fermentation has gradually become the most promising production method due to its advantages of low pollution, high safety and low cost.

[0003] Corynebacterium glutamicum ( Corynebacterium glutamicum ) is a model industrial microorganism with food grade safety (Generally Recognized As Safe, GRAS) and can be used to produce a variety of amino acids including L-serine. At present, the biosynthesis and metabolic pathways directly related to L-serine in Corynebacterium glutamicum have been basically resolved (see Appendix Figure 1 ), glucose undergoes glycolysis to generate the important substrate 3-phosphoglycerate, which is then degraded by 3-phosphoglycerate dehydrogenase (PGDH, serA encoding), phosphoserine aminotransferase (PSAT, serC encoding), phosphoserine phosphatase (PSP, serB In addition, there are two main degradation pathways for L-serine after synthesis. On the one hand, it will be degraded by L-serine dehydratase (SerDH, sA encodes) to pyruvate and on the other hand is degraded by serine hydroxymethyltransferase (SHMT, glyA Encoding) to produce glycine by removing the hydroxymethyl group.

[0004] Although the metabolic pathway of L-serine has gradually become clear, there is still a lack of in-depth understanding of the complex metabolic network between L-serine anabolism and the metabolism of other substances. L-serine is not only a basic amino acid for protein synthesis, but also participates in the synthesis of many other substances as an intermediate metabolite, which makes it difficult to accumulate in large quantities in microorganisms. In addition, due to the existence of feedback inhibition in the metabolic regulation of microorganisms themselves, high concentrations of L-serine will have physiological toxicity to cells, which also makes it difficult to further increase the production of L-serine. Therefore, it is necessary to break through the limitations of the traditional idea of ​​directly transforming the metabolic pathway of L-serine and adopt a blind box breeding method of mutagenesis screening in order to increase the production of L-serine by microbial fermentation.

[0005] Compared with other more traditional mutagenesis techniques (such as ultraviolet mutagenesis and nitrosoguanidine mutagenesis), atmospheric and room temperature plasma (ARTP) mutagenesis breeding technology is a new type of physical mutagenesis method. The plasma used is an ionized gas with roughly equal positive ion and electron densities. It contains a rich variety of active particles that can directly or indirectly act on the genetic material of cells, thereby causing diverse mutations and forming a large-capacity mutation library. This method has the advantages of flexible operation, simple equipment, mild conditions, high safety, diverse mutant types, and fast mutagenesis speed. It has broad application prospects in microbial mutagenesis breeding technology.

[0006] CN 117305152A uses wild-type Corynebacterium glutamicum as a basic strain for ARTP mutagenesis, and the maximum L-serine yield in the fermentation broth of the mutated strain can reach 57.5 g / L. In order to increase the yield, a separation coupling technology of adding a cation exchange resin during the fermentation process is further adopted to release the feedback inhibition of 3-phosphoglycerate dehydrogenase and avoid the conversion of L-serine to glycine by serine hydroxymethyltransferase (SHMT), thereby obtaining a higher yield level. However, since the process needs to add a cation exchange resin in the fermentation system, there are technical problems such as complicated operation steps and high cost, and it is difficult to mass-produce.

[0007] In addition, the existing serine fermentation production process generally has the problem of low sugar-acid conversion rate, which is only about 0.3g / g at most, which leads to high cost of serine production. Therefore, breeding serine production strains with high yield and high sugar-acid conversion rate has become an inevitable requirement for serine fermentation production.

[0008] The multiple breeding methods based on metabolic engineering combined with mutagenesis screening in this application are more effective methods for screening strains with excellent acid production performance. Summary of the invention

[0009] One of the purposes of the present invention is to obtain a mutant strain with high serine production based on metabolic engineering combined with mutagenesis breeding methods.

[0010] To achieve the above object, the present invention selects Corynebacterium glutamicum ( Corynebacterium glutamicum ) ATCC13032 is used as the chassis strain. Based on metabolic engineering, its metabolic pathway is transformed through conventional genetic engineering techniques to enhance the synthesis pathway of L-serine, block the degradation of L-serine to reduce the accumulation of by-products, and enhance the excretion of L-serine. The basic strain for L-serine production is constructed, and then the basic strain is induced by ARTP, and selected by combining analog screening methods to obtain a high-yield strain of L-serine. Fermentation optimization of this strain can further increase the strain's L-serine production.

