Strain for producing lysine using fermentation product of synthesis gas as substrate and application thereof

Abstract

This invention belongs to the field of bioengineering and biorefining technology, specifically relating to a strain and its application for producing lysine using syngas fermentation products as a substrate. The strain is *Corynebacterium glutamicum* (…). Corynebacterium glutamicum Strain 5E16 was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 27, 2025, with accession number GDMCC No: 67371. This strain utilizes syngas fermentation products as substrates to produce lysine. This invention presents a lysine-producing strain designed specifically for the composition of the crude fermentation broth, exhibiting stronger environmental adaptability and robustness, ensuring high and stable yields even under complex raw material conditions.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and biorefining technology, specifically relating to a strain that uses syngas fermentation products as substrates to produce lysine and its application. Background Technology

[0002] Using biomanufacturing as a means and carbon gas as a raw material to produce chemical products is gradually becoming an important technological route to promote the transformation of traditional industries to a green, low-carbon, and circular model.

[0003] One-carbon gases mainly refer to syngas produced from carbon-containing materials such as coal, petroleum, and biomass through oxidation or high-temperature reactions. Their main components include carbon dioxide, carbon monoxide, and hydrogen. This type of syngas can be derived from industries such as iron and steel metallurgy, petroleum refining, coal chemical industry, and biomass gasification, offering advantages such as wide availability and low cost. Through bioconversion processes, syngas can be converted into various organic acids, alcohols, and other chemical intermediates, thus playing a significant role in energy recycling and carbon emission reduction. In recent years, the technology of producing acetic acid (salts) from syngas through bio-fermentation has been continuously developing. This process utilizes anaerobic microorganisms to convert gases such as CO, CO2, and H2 into acetic acid or acetates, thereby achieving efficient biological fixation of one-carbon resources. Acetic acid (salts), as a renewable, non-grain-source carbon source, not only has the advantages of low cost and stable supply but also provides a new substrate option for subsequent biosynthesis. Especially in the biomanufacturing of amino acids and other fine chemicals, using acetic acid (salts) as a carbon source can effectively reduce production costs and improve process sustainability.

[0004] L-Lysine is a basic α-aminocaproic acid, an essential amino acid for humans and animals, and is widely used in feed, pharmaceuticals, cosmetics, and food industries. In 2024, my country's L-lysine production reached 3.058 million tons. With the improvement of living standards and the increase in meat consumption, the market demand for lysine (especially feed-grade lysine) will continue to grow. Currently, industrially, lysine is mainly produced using grains or sugary raw materials such as corn starch and sugarcane molasses as substrates through fermentation by Corynebacterium glutamicum or Escherichia coli. Raw material costs typically account for 40-60% of the total production cost, and raw material prices are greatly affected by factors such as agricultural harvests, international markets, and policies, resulting in significant price fluctuations and thus high cost pressures and operational risks for lysine production. Existing lysine-producing strains (especially Corynebacterium glutamicum) have been modified and domesticated over a long period to adapt to glucose substrates, resulting in a relatively narrow substrate utilization spectrum and a lack of efficient assimilation pathways and regulatory mechanisms for inexpensive non-grain carbon sources (such as acetic acid). This prevents the utilization of this potentially low-cost substrate. In addition, conventional metabolic engineering strategies such as introducing multiple gene copies or using strong promoters are usually adopted to relieve metabolic feedback inhibition or improve the expression level of key metabolic enzymes. However, this will bring a significant metabolic burden to the strain and affect its growth and product synthesis efficiency.

[0005] To address the aforementioned issues, this invention proposes a novel and complete biomanufacturing route, namely, a technical path from syngas to acetic acid (salt) and then to lysine. This process can completely eliminate dependence on expensive grain raw materials, using widely available and low-cost one-carbon resource syngas as the starting material, minimizing intermediate costs, and providing a promising technical route for achieving a revolutionary reduction in lysine production costs. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a strain and its application for the efficient, economical, and sustainable production of lysine using syngas fermentation products as substrates.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A strain that utilizes syngas fermentation products as substrates to produce lysine, the strain being *Corynebacterium glutamicum* (…). Corynebacterium glutamicum )5E16 was deposited on November 27, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, accession number GDMCC No:67371, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The application of a strain that uses syngas fermentation products as a substrate to produce lysine, and the application of the strain in the fermentation production of lysine.

[0009] Application of the strain in the fermentation production of lysine using syngas fermentation products as substrates.

[0010] The fermentation temperature is controlled at 32~35℃, the pH is controlled at 7.0~7.2, and the dissolved oxygen is controlled at 30%~40%.

[0011] The syngas fermentation products contain ammonium acetate or other acetates.

[0012] The fermentation medium consists of the following components added per liter of supernatant obtained from microbial fermentation using syngas as a substrate: glucose 0-40 g / L, soybean meal hydrolysate 20 g / L, molasses 10 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 1 g / L, ferrous sulfate 100 mg / L, manganese sulfate 100 mg / L, zinc sulfate 0.5 mg / L, anhydrous copper sulfate 0.5 mg / L, biotin 0.5 mg / L, and vitamin B3 40 mg / L; wherein the concentration of ammonium acetate in the supernatant obtained from microbial fermentation using syngas as a substrate is 40-60 g / L.

[0013] Furthermore, *Corynebacterium glutamicum* 5E16 was fermented in a fermentation medium under aerobic conditions. The selected control temperature was 28–37°C, preferably 35°C; the selected control pH was 6.5–7.5, preferably 7.0–7.2; the selected acid for adjusting the pH was sulfuric acid or hydrochloric acid, preferably hydrochloric acid; the selected dissolved oxygen concentration was maintained at 20–50%, preferably 30–40%; the feeding strategy involved using an automatic detection instrument to monitor lysine production, acetate consumption, and ammonium consumption. Based on the consumption, simply treated microorganisms were fed in to ferment the supernatant using syngas as a substrate, maintaining the ammonium acetate level in the fermenter at 10–20 g / L. During fermentation, a small amount of glucose could be added to promote lysine production.

[0014] The fermentation broth obtained above is centrifuged / filtered to remove the bacterial cells and then processed using a multi-effect continuous evaporation crystallization device to obtain lysine hydrochloride or lysine sulfate products.

[0015] The recovered residue can be used as feed protein or to extract other components.

[0016] The supernatant obtained by the microorganisms using syngas as a substrate for fermentation is obtained by anaerobic fermentation of the microorganisms in a liquid culture medium in the presence of syngas at 30-60°C, pH 4.5-6.5, and with stirring. The main components of the syngas are H2 and CO2, with a volume ratio of 9:1 to 5:5. The microorganisms are one or more of the aerobic microorganisms.

