Mutation breeding and application of high-serine-producing strains

Abstract

This invention utilizes metabolic engineering combined with atmospheric pressure room temperature plasma (ARTP) mutagenesis to transform Corynebacterium glutamicum, a strain used for serine production, and to generate a high-L-serine-producing mutant strain. Using this mutant strain to produce serine significantly improves serine yield, conversion rate, and production efficiency, effectively reducing production costs. This invention also provides an ARTP-based mutagenesis and breeding technique that effectively improves and optimizes the performance of serine-producing strains, providing a reference and guidance for the 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 high-serine-producing 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. L- The main methods for producing serine include protein hydrolysis, chemical synthesis, enzymatic conversion, and microbial fermentation. Among them, microbial fermentation has gradually become the most promising production method due to its advantages such as low pollution, high safety, and low cost.

[0003] Corynebacterium glutamicum ( Corynebacterium glutamicum ) is a food-grade safe (Generally Recognized As Safe, GRAS) model industrial microorganism that can be used to produce a variety of amino acids including L-serine. Currently, the biosynthesis and metabolic pathways directly related to L-serine in Corynebacterium glutamicum have been basically elucidated (see Appendix Figure 1 ), glucose is converted into 3-phosphoglycerate through glycolysis, which is then degraded by 3-phosphoglycerate dehydrogenase (PGDH, serA encoding), phosphoserine aminotransferase (PSAT, serC encoding), phosphoserine phosphatase (PSP, serB L-serine is catalyzed by L-serine dehydratase (SerDH). sdA ) to pyruvate, and on the other hand is degraded by serine hydroxymethyltransferase (SHMT, glyA encoding) to remove the hydroxymethyl group to produce glycine.

[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 for it 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 produce 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 traditional thinking of directly modifying the L-serine metabolic pathway and adopt a blind box breeding method of mutagenesis screening to increase the production of L-serine by microbial fermentation.

[0005] Compared to other more traditional mutagenesis techniques (such as UV mutagenesis and nitrosoguanidine mutagenesis), atmospheric and room temperature plasma (ARTP) mutagenesis is a novel physical mutagenesis method. The plasma employed is an ionized gas with roughly equal densities of positive ions and electrons. It contains a rich variety of reactive species that can directly or indirectly interact with the genetic material of cells, inducing diverse mutations and generating large-capacity mutant libraries. This method offers advantages such as flexible operation, simple equipment, mild conditions, high safety, diverse mutant types, and rapid mutagenesis, and holds broad application prospects in microbial mutagenesis breeding.

[0006] CN 117305152A uses a wild-type Corynebacterium glutamicum as a base strain for ARTP mutagenesis. The maximum L-serine yield in the fermentation broth of the mutated strain can reach 57.5 g / L. To increase yield, a separation coupling technique is further employed, in which a cation exchange resin is added during the fermentation process. This relieves feedback inhibition of 3-phosphoglycerate dehydrogenase and prevents the conversion of L-serine to glycine by serine hydroxymethyltransferase (SHMT), thereby achieving a higher yield level. However, this process requires the addition of a cation exchange resin to the fermentation system, resulting in technical issues such as complicated operational steps and high costs, making it difficult to implement on a large scale.

[0007] Furthermore, existing serine fermentation production processes generally suffer from low sugar-to-acid conversion rates, with the highest being only around 0.3g / g. This results in high serine production costs. Therefore, breeding serine-producing strains with high yields and high sugar-to-acid conversion rates is essential 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.

[0010] To achieve the above object, the present invention selects Corynebacterium glutamicum ( Corynebacterium glutamicum Using ATCC13032 as the base strain, conventional genetic engineering techniques were used to modify its metabolic pathways based on metabolic engineering. This enhanced the L-serine synthesis pathway, blocked L-serine degradation to reduce byproduct accumulation, and enhanced L-serine excretion, resulting in a basic strain for L-serine production. This basic strain was then subjected to ARTP mutagenesis and selected using analog screening methods to obtain a high-L-serine-producing strain. Fermentation optimization of this strain can further increase L-serine production.

[0011] The invention provides a Corynebacterium glutamicum strain with high L-serine production, and provides a method for inducing 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 Culture Collection Administration Committee on July 1, 2024 (address: No. 3, Yard 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 present 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-serine-yielding strain in serine production.

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

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

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

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

[0021] The fermentation medium comprises (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, and pH 7.0-7.5.