[0011] The invention provides a Corynebacterium glutamicum strain with high L-serine production, and provides a method for inducing mutation and breeding the strain with high L-serine production.

[0012] The present invention provides a Corynebacterium glutamicum ( Corynebacterium glutamicum The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on July 1, 2024 (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), and was classified and named as: Corynebacterium glutamicum ( Corynebacterium glutamicum ), the deposit number is CGMCC No. 31128.

[0013] The invention also provides a mutagenesis and screening technology for a high-yield serine strain.

[0014] The mutagenesis technique is atmospheric and room temperature plasma (ARTP) mutagenesis.

[0015] The screening technique is analog screening and the analog is D-Serine.

[0016] The present invention also provides application of a high-yield serine strain in serine production.

[0017] The present invention also provides a method for producing serine, wherein the method uses a high-yield serine strain.

[0018] The method comprises: culturing the Corynebacterium glutamicum and inoculating it into a fermentation tank for fermentation culture.

[0019] The invention also provides a fermentation optimization process.

[0020] The invention also provides a fermentation medium for producing L-serine by fermentation.

[0021] The composition of the fermentation medium includes (L): glucose 10-50 g, (NH4)2SO4 10-50 g, KH2PO4 0.2-1.0 g, K2HPO4 0.2-1.0 g, MgSO4·7H2O 0.2-1.0 g, CaCl2 5-20 mg, FeSO4·7H2O 10-50 mg, MnSO4·H2O 10-50 mg, ZnSO4·7H2O 0.5-2 mg, CuSO4 0.1-0.5 mg, NiCl2·6H2O 0.01-0.05 mg, biotin 10-50 μg, vitamin B1 0.5-2 mg, vitamin B6 2-10 mg, pH 7.0-7.5.

[0022] The composition of the optimized fermentation medium includes (L): glucose 10-50 g, (NH4)2SO410-50 g, KH2PO41-5g, K2HPO41-5 g, MgSO4·7H2O 0.2-1 g, CaCl2 5-20 mg, FeSO4·7H2O 10-50 mg, MnSO4·H2O10-50 mg, ZnSO4·7H2O 0.5-2 mg, CuSO40.1-1.0 mg, NiCl2·6H2O 0.01-0.05 mg, choline chloride 0.05-0.2 g, betaine 0.2-1.0 g, biotin 50-200 μg, vitamin B1 0.5-2.0 mg, vitamin B6 2-10mg, pH 7.0-7.5.

[0023] The fermentation culture conditions are as follows: the temperature is controlled at 25-35°C, ammonia water is automatically added to stabilize the pH at 7.0-7.5 in the first 30-50 hours of fermentation, the dissolved oxygen is maintained at 20-50%, and when the bacterial growth state remains basically unchanged, the pH is adjusted to 6.5-7.0 and the dissolved oxygen is reduced to 5-15%.

[0024] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: In order to increase the serine yield, the present invention uses atmospheric pressure room temperature plasma (ARTP) to induce mutation on the basic strain, and screens through analogs to obtain the Corynebacterium glutamicum strain GSLW-001 with L-serine yield increased by more than 2 times. After fermentation for 72 h in a 7.5 L fermenter system, the L-serine yield of GSLW-001 reached 78.37 g / L, the sugar-acid conversion rate was 0.438 g / g, and the production intensity was 1.09 g / L / h. By optimizing the fermentation conditions, the L-serine yield was further improved. After fermentation for 72 h in a 7.5 L fermenter system, the L-serine yield could reach 104.12 g / L, the sugar-acid conversion rate was 0.466 g / g, and the production intensity was 1.44 g / L / h. Using this mutant strain to produce serine can greatly improve the yield, conversion rate and production efficiency of serine, and effectively reduce production costs. The present application also provides an effective process and fermentation optimization process for mutagenesis and breeding of serine high-yielding strains based on metabolic engineering, which can further optimize strain performance and provide guidance for the efficient screening of other amino acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 L-serine metabolic pathway in Corynebacterium glutamicum Figure 2 Results of L-serine production in SER-0 shake flask fermentation Figure 3 Lethality curve of SER-0 treated with different ARTP mutagenesis time Figure 4 Results of L-serine production in the initial well plate screening of ARTP-induced strains Figure 5 Results of L-serine production by shake flask screening of ARTP mutant strains Figure 6 Genetic stability of GSLW-001 Figure 7 Results of L-serine production in GSLW-001 fermenter Figure 8 GSLW-001 fermenter L-serine / glucose conversion results DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with specific examples. Where specific experimental conditions are not indicated, they are all based on conventional conditions well known to those skilled in the art.