[0017] The selected synthesis gas flow rate is 1~20 L / min, preferably 5~10 L / min.

[0018] Furthermore, the syngas fermentation broth: Syngas anaerobic fermentation process module for producing crude acetate (ammonium): Syngas is introduced into an anaerobic fermentation reactor containing aerobic microorganisms, and liquid culture medium is added to the fermentation reactor. The selected synthesis gas components and proportions are H2 and CO2, with the volume ratio of H2 to CO2 controlled between 9:1 and 5:5, preferably 7:3 to 6:4; The selected anaerobic microorganism was Clostridium difficile (C. difficile). Clostridium autoethanogenum Clostridium yongdarii ( Clostridium ljungdahlii ), Clostridium carbonmonoxide ( Clostridium carboxidivorans ), Acetobacter wuerii ( Acetobacterium woodii ) and hot acetylcholine ( Moorella thermoacetica Clostridium yunnanense and Acetobacter wuerii are preferred. The liquid culture medium is PETC: per liter of water, add 1 g / L ammonium chloride, 0.1 g / L potassium chloride, 0.2 g / L magnesium sulfate heptahydrate, 0.8 g / L sodium chloride, 0.013 g / L manganese chloride, 0.004 g / L ferrous sulfate, 0.002 g / L cobalt chloride, 0.002 g / L zinc sulfate, 0.0002 g / L nickel chloride, 0.00025 g / L sodium tungstate, 0.0002 g / L sodium selenate, 0.002 g / L sodium molybdate, 0.2 g / L calcium chloride, 0.02 g / L aminotriacetic acid, 0.5 g / L yeast extract, 0.1 g / L cysteine, 0.1 mg / L biotin, 0.02 mg / L folic acid, 0.1 mg / L pyridoxine hydrochloride, 0.25 mg / L thiamine, 0.05 mg / L riboflavin, and 0.05 mg / L niacin. mg / L, calcium pantothenate 0.04 mg / L, cyanocobalamin 0.05 mg / L, para-aminobenzoic acid 0.05 mg / L, lipoic acid 0.05 mg / L.

[0019] Fermentation was carried out under anaerobic conditions, with a controlled temperature of 30–60°C, preferably 35–40°C. The controlled pH was 4.5–6.5, preferably 5.0–5.8, controlled using ammonia. The controlled stirring speed was 100–800 rpm, preferably 300–500 rpm. The controlled synthesis gas flow rate was 1–20 L / min, preferably 5–10 L / min. Parameter optimization was used to improve the yield and concentration of acetate (salt). The selected fermenter is equipped with a membrane retention device containing a 50 μm ceramic membrane. After fermentation, the membrane retention device is used for concentration and separation. The concentrated liquid is returned to the fermenter, and the clear liquid is sent to the crude acetate (ammonium) fermentation broth treatment module. When fermentation is complete or reaches a stable state, crude acetate (ammonium) fermentation broth (the clear liquid obtained above) is obtained, wherein the crude fermentation broth contains the target product acetate ions (CH3COO). - ) and ammonium ions (NH4) + The contents include microbial cells, residual substrates, trace fermentation byproducts (such as ethanol and organic acids), and culture medium components, wherein the concentration of ammonium acetate is not less than 50 g / L. This invention has the following significant advantages and positive effects: 1. Achieving integrated co-production: This invention integrates the process of producing acetate (ammonium) from syngas fermentation with the production process of lysine, forming a continuous biorefining chain from one-carbon gas to high-value amino acids. The acetate (ammonium) produced by syngas fermentation is used to produce lysine, resulting in a high-value-added product.

[0020] 2. Significantly reduce production costs (a) Simplified acetate purification: This invention directly uses the crude acetate (ammonium) fermentation broth produced by syngas fermentation as the raw material for lysine fermentation, avoiding the expensive and energy-consuming acetate / acetate separation and purification steps (such as distillation and extraction) in traditional processes, and significantly reducing equipment investment and operating costs (energy consumption and chemical consumption). (b) Improve nitrogen source utilization efficiency: During the syngas fermentation stage, the ammonia water added to adjust the pH first forms ammonium acetate, which is then used by lysine-producing bacteria as a carbon and nitrogen source to produce amino acids, thereby improving nitrogen utilization.

[0021] (c) Reduced material loss: The integrated process reduces the transfer and purification steps of intermediate products, thus reducing the loss of carbon and nitrogen.

[0022] 3. Improved resource utilization efficiency and sustainability: Maximizes the use of carbon and hydrogen in syngas, as well as the added nitrogen source, in line with the principles of green chemistry and circular economy. Utilizes non-grain raw materials to reduce carbon footprint.

[0023] 4. Specialized tolerant strains: Lysine production strains designed specifically for the components of crude fermentation broth have stronger environmental adaptability and robustness, ensuring high and stable production under complex raw material conditions. Attached Figure Description

[0024] Figure 1 The diagram shows the yield effect of each substance in the syngas fermentation liquid provided in the embodiment of the present invention.

[0025] Figure 2 The growth curve of Corynebacterium glutamicum provided in the embodiments of the present invention.

[0026] Figure 3 The lethality curve of Corynebacterium glutamicum during the mutagenesis process provided in the embodiments of the present invention.

[0027] Figure 4 The graph shows a comparison of the growth and lysine yield of the Corynebacterium glutamicum mutant strain and the high-lysine-producing strain provided in this embodiment of the invention.

[0028] Figure 5 The above is a flowchart of the integrated lysine fermentation process provided in an embodiment of the present invention.

[0029] Figure 6 The diagram shows the effect of using the crude product obtained by syngas fermentation as a substrate to produce lysine, as provided in an embodiment of the present invention. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and should not be used to limit the scope of protection of the present invention.

[0031] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0032] This invention employs an integrated process method that enables the direct co-production of syngas into acetate (ammonium) and lysine. The process unit for anaerobic fermentation of syngas to produce acetate (ammonia) can be deeply coupled physically and / or in terms of material flow with the process unit for aerobic fermentation of the same acetate (ammonia) to produce lysine. The core is to directly use the fermentation broth containing crude acetate (ammonia) produced by syngas fermentation without complex purification (or after only simple pretreatment) as the main carbon and nitrogen source for lysine fermentation. This process enables highly efficient synergistic conversion of carbon and nitrogen streams, maximizing the conversion of carbon (CO, CO2) in syngas into carbon in acetate, and further efficiently into carbon in lysine, reducing carbon loss caused by intermediate separation. The inorganic nitrogen source (ammonia) added during the syngas fermentation stage is converted into organic nitrogen (ammonium acetate). This organic nitrogen is utilized simultaneously as both a carbon and nitrogen source during the lysine fermentation stage. The nitrogen required for lysine synthesis is provided by ammonium acetate, significantly reducing the total amount of exogenous nitrogen added.