[0022] The composition of the optimized fermentation medium includes (L): glucose 10-50 g, (NH4)2SO410-50 g, KH2PO41-5 g, 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 2-10 mg, 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, and the dissolved oxygen is maintained at 20-50%. 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:

[0025] To increase serine production, the present invention used atmospheric pressure room temperature plasma (ARTP) mutagenesis on a base strain and screened with analogs to obtain the Corynebacterium glutamicum strain GSLW-001, which exhibits more than a two-fold increase in L-serine production. After 72 hours of fermentation in a 7.5-L fermentor, GSLW-001 achieved an L-serine yield of 78.37 g / L, a sugar-acid conversion rate of 0.438 g / g, and a production rate of 1.09 g / L / h. By optimizing fermentation conditions, L-serine production was further increased, reaching 104.12 g / L, a sugar-acid conversion rate of 0.466 g / g, and a production rate of 1.44 g / L / h in a 7.5-L fermentor for 72 hours. Using this mutant strain to produce serine significantly improves serine yield, conversion rate, and production efficiency, effectively reducing production costs. This application also provides an effective process and fermentation optimization process for mutagenesis and breeding of high-yielding serine 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

[0026] Figure 1 L-serine metabolic pathway in Corynebacterium glutamicum

[0027] Figure 2Results of L-serine production in shake flask fermentation of SER-0

[0028] Figure 3 Lethality curves of SER-0 cells treated with different ARTP mutagenesis times

[0029] Figure 4 Results of L-serine production in the initial well plate screening of ARTP-induced strains

[0030] Figure 5 Results of L-serine production by shake flask screening of ARTP-induced strains

[0031] Figure 6 Genetic stability of GSLW-001

[0032] Figure 7 L-serine production results of GSLW-001 fermenter

[0033] Figure 8 Results of L-serine / glucose conversion rate in GSLW-001 fermenter DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below with reference to specific examples. Where specific experimental conditions are not specified, they are based on conventional conditions well known to those skilled in the art.

[0035] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples were performed according to conventional experimental conditions, such as those described in Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or according to the conditions recommended in the manufacturer's instructions.

[0036] Example 1: Construction of basic strains using metabolic engineering

[0037] 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 avtA ΔC-T ilvN) was constructed by replacing the strain SYPS-062-33a in the literature with Corynebacterium glutamicum ATCC13032 as the starting strain, and knocking out serA The C-terminal 591 bp of the 3-phosphoglycerate dehydrogenase (PGDH) was removed to relieve the feedback inhibition of L-serine on 3-phosphoglycerate dehydrogenase (PGDH) and enhance L-serine synthesis. sdaA、alaT、avtA The gene blocks serine degradation 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 removed to obtain S1.

[0038] According to reference 2 (Zhang X, and, et al. Integration of ARTP mutagenesis with biosensor-mediated high-throughput screening to improve L-serine yieldin 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.

[0039] 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 as the starting strain to overexpress the serine transporter encoding gene. serE , promoting L-serine excretion, and finally obtaining an L-serine-producing strain, recorded as SER-0, as the basic strain for mutagenesis initiation.

[0040] Table 1 Strains used in the present invention

[0041]

[0042] Example 2: Shake flask test of basic strains

[0043] 1. Culture medium preparation

[0044] LBB activation medium (L): 5 g yeast extract, 10 g peptone, 10 g brain heart infusion, 10 g NaCl, and 20 g agar powder for solid medium.

[0045] 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.

[0046] 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 1 mg, 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.

[0047] 2. Shake flask fermentation yield test

[0048] Streak SER-0 glycerol-frozen bacteria onto LBB solid medium and culture overnight at 30°C. Inoculate a fresh single colony into 5 mL of LBB liquid medium at 30°C and 200 rpm overnight. Transfer a 1% inoculum to a 10 mL shake flask seed medium and culture for 16-18 hours. Then, control the initial inoculum OD to 1 and inoculate into a 20 mL shake flask fermentation medium. Culture at 30°C and 200 rpm for 48 hours.

[0049] 1 mL of the fermentation broth sample was pipetted into a 1.5 mL centrifuge tube and centrifuged at 12,000 rpm for 2 min. The supernatant was filtered through a 0.22 μm PES filter membrane and the serine production was detected by HPLC.

[0050] HPLC instrumentation 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.

[0051] 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, it can produce 1.05 g / L of L-serine.

[0052] Example 3: Determination of ARTP mortality

[0053] (1) Preparation of bacterial suspension: Streak the 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 and culture at 30℃ and 200 rpm overnight. Then transfer the inoculum to 20 mL LBB liquid medium at 1% inoculum and culture at 30℃ and 200 rpm until the logarithmic growth phase. Wash with sterile saline three times and resuspend the bacterial suspension to 10 8 cells / mL for mutagenesis.