[0027] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), etc., or according to the conditions recommended by the manufacturer's instructions.

[0028] Example 1: Construction of basic strains using metabolic engineering According to reference 1 (Zhu Q, and, et al. L-Serine overproduction withminimization of by-product synthesis by engineered Corynebacterium glutamicum . Appl Microbiol Biotechnol. 2015, 99(4):1665-73. doi: 10.1007 / s00253-014-6243-0.) aLaT Δ aV ΔC-T ilvN ) was constructed by using Corynebacterium glutamicum ATCC13032 instead of the strain SYPS-062-33a in the literature as the starting strain, and knocking out serA The C-terminal 591 bp of sdaA、alaT、avtA The gene blocks the degradation of serine and reduces the accumulation of byproducts pyruvate and L-alanine. In order to further reduce the content of the byproduct L-valine, the activity of acetohydroxyacid synthase (AHAS) is directly reduced and the ilvN The C-terminal 249 bp were obtained to obtain S1.

[0029] According to reference 2 (Zhang X, and, et al. Integration of ARTP mutagenesis with biosensor-mediated high-throughput screening to improve L-serine yield Corynebacterium glutamicum . Appl Microbiol Biotechnol. 2018, 102(14):5939-5951. doi: 10.1007 / s00253-018-9025-2.) was used to construct the engineered strain (ΔSSAAI-pDser(pabAB426)) described in the literature, with S1 replacing the strain ΔSSAAI-pDser in the literature as the starting strain. pabAB The threonine at position 426 encoded by the gene was mutated to isoleucine (T426I), blocking the degradation of L-serine to glycine and obtaining S1-pabAB.

[0030] According to reference 3 (Zhang X, and, et al. High-yield production of L-serinethrough a novel identified exporter combined with synthetic pathway in Corynebacterium glutamicum . Microb Cell Fact. 2020, 19(1):115. doi: 10.1186 / s12934-020-01374-5.) was used to construct an engineered strain (SSAAI-serE) described in the literature, using S1-pabAB instead of the strain SSAAI in the literature as the starting strain to overexpress the serine transporter encoding gene sE , promoting the excretion of L-serine, and finally obtaining an L-serine-producing strain, denoted as SER-0, as the basic strain for the initiation of mutagenesis.

[0031] Table 1 Strains used in the present invention

[0032] Example 2: Shake flask test of basic strains 1. Culture medium preparation LBB activation medium (L): 5 g yeast extract, 10 g peptone, 10 g brain heart infusion, 10 g NaCl, and 20 g agar powder added to the solid medium.

[0033] Shake flask seed medium (L): glucose 20 g, brain heart infusion 37 g, (NH4)2SO4 10 g, K2HPO4 0.2 g, NaH2PO4 0.3 g, MgSO4·7H2O 0.5 g, pH 7.2.

[0034] Shake flask fermentation medium (L): glucose 20 g, ammonium sulfate 20 g, urea 5 g, KH2PO4 3 g, K2HPO4 1 g, MgSO4·7H2O 0.5 g, CaCl2 10 mg, FeSO4·7H2O 10 mg, MnSO4·H2O 10 mg, ZnSO4·7H2O 1mg, CuSO4 0.2 mg, NiCl2·6H2O 0.02 mg / L, protocatechuic acid 30 mg, biotin 25 μg, vitamin B1 1 mg, vitamin B6 5 mg, MOPS 42 g, pH 7.2.

[0035] 2. Shake flask fermentation yield test Streak SER-0 glycerol frozen bacteria on LBB solid medium and culture at 30℃ overnight. Inoculate a fresh single colony into 5 mL LBB liquid medium at 30℃ and 200 rpm overnight, then transfer to 10 mL shake flask seed medium at 1% inoculation volume and culture for 16-18 h. Then control the initial inoculation OD=1 and inoculate into 20 mL shake flask fermentation medium and culture at 30℃ and 200 rpm for 48 h.

[0036] 1 mL of the fermentation broth sample was pipetted into a 1.5 mL centrifuge tube and centrifuged at 12000 rpm for 2 min. The supernatant was filtered with a 0.22 μm PES filter membrane and the serine production was detected by HPLC.