[0033] Example 1 Preparation of syngas fermentation broth Bacterial strain: using Clostridium yongdarii ( Clostridium ljungdahlii SL40 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:67679, deposit date of January 15, 2026, and deposit address of Building 59, No. 100 Xianlie Middle Road, Guangzhou. Reactor: 10 L anaerobic stirred tank; Culture medium PETC: Add the following per liter of water: ammonium chloride 1 g / L, potassium chloride 0.1 g / L, magnesium sulfate heptahydrate 0.2 g / L, sodium chloride 0.8 g / L, manganese chloride 0.013 g / L, ferrous sulfate 0.004 g / L, cobalt chloride 0.002 g / L, zinc sulfate 0.002 g / L, nickel chloride 0.0002 g / L, sodium tungstate 0.00025 g / L, sodium selenate 0.0002 g / L, sodium molybdate 0.002 g / L, calcium chloride 0.2 g / L, aminotriacetic acid 0.02 g / L, yeast extract 0.5 g / L, cysteine ​​0.1 g / L, biotin 0.1 mg / L, folic acid 0.02 mg / L, pyridoxine hydrochloride 0.1 mg / L, thiamine 0.25 mg / L, riboflavin 0.05 mg / L, and niacin 0.05 mg / L. mg / L, calcium pantothenate 0.04 mg / L, cyanocobalamin 0.05 mg / L, para-aminobenzoic acid 0.05 mg / L, lipoic acid 0.05 mg / L.

[0034] Fermentation conditions: temperature 37℃, pH controlled at around 5.8 (adjusted with ammonia), stirring speed 500 rpm, syngas introduced (H2:CO2 = 7:3, v / v), flow rate controlled at 1000 mL / min.

[0035] Fermentation Process: The culture medium was sterilized in an anaerobic fermenter. After sterilization, nitrogen was purged for 30 minutes to achieve an anaerobic state. Syngas was then introduced for 30 minutes to displace the nitrogen, followed by inoculation at a 10% inoculum size. During fermentation, the stirring speed and gas flow rate were gradually increased as the biomass increased. Figure 1 It can be seen that after 132 h of culture, the ammonium acetate concentration can reach 64 g / L.

[0036] Example 2: Mutagenesis Breeding Method of Corynebacterium glutamicum 1. Determination of growth curve and optimal growth time of Corynebacterium glutamicum Bacterial strain: Corynebacterium glutamicum ATCC 13032 (purchased from Beina Chuanglian Biotechnology Co., Ltd.); Culture medium: 30 mL BHI medium, initial pH 7.1; Culture conditions: cultured at 30℃ in a shaker at 250 rpm. Inoculum size: 5%, to achieve initial OD... 600 Around 0.2; Results: After 18 h of culture in a shaker, the growth curve of Corynebacterium glutamicum was obtained (see...). Figure 2 The 10-hour period represents the mid-logarithmic growth phase. In subsequent mutation breeding, *Corynebacterium glutamicum* that had grown for 10 hours were used as the mutation target.

[0037] 2. Mortality curve and determination of optimal mutagenesis time Bacterial strain: Corynebacterium glutamicum ATCC 13032; Culture medium: 30 mL BHI medium, initial pH 7.1; Culture conditions: cultured at 30℃ in a shaker at 250 rpm; Inoculum size: 5%, to achieve an initial OD600 of approximately 0.2; Chemical mutagen: ethyl methanesulfonate (EMS).

[0038] Mutagenesis steps: (1) Take an appropriate amount of Corynebacterium glutamicum bacterial culture after 10 h of growth, measure its OD600 (approximately 5-6), dilute with sterile water, and adjust its OD600. 600 It is around 1.

[0039] (2) Set the mutagenesis times to 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min respectively. Add EMS to the diluted bacterial solution to make the final EMS concentration 5%. Incubate at 30℃ and 250 rpm for the above mutagenesis times respectively.

[0040] (3) After the mutagenesis time is over, add 5% sodium thiosulfate solution to the bacterial culture and react for 10 min. Then centrifuge at 12000 rpm for 10 min, remove the supernatant, and resuspend in sterile water.

[0041] (4) Dilute the resuspended bacterial solution by 100 to 2000 times, take 50 μL of bacterial solution, spread it on BHI solid medium, and control the number of colonies on the solid medium to be around 200.

[0042] (5) Calculate the mortality rate: .

[0043] Depend on Figure 3 The results show that under 5% EMS mutagenesis, the mutagenesis lethality rate was 90% after 50 minutes, which was determined to be the optimal mutagenesis time.

[0044] 3. Screening of single strains (1) Select a single colony that has grown on a BHI plate after 50 min of mutagenesis with 5% EMS and transfer it to BHI liquid medium for further culture.

[0045] (2) After 24 hours of incubation, OD was measured. 600 The samples were then inoculated into 30 mL of selection medium to achieve an initial OD600 of approximately 0.2. The selection medium consisted of: ammonium acetate 40 g / L, glucose 10 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, and yeast extract 5 g / L.

[0046] (3) The mutant strain was cultured continuously for 120 h in a shaker at 30℃ and 250 rpm. OD was monitored every 24 h.600 And lysine production. Screen for mutant strains that can produce lysine (the original strain ATCC 13032 cannot produce lysine).

[0047] (4) A lysine-producing strain, Corynebacterium glutamicum CK, was obtained through mutagenesis and screening.

[0048] (5) Mutagenesis and laboratory adaptive evolution were performed on Corynebacterium glutamicum CK to further improve its acetic acid utilization and lysine production capacity. The mutagenesis method was the same as before. Laboratory adaptive evolution: The bacteria were continuously passaged every 48 h in 30 mL of selection medium at 30℃ and 250 rpm, with an inoculum size of 10%.

[0049] (6) After 30 generations of continuous subculturing, the high-lysine-producing strain Corynebacterium glutamicum 5E16 was obtained.

[0050] The obtained strain Corynebacterium glutamicum ( Corynebacterium glutamicum )5E16 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 27, 2025, with accession number GDMCC No:67371.