[0054] (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.

[0055] (3) Mutagenesis treatment process: Sterilize the ARTP-specific metal slide with high-pressure steam in advance and dry and cool. Take 10 μL of bacterial solution and evenly spread it on the surface of the slide. Treat according to the above parameters. 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.

[0056] (4) Sample elution and coating: 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, dilute the obtained bacterial suspension to a suitable gradient, draw 200 μL and coat it on LBB solid culture medium, culture overnight at 30°C for 48 h and count to determine the lethality.

[0057] (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%

[0058] The results showed that (see Figure 2 SER-0 survival decreased with increasing mutagenesis time. At treatment times of 0, 15, 30, 45, 60, 75, 90, and 105 s, lethality rates reached 0, 10%, 67.7%, 88.3%, 95.6%, 99.4%, 100%, and 100%, respectively. Given that a lethality rate of 80-90% makes it easier to screen for effective mutations and to ensure growth on the screening plate, the optimal mutagenesis conditions were set at 120 W, 10 SLM, and 45 s.

[0059] Example 4: ARTP mutagenesis of L-serine producing strains

[0060] 1. Selection of structural analogs and determination of screening concentration

[0061] Although the knockout serA The C-terminus of L-serine is activated, relieving the feedback inhibition of L-serine. However, high concentrations of L-serine still have a certain degree of cytotoxicity to bacteria, making it difficult to accumulate L-serine in large quantities. As the dextrorotatory isomer of L-serine, D-serine has advantages over other structural analogs of L-serine, such as having the same molecular weight and essentially the same chemical structure. Moreover, because D-serine does not exist in natural bacterial cells, it has unparalleled advantages as a resistance screening marker for L-serine-producing strains. By screening strains with enhanced D-serine resistance and improving the strain's product tolerance, high-L-serine-producing strains can be screened.

[0062] Resistance screening was conducted on the base strain SER-0 using solid culture media supplemented with different concentrations of D-serine (0, 30, 60, 90, 120, and 150 g / L). As the D-serine concentration increased, the growth of the test strain slowed and the colony morphology decreased. Growth ceased at a D-serine concentration of 150 g / L. Therefore, a D-serine concentration of 120 g / L was selected for post-mutagenesis screening.

[0063] 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.

[0064] 3. ARTP mutagenesis

[0065] (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, cultured at 30°C and 200 rpm until the logarithmic growth phase, washed three times with sterile saline, and then resuspended in saline containing 5% glycerol to 10 8 cells / mL for mutagenesis.

[0066] (2) ARTP mutagenesis conditions: working power 120 W, gas flow 10 SLM, and mutagenesis time 45 s.

[0067] (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 spread it on the surface of the slide. Set the parameters according to the mutagenesis conditions for treatment.

[0068] (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 onto screening solid culture medium with a D-serine concentration of 120 g / L and cultured at 30°C for 48 h.

[0069] Example 5: Screening of mutant strains

[0070] 1. Orifice plate initial screening

[0071] Single colonies that grew well on the screening solid medium were picked and transferred to 5 mL of LBB liquid medium for 16-18 h. The culture was then inoculated into 1 mL of shake flask fermentation medium at 10% of the initial inoculum 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.

[0072] After centrifuging the bacterial suspension at 12,000 rpm for 2 minutes, the supernatant was filtered through a 0.22 μm filter and assayed for L-serine production by HPLC. A two-fold increase in L-serine production compared to the baseline strain (L-serine production ≥ 2.1 g / L) was used as the screening criterion.

[0073] The results of the initial screening of the orifice plate show (see Figure 4 96 individual colonies were isolated on solid screening medium. Seven strains, including SER-18, SER-50, SER-51, SER-53, SER-54, SER-55, and SER-56, produced L-serine production exceeding 2.1 g / L, double that of the pre-mutagenized SER-0 strain. Seven strains were identified in the initial screening, yielding 7.3% of the strain. These seven strains were then rescreened using shake flask fermentation.

[0074] 2. Shake flask rescreening

[0075] Since the initial screening was conducted in a small-volume well plate, it was easily affected by various factors such as the environment, which could lead to deviations in the results. To further ensure the accuracy of the screening results, a larger-volume, more stable shake flask fermentation was used for secondary screening based on the initial screening.