[0037] HPLC instrument and parameters: high performance liquid chromatography (Agilent LC-40D); chromatographic column: Agilent AdvanceBio AAA, C18, (4.6 × 100 mm, 2.7 μm); mobile phase A: 10 mmol / L disodium hydrogen phosphate and 10 mM sodium borate solution, pH adjusted to 8.2 with hydrochloric acid, mobile phase B: acetonitrile, methanol and water (45:45:10, v:v:v); flow rate: 1.0 mL / min, column temperature: 40 °C, detection wavelength: 338 nm, injection volume: 2 μL.

[0038] HPLC results showed that (see Figure 2 ), after 48 h of fermentation of the basic strain SER-0 before mutagenesis, OD 600 Reaching 10.56, 1.05 g / L of L-serine can be produced.

[0039] Example 3: Determination of ARTP mortality (1) Preparation of bacterial suspension: Streak SER-0 glycerol-frozen bacteria on LBB solid medium and culture at 30°C overnight. Inoculate a fresh single colony into 5 mL LBB liquid medium and culture at 30°C and 200 rpm overnight. Then transfer the inoculum to 20 mL LBB liquid medium at 1% and culture at 30°C and 200 rpm until the logarithmic growth phase. Wash with sterile saline three times and resuspend the bacterial suspension to 10% in saline containing 5% glycerol. 8 cells / mL for mutagenesis.

[0040] (2) Instrument parameter settings: working power 120 W, gas source He, gas flow rate 10 SLM, processing time gradient 0, 15, 30, 45, 60, 75, 90, 105 s.

[0041] (3) Mutagenesis treatment process: sterilize the ARTP special metal slide with high pressure steam in advance and dry and cool it, take 10 μL of bacterial solution and evenly apply it on the surface of the slide, and treat it according to the above parameter settings. The treatment time of 0 s is the control group, and the treatment time of 15, 30, 45, 60, 75, 90, and 105 s is the treatment group.

[0042] (4) Sample elution and spreading: Use sterile tweezers to place the slide in a 2 mL EP tube containing 1 mL of sterile saline. Elute by shaking for 1 min. Dilute the obtained bacterial suspension to an appropriate gradient. Pipette 200 μL and spread it on LBB solid culture medium. Incubate overnight at 30°C for 48 h and count the cells to determine the lethality.

[0043] (5) Calculation of lethality: Lethality (%) = (number of monoclonal clones in the control group - number of monoclonal clones in the treatment group) / number of monoclonal clones in the control group × 100% The results showed that (see Figure 2 ), the survival rate of SER-0 decreased with the increase of mutagenesis time. When the treatment time was 0, 15, 30, 45, 60, 75, 90, and 105 s, the lethality reached 0, 10%, 67.7%, 88.3%, 95.6%, 99.4%, 100%, and 100%, respectively. Considering that it is easier to screen effective mutations when the lethality rate is 80-90%, and in order to ensure that the strain can grow on the screening plate, the optimal mutagenesis conditions were set to 120 W, 10 SLM, and 45 s.

[0044] Example 4: ARTP mutagenesis of L-serine producing strains 1. Selection of structural analogs and determination of screening concentration Although in Example 1, the serA The C-terminus of L-serine is removed, but high concentrations of L-serine still have certain cytotoxicity to the bacteria, making it difficult for L-serine to accumulate in large quantities. D-serine, as the right-handed isomer of L-serine, has the advantages of the same molecular weight and basically the same chemical structure compared to other structural analogs of L-serine. Moreover, since D-serine does not exist in natural bacterial cells, it has unparalleled advantages as a resistance screening marker for L-serine production strains compared to other analogs. By screening strains with enhanced D-serine resistance to improve the product tolerance of the strains, strains with high L-serine production can be screened out.

[0045] The solid culture medium with different concentrations of D-serine (0, 30, 60, 90, 120, 150 g / L) was used to screen the basic strain SER-0 for resistance. With the increase of D-serine concentration, the growth of the tested strain slowed down and the colony morphology decreased. When the D-serine concentration reached 150 g / L, the strain stopped growing. Therefore, D-serine with a concentration of 120 g / L was selected for screening after mutagenesis.