[0051] 4. The high-lysine-producing strain Corynebacterium glutamicum 5E16 obtained through the above screening was characterized. (1) Comparison of growth and lysine yield between the high-lysine-producing strain Corynebacterium glutamicum 5E16 and the starting strain.

[0052] Under conditions of 30℃ and 250 rpm shaking incubator, the starting strain *Corynebacterium glutamicum* ATCC 13032, the mutant strain *Corynebacterium glutamicum* CK, and the high-lysine-producing strain *Corynebacterium glutamicum* 5E16 were inoculated into 30 mL of selection medium at a 5% inoculum size and cultured. *Corynebacterium glutamicum* ATCC 13032 could not produce lysine; *Corynebacterium glutamicum* CK and *Corynebacterium glutamicum* 5E16 could produce lysine. The biomass and growth rate of *Corynebacterium glutamicum* ATCC 13032 were slightly higher than those of *Corynebacterium glutamicum* CK and *Corynebacterium glutamicum* 5E16.

[0053] (2) Comparison of growth and lysine production of Corynebacterium glutamicum mutant strain in simulated crude fermentation broth.

[0054] Under conditions of 30℃ and 250 rpm shaking in a shaker, the mutant strain *Corynebacterium glutamicum* CK and the high-lysine-producing strain *Corynebacterium glutamicum* 5E16 were inoculated into 30 mL of simulated crude fermentation broth at a 5% inoculation rate and cultured. The simulated crude fermentation broth consisted of: syngas crude fermentation broth (containing 40 g / L ammonium acetate), 10 g / L glucose, 1 g / L potassium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, and 5 g / L yeast extract. Figure 4 It can be seen that the lysine production of the mutant strain was 15.4 g / L, which was 50% higher than that of the original strain CK.

[0055] (3) Comparison of gene and amino acid sequences between the high-lysine-producing strain Corynebacterium glutamicum 5E16 and the originating strain Corynebacterium glutamicum ATCC 13032, including ABC transporter permease CGL_RS00185, aspartate kinase CGL_RS01330, ABC transporter ATP-binding protein CGL_RS03325, pyruvate carboxylase CGL_RS03440, polyphosphate kinase 2 CGL_RS04595, 1,4-α-glucan branched protein GlgBCGL_RS06100, and large subunit of isopropyl malate isomerase CGL_RS06555. Succinyldiaminopimelic acid transaminase CGL_RS05505, homoserine dehydrogenase CGL_RS05895, acetylglutamate kinase CGL_RS06975, NADP-dependent phosphoglucate dehydrogenase CGL_RS07265, phosphoenolpyruvate carboxylase CGL_RS07925, malate dehydrogenase (quinone) CGL_RS09955, malate dehydrogenase CGL_RS11800, LysE family transporter CGL_RS13250: The original gene sequence of the ABC transporter permease CGL_RS00185 of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO.1. SEQ ID NO.1

[0056] The amino acid sequences corresponding to the above sequences are shown in SEQ ID NO.2. SEQ ID NO.2 MTKIKSGEASTSIVERALKRPELTSLLGAVLVFTLFMVVAPAFRSWDSMATVLYASSTIGIMAVAVGLLMIADEFDLSTGVAVTTAALAASMFSYNLWLNTWVGALIALVISLAIGFFNGFLVVKTKIASFLITLATFLMLQGINLAVTKLISGTVATPTIADMEGFPSAR AVFASSIPIFGVNIRITVFWWLLFVIVGTFVLFKTRIGNWIFAVGGDEEAARAVGVPVRGVKIGLFMFVGFAAWFVGMHNLFLFDSIQAGQGVGNEFLYIIAAVIGGISMTGGRGTVVGTMIGALIFGMTNQGIVYAGWNPDWFMFFLGGTLLLAVLLNHRFERFNKERS.

[0057] Mutant 5E16 was obtained through the above-described mutation treatment. It is a mutation of A to T at position 551 of the gene sequence of the ABC transporter permease CGL_RS00185 from the starting strain. The gene sequence is shown in SEQ ID NO.3. SEQ ID NO.3

[0058] The amino acid sequence corresponding to SEQ ID NO.3 above (with the mutation of Asn to Ile at position 184) is shown in SEQ ID NO.4: SEQ ID NO.4 MTKIKSGEASTSIVERALKRPELTSLLGAVLVFTLFMVVAPAFRSWDSMATVLYASSTIGIMAVAVGLLMIADEFDLSTGVAVTTAALAASMFSYNLWLNTWVGALIALVISLAIGFFNGFLVVKTKIASFLITLATFLMLQGINLAVTKLISGTVATPTIADMEGFPSAR AVFASSIPIFGVIIRITVFWWLLFVIVGTFVLFKTRIGNWIFAVGGDEEAARAVGVPVRGVKIGLFMFVGFAAWFVGMHNLFLFDSIQAGQGVGNEFLYIIAAVIGGISMTGGRGTVVGTMIGALIFGMTNQGIVYAGWNPDWFMFFLGGTLLLAVLLNHRFERFNKERS.

[0059] The original gene sequence of the aspartate kinase CGL_RS01330 of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO. 5. SEQ ID NO.5

[0060] The original amino acid sequence of the aspartate kinase CGL_RS01330 of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO. 6. SEQ ID NO.6 MGDTTDELLELAAAVNPVPPAREMDMLLTAGERISNALVAMAIESLGAEAQSFTGSQAGVLTTERHGNARIVDVTPGRVREALDEGKICIVAGFQGVNKETRDVTTLGRGGSDTTAVALAAALNADVCEIYSDVDGVYTADPRIVPNAQKLEKLSFEEMLELAAVGSKILVLRSVEYARAFNVPLRVRSS YSNDPGTLIAGSMEDIPVEEAVLTGVATDKSEAKVTVLGISDKPGEAAKVFRALADAEINIDMVLQNVSSVEDGTTDITFTCPRSDGRRAMEILKKLQVQGNWTNVLYDDQVGKVSLVGAGMKSHPGVTAEFMEALRDVNVNIELISTSEIRISVLIREDDLDAAARALHEQFQLGGEDEAVVYAGTGR.