[0076] The 7 mutant strains obtained in the initial screening were streaked onto LBB plates and cultured at 30°C overnight. Single colonies were picked and cultured in 5 mL of LBB liquid medium for 16-18 h. The initial inoculation OD was controlled to be 1 and inoculated into 20 mL of shake flask fermentation medium. The culture was cultured at 30°C and 200 rpm for 48 h for shake flask rescreening.

[0077] The shake flask fermentation results showed that (see Figure 5 ), the yields of SER-18, SER-53, and SER-55 were significantly increased, more than 2 times that of 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 basic strain serine. This strain was named GSLW-001.

[0078] Example 6: Stability test of mutagenized strains

[0079] The screened mutant strain GSLW-001 was serially passaged five times on LBB solid medium, fermented in shake flasks for 48 h, and the growth ability and acid production capacity of the first and fifth generations were determined.

[0080] See the 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 600The growth rate of the mutant strain GSLW-001 was 10.32±0.12, and the L-serine production was 2.686±0.055 g / L, showing no significant difference in growth and acid production compared to the first generation. This indicates that the 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 industrial serine production.

[0081] Example 7: Fermentation tank test of mutagenic bacteria

[0082] 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.

[0083] 1. Culture medium preparation:

[0084] Primary seed culture 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.

[0085] 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.

[0086] Fermentation medium in the fermenter (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.

[0087] 2. Fermentation culture in fermentation tank:

[0088] (1) The mutant strain GSLW-001 was streaked onto LBB medium and cultured at 30°C overnight.

[0089] (2) Inoculate a well-grown single colony into 10 mL of primary seed culture medium and culture at 30°C and 200 rpm for 16-18 hours.

[0090] (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 hours.

[0091] (4) The secondary seed culture obtained in step (3) was transferred to a 7.5 L fermenter containing 2.5 L fermenter culture medium. The temperature was controlled at 30 °C, and ammonia 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.

[0092] 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 yield and conversion rate of L-serine produced by this mutant strain were significantly increased, with a production intensity of 1.09 g / L / h and a sugar-acid conversion rate of 0.438 g / g. This significantly improved production efficiency and reduced production costs by fermenting L-serine.

[0093] Table 2 Fermentation performance of GSLW-001

[0094]

[0095] Example 8: Optimization of fermentation conditions

[0096] In order to further improve the fermentation performance of GSLW-001, the fermentation conditions (fermentation medium, fermentation process) of its fermenter were optimized.

[0097] 1. Fermentation medium optimization

[0098] Phosphorus is a component of ADP, ATP, and phospholipids, and potassium ions are activators of many enzymes. Appropriate amounts of K2HPO4 and KH2PO4 promote bacterial growth and acid production. Biotin, as a crucial cofactor, not only influences key pathways such as glycolysis, the glyoxylate cycle, and nitrogen metabolism, but also impacts cell membrane synthesis and alters cell membrane permeability. Therefore, the addition of appropriate amounts of biotin may promote L-serine synthesis.

[0099] 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, providing 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.

[0100] The concentrations of phosphate, biotin, choline chloride, and betaine in the fermentation medium were adjusted, and the optimized medium was as follows:

[0101] 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.

[0102] 2. Fermentation process optimization

[0103] A secondary seed culture was obtained using the culture method described in Example 7. The entire secondary seed culture was transferred to a 7.5 L fermentor containing 2.5 L of optimized fermentation medium. The temperature was controlled at 30°C, and ammonia was automatically fed to maintain a pH of approximately 7.2 and a dissolved oxygen level of approximately 30% for the first 42 hours of fermentation. After 42 hours, the bacterial growth state remained essentially unchanged. The pH was adjusted to approximately 6.8, and the dissolved oxygen level was reduced to 10% for L-serine production.

[0104] 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 increased from 78.37 g / L to 104.12 g / L, the sugar-acid conversion rate further increased from 0.438 g / g to 0.466 g / g, and the production intensity reached 1.44 g / L / h.

[0105] 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-serine-yielding strains, and also provides a fermentation optimization process, which can further optimize strain performance and provide guidance for the efficient screening of other amino acids.

[0106] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A Corynebacterium glutamicum ( Corynebacterium glutamicum ), characterized in that The glutamicum bacteria were deposited in the General Microbiology Center of China 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 according to claim 1 and inoculate it into a fermentation tank for fermentation.

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, which are choline chloride and betaine.

6. The method for producing L-serine according to any one of claims 3 to 4, wherein: 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.

7. The method for producing L-serine according to claim 5, wherein 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 0.2-1 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, 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.

8. The method for producing L-serine according to claim 7, 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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