[0046] Screening solid culture medium (L): glucose 20 g, (NH4)2SO4 20 g, KH2PO4 0.5 g, K2HPO4 0.5 g, MgSO4·7H20 0.25 g, FeSO4·7H2O 10 mg, MnSO4·H2O 10 mg, ZnSO4·7H2O 1 mg, CuSO40.2 mg, NiCl2·6H2O 0.02 mg, biotin 25 μg, agar powder 20 g, D-serine 0, 30, 60, 90, 120, 150 g / L.

[0047] 3. ARTP mutagenesis (1) Preparation of bacterial suspension: The basic strain SER-0 activated in LBB liquid medium was transferred to 20 mL LBB liquid medium at a 1% inoculum volume and cultured at 30°C and 200 rpm until the logarithmic growth phase. After washing with sterile saline three times, the bacterial suspension was resuspended in saline containing 5% glycerol to 10 8 cells / mL for mutagenesis.

[0048] (2) ARTP mutagenesis conditions: working power 120 W, gas flow 10 SLM, and mutagenesis time 45 s.

[0049] (3) Mutagenesis treatment process: sterilize the ARTP-specific metal slide with high-pressure steam in advance and dry and cool it. Take 10 μL of bacterial solution and evenly apply it on the surface of the slide. Set the parameters according to the mutagenesis conditions and perform the treatment.

[0050] (4) Sample elution and coating screening: Use sterile tweezers to place the slide in a 2 mL EP tube containing 1 mL of sterile saline, shake and elute for 1 min, and dilute the obtained bacterial suspension to 10 4 , 10 5 , 10 6 , respectively, spread on the screening solid culture medium with a D-serine concentration of 120 g / L and cultured at 30°C for 48 h.

[0051] Example 5: Screening of mutant strains 1. Orifice plate initial screening A single colony that grew well on the screening solid medium was picked up and placed in 5 mL of LBB liquid medium for culture for 16-18 h. Then, 10% of the initial inoculum was inoculated into 1 mL of shake flask fermentation medium and mixed evenly. 100 μL was transferred to a 96-well plate and cultured at 30°C and 800 rpm for 48 h using a microbial growth curve analyzer.

[0052] After centrifuging the bacterial solution at 12,000 rpm for 2 min, the supernatant was filtered through a 0.22 μm filter membrane and then tested for L-serine production by HPLC. The L-serine production was 2 times higher than that of the basic strain (L-serine production ≥ 2.1 g / L) as the screening standard.

[0053] The results of the initial screening of the well plate show (see Figure 4 ), a total of 96 single colonies were picked on the screening solid medium, of which 7 strains, SER-18, SER-50, SER-51, SER-53, SER-54, SER-55 and SER-56, had an L-serine production of more than 2.1 g / L, reaching twice that of the basic strain SER-0 before mutagenesis. Seven strains were screened in the initial screening, with a screening yield of 7.3%. These 7 strains were screened again by shake flask fermentation.

[0054] 2. Shake bottle rescreening Since the initial screening is carried out in a small-volume well plate, it is easily affected by various factors such as the environment, resulting in deviations in the results. In order to further ensure the accuracy of the screening results, a larger-volume, more stable shake flask fermentation is used for rescreening based on the initial screening.

[0055] The 7 mutant strains obtained in the initial screening were streaked on LBB plates and cultured at 30°C overnight. Single colonies were picked and cultured in 5 mL LBB liquid culture medium for 16-18 h. The initial inoculation OD was controlled to be 1 and inoculated into 20 mL shake flask fermentation medium. The culture was carried out at 30°C and 200 rpm for 48 h for shake flask rescreening.

[0056] The shake flask fermentation results showed (see Figure 5 ), the yields of SER-18, SER-53, and SER-55 were significantly increased, more than 2 times compared with the basic strain SER-0. Among them, SER-18 had the highest yield, reaching 2.78 g / L, which was nearly 3 times that of the serine in the basic strain. The strain was named GSLW-001.

[0057] Example 6: Stability test of mutagenized strains The screened mutant strain GSLW-001 was subcultured five times on LBB solid medium, fermented in shake flasks for 48 h, and the growth ability and acid production ability of the first and fifth generations of strains were determined.

[0058] Results Figure 6 , OD of the first generation of mutant strain GSLW-001 600 was 10.45±0.06, and the L-serine production was 2.575±0.0676 g / L. After 5 passages, OD 600 The growth rate and L-serine production of the mutant strain GSLW-001 were 10.32±0.12, and the L-serine production was 2.686±0.055 g / L, which had no significant difference from the growth and acid production of the first generation. It can be seen that the bacterial growth and L-serine production of the mutant strain GSLW-001 were very stable during the passage process, indicating that it has good genetic stability and is suitable for the industrial production of serine.