[0061] Mutant 5E16 is a mutation of C to T at position 932 of the aspartate kinase CGL_RS01330 gene sequence of the originating strain Corynebacterium glutamicum ATCC 13032, as shown in SEQ ID NO. 7. SEQ ID NO.7

[0062] The amino acid sequence corresponding to SEQ ID NO.7 above (Th at position 269 is mutated to Ile) is shown in SEQ ID NO.8. SEQ ID NO.8 MGDTTDELLELAAAVNPVPPAREMDMLLTAGERISNALVAMAIESLGAEAQSFTGSQAGVLTTERHGNARIVDVTPGRVREALDEGKICIVAGFQGVNKETRDVTTLGRGGSDTTAVALAAALNADVCEIYSDVDGVYTADPRIVPNAQKLEKLSFEEMLELAAVGSKILVLRSVEYARAFNVPLRVRSS YSNDPGTLIAGSMEDIPVEEAVLTGVATDKSEAKVTVLGISDKPGEAAKVFRALADAEINIDMVLQNVSSVEDGTTDIIFTCPRSDGRRAMEILKKLQVQGNWTNVLYDDQVGKVSLVGAGMKSHPGVTAEFMEALRDVNVNIELISTSEIRISVLIREDDLDAAARALHEQFQLGGEDEAVVYAGTGR.

[0063] The original gene sequence of the ABC transporter ATP-binding protein CGL_RS03325 of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO. 9. SEQ ID NO.9 CTAGTGAAGTGCGGTGATGTGGTGTCGTCCCATGGGCATGACCAGTGGTGCGCCGGAGACTGGGTCGGCGATAATGCGGGCGTCGGTGTGGAAGACCTCGCTCATCATGGTTTCGGTGATGACGTTGGTGGGGGTGCCTTGGGCGTAGATTTTTCCGGCGTTCATGGCGATGAGGTGGTCAGAGTAACGTGCGGCAAGTCCCAATTCGTGGAGCACCATGACGATGGTGGTGCCGTGGTTGTGGTTGAGGTCAGTGAGGAGATCAAGCACTTCGAGCTGGTTGGCGATGTCGAGGTAGGTGGTGGGTTCATCAAGAAGCAGGATGTCTGTTTCTTGGGCAAGGGCCATGGCGATCCAGACGCGTTGGCGTTGGCCGCCGGAGAGTTCGTCGATGGGGCGTTCGGCAAGCTCGGTGGTGTTGGTCATTTCCAATGCTTGGGCCACTACTTCGTAGTCGCGTGTGGACCATCTGCCCATGAGTCCTTGGTGGGGGTGGCGGCCGCGGCCCACGAGGTCGGCGACGACGATGCCTTCAGGTGCGGTGGGGGATTGCGGTAACAGCCCGAGCATGCGAGCTAGTTCTTTGCCTGGCAGTGAAGGAAGGGGGTGGGCGTCGATAAGCGCTTGCCCGGCGCTAGGTTTAAGGAGGCGCGCAAAGGCGCGCAGCAGCGTTGATTTGCCGCATCCGTTGGGGCCGACGATGGAGGTGATTTTGCCGGGGACGATGTCGACGCTGAGCGAATCGATGATGGTGCGCTCGCCGTACGCCAGGGAAATTTCTTCGGCGGATAGTTGATGGTTGGTGGTCAC。

[0064] The original amino acid sequence of the ABC transporter ATP-binding protein CGL_RS03325 of the starting strain Corynebacterium glutamicum ATCC 13032 is as shown in SEQ ID NO.10 SEQ ID NO.10 MRPDRIRGVCVWPHCRAHFRLRRIAHHPLRTHRRVDRAHRRPNWPILPRHPLPRRSCHRRIRRPIPYLFTHSFQPRGSNPVTTNHQLSAEEISLAYGERTIIDSLSVDIVPGKITSIVGPNGCGKSTLLRAFARLLKPSAGQALIDAHPLPSLPGKELARMLGLLPQSPTAPEGI VVADLVGRGRHPHQGLMGRWSTRDYEVVAQALEMTNTTELAERPIDELSGGQRQRVWIAMALAQETDILLLDEPTTYLDIANQLEVLDLLTDLNHNHGTTIVMVLHELGLAARYSDHLIAMNAGKIYAQGTPTNVITETMMSEVFHTDARIIADPVSGAPLVMPMGRHHITALH.

[0065] Mutant 5E16 is derived from the mutation of C to T at position 216 of the gene sequence of the ABC transporter ATP-binding protein CGL_RS03325 from the originating strain Corynebacterium glutamicum ATCC 13032, as shown in SEQ ID NO. 11. SEQ ID NO.11 CTAGTGAAGTGCGGTGATGTGGTGTCGTCCCATGGGCATGACCAGTGGTGCGCCGGAGACTGGGTCGGCGATAATGCGGGCGTCGGTGTGGAAGACCTCGCTCATCATGGTTTCGGTGATGACGTTGGTGGGGGTGCCTTGGGCGTAGATTTTTCCGGCGTTCATGGCGATGAGGTGGTCAGAGTAACGTGCGGCAAGTCCCAATTCGTGGAGCATCATGACGATGGTGGTGCCGTGGTTGTGGTTGAGGTCAGTGAGGAGATCAAGCACTTCGAGCTGGTTGGCGATGTCGAGGTAGGTGGTGGGTTCATCAAGAAGCAGGATGTCTGTTTCTTGGGCAAGGGCCATGGCGATCCAGACGCGTTGGCGTTGGCCGCCGGAGAGTTCGTCGATGGGGCGTTCGGCAAGCTCGGTGGTGTTGGTCATTTCCAATGCTTGGGCCACTACTTCGTAGTCGCGTGTGGACCATCTGCCCATGAGTCCTTGGTGGGGGTGGCGGCCGCGGCCCACGAGGTCGGCGACGACGATGCCTTCAGGTGCGGTGGGGGATTGCGGTAACAGCCCGAGCATGCGAGCTAGTTCTTTGCCTGGCAGTGAAGGAAGGGGGTGGGCGTCGATAAGCGCTTGCCCGGCGCTAGGTTTAAGGAGGCGCGCAAAGGCGCGCAGCAGCGTTGATTTGCCGCATCCGTTGGGGCCGACGATGGAGGTGATTTTGCCGGGGACGATGTCGACGCTGAGCGAATCGATGATGGTGCGCTCGCCGTACGCCAGGGAAATTTCTTCGGCGGATAGTTGATGGTTGGTGGTCAC。

[0066] The amino acid sequence corresponding to SEQ ID NO.11 above (Val at position 279 is mutated to Met) is shown in SEQ ID NO.12 SEQ ID NO.12 MRPDRIRGVCVWPHCRAHFRLRRIAHHPLRTHRRVDRAHRRPNWPILPRHPLPRRSCHRRIRRPIPYLFTHSFQPRGSNPVTTNHQLSAEEISLAYGERTIIDSLSVDIVPGKITSIVGPNGCGKSTLLRAFARLLKPSAGQALIDAHPLPSLPGKELARMLGLLPQSPTAPEGI VVADLVGRGRHPHQGLMGRWSTRDYEVVAQALEMTNTTELAERPIDELSGGQRQRVWIAMALAQETDILLLDEPTTYLDIANQLEVLDLLTDLNHNHGTTIVMMLHELGLAARYSDHLIAMNAGKIYAQGTPTNVITETMMSEVFHTDARIIADPVSGAPLVMPMGRHHITALH.

[0067] The original gene sequence of pyruvate carboxylase CGL_RS03440 of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO. 13. SEQ ID NO.13

[0068] The amino acid sequence corresponding to SEQ ID NO.13 above is shown in SEQ ID NO.14. SEQ ID NO.14 。

[0069] Mutant 5E16 is a mutation of C to T at position 1372 of the pyruvate carboxylase CGL_RS03440 gene sequence of the originating strain Corynebacterium glutamicum ATCC 13032, as shown in SEQ ID NO. 15. SEQ ID NO.15

[0070] The amino acid sequence corresponding to the above-mentioned mutated bases (Pro at position 281 is mutated to Ser) is shown in SEQ ID NO.16. SEQ ID NO.16 。

[0071] The original gene sequence of polyphosphate kinase 2CGL_RS04595 of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO.17. SEQ ID NO.17 ATGCGAAAGAAAAAAGACGGTCAAAATCTCCCAGACTTCCGGAAAAATCCGCCAAAGCTGGATAAAAAGGCTTATGAAAAAGAACTAAAAAGACTTCAAGCCGAACTCGTCGATTTGCAACAATGGGTTGTGGAAACCGGTGCGCGCGTGGTCATCGTCATGGAAGGCCGCGACGCCGCTGGTAAAGGTTCTGCGATCAAGCGCATTACGCAGTACCTCAACCCCCGGTCCGCAAGGATCGAAGCGCTGCCCACCCCAAACTCTCGGGAAAAAGGGCAGTGGTATTTCCAGCGCTACATCGAAAAATTGCCGACTGCTGGTGAGATCGTTATCTTTGACCGCTCCTGGTACAACCGTGCAGGAGTCGAGCGCGTCATGGGATTTTGCACCTCCCAGGAGTACCGCCGATTCCTTCACCAGGCACCAATCTTTGAACGCCTGTTGGTGGAAGATGGCATTCACCTGCGTAAATACTGGTTCTCTGTATCTGATGAAGAGCAGATTGAGCGTTTCGAAGACCGCCTGAGCGATCCGCTGCGCCGGTGGAAGTTGTCGCCAATGGATTTACAATCGATCACCCGCTGGGAAGATTACTCACGCGCAAAAGATGAGATGTTCATCCACACGGACATCCCGTCAGCACCGTGGTACACGGTGGAATCTGAGGACAAGAAGCGTTCCCGCATCAACGTCATTTCGCATCTGCTCTCGACGATTCCTTATGAGAAGATCGATCGTCCATTGCCGGAAATCCCTCATCGCCCAGATTCTGAATCTGATTATGTACGTCCCCCTCGCGATGAGTTCCGTTATGTTCCAGATGTGGCAGCACACTTGGAAGAAGAGCGCATCAAGAAAGAAGAAAAAGCCAAGAAGGCAAAGAAGCCAGCTAAGGCTGCAGGAAAGAACTCGGATAAGCAGAAGTCTTCCGGAGGAAAAGGCAAGAAGAAGTCCAAGAAATAG。

[0072] The amino acid sequence corresponding to the original bases of the above-mentioned polyphosphoric acid kinase 2CGL_RS04595 is shown in SEQ ID NO.18. SEQ ID NO.18 MRKKKDGQNLPDFRKNPPKLDKKAYEKELKRLQAELVDLQQWVVETGARVVIVMEGRDAAGKGSAIKRITQYLNPRSARIEALPTPNSREKGQWYFQRYIEKLPTAGEIVIFDRSWYNRAGVERVMGFTCTSQEYRRFLHQAPIFERLLVEDGIHLRKYWF SVSDEEQIERFEDRLSDPLRRWKLSPMDLQSITRWEDYSRAKDEMFIHTDIPSAPWYTVESEDKKRSRINVISHLLSTIPYEKIDRPLPEIPHRPDSESDYVRPPRDEFRYVPDVAAHLEEERIKKEEKAKKAKKPAKAAGKNSDKQKSSGGKGKKKSKK.

[0073] Mutant 5E16 is a mutation of C to T at position 226 of the polyphosphate kinase 2CGL_RS04595 gene sequence of the originating strain Corynebacterium glutamicum ATCC 13032, as shown in SEQ ID NO. 19. SEQ ID NO.19 ATGCGAAAGAAAAAAGACGGTCAAAATCTCCCAGACTTCCGGAAAAATCCGCCAAAGCTGGATAAAAAGGCTTATGAAAAAGAACTAAAAAGACTTCAAGCCGAACTCGTCGATTTGCAACAATGGGTTGTGGAAACCGGTGCGCGCGTGGTCATCGTCATGGAAGGCCGCGACGCCGCTGGTAAAGGTTCTGCGATCAAGCGCATTACGCAGTACCTCAACCCCTGGTCCGCAAGGATCGAAGCGCTGCCCACCCCAAACTCTCGGGAAAAAGGGCAGTGGTATTTCCAGCGCTACATCGAAAAATTGCCGACTGCTGGTGAGATCGTTATCTTTGACCGCTCCTGGTACAACCGTGCAGGAGTCGAGCGCGTCATGGGATTTTGCACCTCCCAGGAGTACCGCCGATTCCTTCACCAGGCACCAATCTTTGAACGCCTGTTGGTGGAAGATGGCATTCACCTGCGTAAATACTGGTTCTCTGTATCTGATGAAGAGCAGATTGAGCGTTTCGAAGACCGCCTGAGCGATCCGCTGCGCCGGTGGAAGTTGTCGCCAATGGATTTACAATCGATCACCCGCTGGGAAGATTACTCACGCGCAAAAGATGAGATGTTCATCCACACGGACATCCCGTCAGCACCGTGGTACACGGTGGAATCTGAGGACAAGAAGCGTTCCCGCATCAACGTCATTTCGCATCTGCTCTCGACGATTCCTTATGAGAAGATCGATCGTCCATTGCCGGAAATCCCTCATCGCCCAGATTCTGAATCTGATTATGTACGTCCCCCTCGCGATGAGTTCCGTTATGTTCCAGATGTGGCAGCACACTTGGAAGAAGAGCGCATCAAGAAAGAAGAAAAAGCCAAGAAGGCAAAGAAGCCAGCTAAGGCTGCAGGAAAGAACTCGGATAAGCAGAAGTCTTCCGGAGGAAAAGGCAAGAAGAAGTCCAAGAAATAG。