[0059] Example 7: Fermentation tank test of mutagenic bacteria To further investigate the fermentation performance of the mutagenized strain GSLW-001, a fed-batch fermentation test was carried out in a 7.5 L fermenter.

[0060] 1. Culture medium preparation: Primary seed medium (L): glucose 20 g, brain heart infusion 37 g, (NH4)2SO4 10 g, K2HPO4 0.2 g, NaH2PO4 0.3 g, MgSO4·7H2O 0.5 g, pH 7.2.

[0061] Secondary seed medium (L): glucose 20 g, (NH4)2SO4 10 g, KH2PO4 0.5 g, MgSO4·7H2O0.5 g, FeSO4·7H2O 20 mg, MnSO4·H2O 20 mg, pH 7.2.

[0062] Fermentation tank fermentation medium (L): glucose 20 g, (NH4)2SO4 30 g, KH2PO4 0.5 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, CaCl2 10 mg, FeSO4·7H2O 20 mg, MnSO4·H2O 20 mg, ZnSO4·7H2O 1 mg, CuSO4 0.2 mg, NiCl2·6H2O 0.02 mg, biotin 25 μg, vitamin B1 1 mg, vitamin B6 5 mg, pH 7.2.

[0063] 2. Fermentation culture in fermentation tank: (1) Streak the mutant strain GSLW-001 onto LBB medium and culture at 30°C overnight.

[0064] (2) Inoculate a single colony with good growth into 10 mL of primary seed culture medium and culture at 30°C and 200 rpm for 16-18 h.

[0065] (3) Take 5 mL of the first-level seed solution and inoculate it into 50 mL of the second-level seed culture medium. Culture it at 30°C and 200 rpm for 16-18 h.

[0066] (4) The secondary seed culture liquid obtained in step (3) was transferred to a 7.5 L fermenter containing 2.5 L fermenter fermentation medium. The temperature was controlled at 30°C, and ammonia water was automatically added to stabilize the pH at about 7.2. The dissolved oxygen was maintained at about 30%, and the fermentation was continued for 72 h.

[0067] The fermentation results showed (see Figure 7-8 ), strain GSLW-001 fermented in a fermenter for 72 h, the maximum L-serine yield could reach 78.37 g / L, and the maximum biomass OD 600 The production intensity was 1.09 g / L / h, and the sugar-acid conversion rate was 0.438 g / g. The production of L-serine by fermentation with this mutant strain can greatly increase the yield and conversion rate of L-serine, improve production efficiency, and effectively reduce production costs.

[0068] Table 2 Fermentation performance of GSLW-001

[0069] Example 8: Optimization of fermentation conditions In order to further improve the fermentation performance of GSLW-001, the fermentation conditions (fermentation medium, fermentation process) of its fermenter were optimized.

[0070] 1. Fermentation medium optimization Phosphorus is a component of ADP, ATP and phospholipids, and potassium ions are activators of many enzymes. Appropriate amounts of K2HPO4 and KH2PO4 are beneficial to bacterial growth and acid production. Biotin, as an important cofactor, not only affects important pathways such as glycolysis, glyoxylate cycle and nitrogen metabolism, but also affects the synthesis of cell membranes and changes the permeability of cell membranes. Therefore, the addition of an appropriate amount of biotin may promote the synthesis of L-serine.

[0071] Choline chloride and betaine are alkaloids. They not only serve as precursors in the synthesis of phospholipids and regulate cell membrane permeability, but also act as methyl donors to provide the required active methyl groups for various methylation reactions in bacterial metabolism, thereby improving bacterial vitality. Therefore, an appropriate amount of alkaloids is beneficial to bacterial growth and acid production.

[0072] The concentrations of phosphate, biotin, choline chloride and betaine in the fermentation medium were adjusted, and the optimized medium was as follows: Optimized fermentation medium (L): glucose 20 g, (NH4)2SO4 30 g, KH2PO4 3 g, K2HPO4 3 g, MgSO4·7H2O 0.5 g, CaCl2 10 mg, FeSO4·7H2O 20 mg, MnSO4·H2O 20 mg, ZnSO4·7H2O 1 mg, CuSO4 0.2 mg, NiCl2·6H2O 0.02 mg, choline chloride 0.1 g, betaine 0.5 g, biotin 100 μg, vitamin B1 1 mg, vitamin B6 5 mg, pH 7.2.