[0074] The amino acid sequence corresponding to the above-mentioned mutated base sequence (Arg at position 184 is mutated to Trp) is shown in SEQ ID NO. 20. SEQ ID NO.20 MRKKKDGQNLPDFRKNPPKLDKKAYEKELKRLQAELVDLQQWVVETGARVVIVMEGRDAAGKGSAIKRITQYLNPWSARIEALPTPNSREKGQWYFQRYIEKLPTAGEIVIFDRSWYNRAGVERVMGFTCTSQEYRRFLHQAPIFERLLVEDGIHLRKYWF SVSDEEQIERFEDRLSDPLRRWKLSPMDLQSITRWEDYSRAKDEMFIHTDIPSAPWYTVESEDKKRSRINVISHLLSTIPYEKIDRPLPEIPHRPDSESDYVRPPRDEFRYVPDVAAHLEEERIKKEEKAKKAKKPAKAAGKNSDKQKSSGGKGKKKSKK.

[0075] The original gene sequence of the 1,4-α-glucan branched protein GlgBCGL_RS06100 of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO. 21. SEQ ID NO.21

[0076] The amino acid sequence corresponding to the original gene of the 1,4-α-glucan branching protein GlgBCGL_RS06100 is shown in SEQ ID NO.22 SEQ ID NO.22 MRSPAPVTCGQRRTLSASSPYAMSPTSLFFLNFQRLAVSVSRGAKSRPTARNFPSLYLLSRIIIMTVDPASHITIPEADLARLRHCNHHDPHGFYGWHETEAGSVIRTRQVGATQVNLLIDDTSHVMTPIGDDIFAIDLGHRERADYRLEVTWPDQEPQVKADPYYFLPTVGEMDIYLFSEGRHERLWEILGANIKTYQTALGTVRGTAFTVWAPNAIGCAVVGGFNGWNASQHPMRSMGGSGLWELFIPGIEEGEVYKFAVQTREGQRRDKADPMARRAELAPATGSIVASSEYQWQDSEWLRERSQTDLASKPMSVYEVHLGSWRWGKNYEDLATELVDYVADLGYTHVEFLPVAEHPFGGSWGYQVTGYYAPTSRWGTPDQFRALVDAFHARGIGVIMDWVPAHFPKDDWALARFDGEALYEHPDWRRGEQKDWGTLVFDFGRNEVRNFLVANALYWIEEFHIDGLRVDAVASMLYLDYSREHGEWEPNIYGGRENLEAVQFLQEMNATVLRLHPGALTIAEESTSWPGVTAPTWDGGLGFSLKWNMGWMHDTLEYFSKNPVHRAFHHSELTFSLVYAFSERFVLPISHDEVVHGKGSLWDRMPGDTWNKAAGLRTFLAYMWSHPGKKLLFMGQEFGQREEWAEGQGLPWDIVDGWQGEYHEAIRTLTRSLNGVYSDSPALHTQDFTGEGFTWNKGDDATNNILAFTRFGSDGSQMLCVFNLSGTSQPEYQLGVAAGGEWKLVLNTDDAEFLGAENDIATSVQAAATPRDNFAYSLSLHVPAMSAQFYSLQK。

[0077] Mutant 5E16 is derived by mutating G to A at position 996 of the 1,4-α-glucan branch protein GlgBCGL_RS06100 gene sequence of the originating strain Corynebacterium glutamicum ATCC 13032, as shown in SEQ ID NO. 23. SEQ ID NO.23

[0078] The amino acid sequence corresponding to the above-mentioned base gene (His is mutated to Tyr at position 465) is shown in SEQ ID NO.24. SEQ ID NO.24 .

[0079] The original gene sequence of the large subunit CGL_RS06555 of the isopropyl malate isomerase of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO. 25. SEQ ID NO.25

[0080] The amino acid sequence corresponding to the original gene of the large subunit CGL_RS06555 of the isopropyl malate isomerase is shown in SEQ ID NO. 26. SEQ ID NO.26 MTSPVENSTSTEKLTLAEKVWRDHVVSKGENGEPDLLYIDLQLLHEVTSPQAFDGLRMTGRKLRHPELHLATEDHNVPTEGIKTGSLLEINDKISRLQVSTLRDNCEEFGVRLHPMGDVR QGIVHTVGPQLGATQPGMTIVCGDSHTSTHGAFGSMAFGIGTSEVEHVMATQTLPLKPFKTMAIEVTGELQPGVSSKDLILAIIAKIGTGGGQGYVLEYRGEAIRKMSMDARMTMCNMSIE AGARAGMIAPDQTTFDYVEGREMAPKGADWDEAVAYWKTLPTDEGATFDKVVEIDGSALTPFITWGTNPGQGLPLGESVPSPEDFTNDNDKAAAEKALQYMDLVPGTPLRDIKIDTVFLG SCTNARIEDLQIAADILKGHKIADGMRMMVVPSSTWIKQEAEALGLDKIFTDAGAEWRTAGCSMCLGMNPDQLKPGERSASTSNRNFEGRQGPGGRTHLVSPAVAAATAIRGTLSSPADI.

[0081] Mutant 5E16 is derived from the above-mentioned mutation pathway, specifically the mutation of C to A at position 1351 and G to A at position 1367 of the CGL_RS06555 gene sequence of the large subunit of isopropyl malate isomerase from the starting strain Corynebacterium glutamicum ATCC 13032. The gene sequence is shown in SEQ ID NO. 27. SEQ ID NO.27

[0082] The amino acid sequences corresponding to the above-mentioned mutated genes (Arg at position 451 is mutated to Ser and Gly at position 456 is mutated to Asp) are shown in SEQ ID NO. 28. SEQ ID NO.28 MTSPVENSTSTEKLTLAEKVWRDHVVSKGENGEPDLLYIDLQLLHEVTSPQAFDGLRMTGRKLRHPELHLATEDHNVPTEGIKTGSLLEINDKISRLQVSTLRDNCEEFGVRLHPMGDVR QGIVHTVGPQLGATQPGMTIVCGDSHTSTHGAFGSMAFGIGTSEVEHVMATQTLPLKPFKTMAIEVTGELQPGVSSKDLILAIIAKIGTGGGQGYVLEYRGEAIRKMSMDARMTMCNMSIE AGARAGMIAPDQTTFDYVEGREMAPKGADWDEAVAYWKTLPTDEGATFDKVVEIDGSALTPFITWGTNPGQGLPLGESVPSPEDFTNDNDKAAAEKALQYMDLVPGTPLRDIKIDTVFLG SCTNARIEDLQIAADILKGHKIADGMRMMVVPSSTWIKQEAEALGLDKIFTDAGAEWRTAGCSMCLGMNPDQLKPGERSASTSNRNFEGSQGPGDRTHLVSPAVAAATAIRGTLSSPADI.