[0073] 2. Fermentation process optimization The secondary seed culture solution was obtained according to the culture method described in Example 7, and all the secondary seed solution was transferred to a 7.5 L fermentation tank filled with 2.5 L optimized fermentation medium, the temperature was controlled at 30°C, and ammonia water was automatically added to stabilize the pH at about 7.2 in the first 42 hours of fermentation, and the dissolved oxygen was maintained at about 30%. After 42 hours, the growth state of the bacteria remained basically unchanged, the pH was adjusted to about 6.8, and the dissolved oxygen was reduced to 10% for the production of L-serine.

[0074] Compared with the fermentation results of Example 7, adjusting the fermentation conditions is beneficial to increasing the yield of L-serine and the sugar-acid conversion rate. The serine yield is increased from 78.37 g / L to 104.12 g / L, and the sugar-acid conversion rate is further increased from 0.438 g / g to 0.466 g / g, and the production intensity reaches 1.44 g / L / h.

[0075] This application uses atmospheric pressure room temperature plasma (ARTP) mutagenesis and fermentation optimization to obtain a mutant strain of Corynebacterium glutamicum with a maximum L-serine yield of 104.12 g / L, a production intensity of 1.44 g / L / h, and a sugar-acid conversion rate of 0.466 g / g. It provides an effective process for mutagenesis and breeding of high-yield serine strains, and also provides a fermentation optimization process, which can further optimize strain performance in this way and provide guidance for the efficient screening of other amino acids.

[0076] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A Corynebacterium glutamicum ( Corynebacterium glutamicum ), characterized in that The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on July 1, 2024, with the deposit number CGMCC No.31128.

2. Use of Corynebacterium glutamicum as claimed in claim 1 in producing L-serine.

3. A method for producing L-serine, characterized in that: Cultivate the Corynebacterium glutamicum as claimed in claim 1, and inoculate it into a fermentation tank for fermentation culture.

4. The method for producing L-serine according to claim 3, wherein The fermentation medium used in the fermentation culture contains biotin.

5. The method for producing L-serine according to claim 4, wherein The fermentation medium also contains alkaloids.

6. The method for producing L-serine according to claim 5, wherein The alkaloids are choline chloride and betaine.

7. The method for producing L-serine according to any one of claims 3 to 4, characterized in that: Each liter of the fermentation medium contains: glucose 10-50 g, (NH4)2SO4 10-50 g, KH2PO4 0.2-1.0 g, K2HPO4 0.2-1.0 g, MgSO4·7H2O 0.2-1.0 g, CaCl2 5-20 mg, FeSO4·7H2O 10-50 mg, MnSO4·H2O 10-50 mg, ZnSO4·7H2O 0.5-2 mg, CuSO4 0.1-0.5 mg, NiCl2·6H2O 0.01-0.05 mg, biotin 10-50 μg, vitamin B1 0.5-2.0 mg, and vitamin B6 2-10 mg.

8. The method for producing L-serine according to any one of claims 5 to 6, characterized in that: Each liter of the fermentation medium contains: glucose 10-50 g, (NH4)2SO4 10-50 g, KH2PO4 1-5 g, K2HPO4 1-5 g, MgSO4·7H2O 0.2-1g, CaCl2 5-20 mg, FeSO4·7H2O 10-50 mg, MnSO4·H2O 10-50 mg, ZnSO4·7H2O 0.5-2 mg, CuSO4 0.1-0.5mg, NiCl2·6H2O 0.01-0.05 mg, choline chloride 0.05-0.2 g, betaine 0.2-1.0 g, biotin 50-200 μg, vitamin B1 0.5-2.0 mg, and vitamin B6 2-10 mg.

9. The method for producing L-serine according to claim 8, wherein After inoculation into the fermentation tank, the temperature is controlled at 25-35℃, and ammonia water is automatically added to stabilize the pH at 7.0-7.5 in the first 30-50 hours of fermentation, and the dissolved oxygen is maintained at 20-50%. When the growth state of the bacteria remains basically unchanged, the pH is adjusted to 6.5-7.0 and the dissolved oxygen is reduced to 5-15%.

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