[0083] The original gene sequence of succinyldiaminopimelic acid transaminase CGL_RS05505 of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO. 29. SEQ ID NO.29

[0084] The amino acid sequence corresponding to the original gene of the succinyldiaminopimelic acid transaminase CGL_RS05505 is shown in SEQ ID NO. 30. SEQ ID NO.30 MTSRTPLVSVLPDFPWDSLASAKAKAASHPDGIVNLSVGTPVDPVAPSIQIALAEAAGFSGYPQTIGTPELRAAIRGALERRYNMTKLVDASLLPVVGTKEAIALLPFALGISGTVVIPEIAYPTYEVAVVAAGCTVLRSDSLFKLGPQIPSMMFINSPSNPTGKVLGIPHLRKVVKWAQENNV ILAADECYLGLGWDDENPPISILDPRVCDGDHTNLIAIHSLSKTSNLASYRAGYLVGDPALIGELTEVRKNLGLMVPFPIQQAMIAALNDDDQEAGQKLTYAIRRAKLMRALLESGFQVDNSEAGLYLWATREEPCRDTVDWFAERGILVAPGDFYGPRGAQHVRVAMTETDERVDAFVSRLS.

[0085] Mutant 5E16 is a mutation of C to T at position 626 of the succinyldiaminopimelic acid transaminase CGL_RS05505 gene sequence of the originating strain Corynebacterium glutamicum ATCC 13032, as shown in SEQ ID NO. 31. SEQ ID NO.31

[0086] The amino acid sequence corresponding to the above-mentioned mutated gene (Pro at position 209 is mutated to Leu) is shown in SEQ ID NO.32. SEQ ID NO.32 MTSRTPLVSVLPDFPWDSLASAKAKAASHPDGIVNLSVGTPVDPVAPSIQIALAEAAGFSGYPQTIGTPELRAAIRGALERRYNMTKLVDASLLPVVGTKEAIALLPFALGISGTVVIPEIAYPTYEVAVVAAGCTVLRSDSLFKLGPQIPSMMFINSPSNPTGKVLGIPHLRKVVKWAQENNV ILAADECYLGLGWDDENPPISILDLRVCDGDHTNLIAIHSLSKTSNLASYRAGYLVGDPALIGELTEVRKNLGLMVPFPIQQAMIAALNDDDQEAGQKLTYAIRRAKLMRALLESGFQVDNSEAGLYLWATREEPCRDTVDWFAERGILVAPGDFYGPRGAQHVRVAMTETDERVDAFVSRLS.

[0087] The original gene sequence of the homoserine dehydrogenase CGL_RS05895 of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO.33. SEQ ID NO.33

[0088] The amino acid sequence corresponding to the original gene of the homoserine dehydrogenase CGL_RS05895 is shown in SEQ ID NO. 34. SEQ ID NO.34 MTSASAPSFNPGKGPGSAVGIALLGFGTVGTEVMRLMTEYGDELAHRIGGPLEVRGIAVSDISKPREGVAPELLTEDAFALIEREDVDIVVEVIGGIEYPREVVLAALKAG KSVVTANKALVAAHSAELADAAEAANVDLYFEAAVAGAIPVVGPLRRSLAGDQIQSVMGIVNGTTNFILDAMDSTGADYADSLAEATRLGYAEADPTADVEGHDAASKAAIL ASIAFHTRVTADDVYCEGISNISAADIEAAQQAGHTIKLLAICEKFTNKEGKSAISARVHPTLLPVSHPLASVNKSFNAIFVEAEAAGRLMFYGNGAGGAPTASAVLGDVV GAARNKVHGGRAPGESTYANLPIADFGETTTRYHLDMDVEDRVGVLAELASLFSEQGISLRTIRQEERDDDDARLIVVTHSALESDLSRTVELLKAKPVVKAINSVIRLERD.

[0089] Mutant 5E16 is a mutation in the gene sequence of the homoserine dehydrogenase CGL_RS05895 from the originating strain Corynebacterium glutamicum ATCC 13032, where a T-to-C mutation occurs at position 176. The gene sequence is shown in SEQ ID NO. 35. SEQ ID NO.35

[0090] The amino acid sequence corresponding to the above-mentioned mutated gene (Val at position 59 is mutated to Ala) is shown in SEQ ID NO.36. SEQ ID NO.36 MTSASAPSFNPGKGPGSAVGIALLGFGTVGTEVMRLMTEYGDELAHRIGGPLEVRGIAASDISKPREGVAPELLTEDAFALIEREDVDIVVEVIGGIEYPREVVLAALKAG KSVVTANKALVAAHSAELADAAEAANVDLYFEAAVAGAIPVVGPLRRSLAGDQIQSVMGIVNGTTNFILDAMDSTGADYADSLAEATRLGYAEADPTADVEGHDAASKAAIL ASIAFHTRVTADDVYCEGISNISAADIEAAQQAGHTIKLLAICEKFTNKEGKSAISARVHPTLLPVSHPLASVNKSFNAIFVEAEAAGRLMFYGNGAGGAPTASAVLGDVV GAARNKVHGGRAPGESTYANLPIADFGETTTRYHLDMDVEDRVGVLAELASLFSEQGISLRTIRQEERDDDDARLIVVTHSALESDLSRTVELLKAKPVVKAINSVIRLERD.

[0091] The original gene sequence of acetylglutamate kinase CGL_RS06975 of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO.37. SEQ ID NO.37 ATGAATGACTTGATCAAAGATTTAGGCTCTGAGGTGCGCGCAAATGTCCTCGCTGAGGCGTTGCCATGGTTGCAGCACTTCCGCGACAAGATTGTTGTCGTGAAATATGGCGGAAACGCCATGGTGGATGATGATCTCAAGGCTGCTTTTGCTGCCGACATGGTCTTCTTGCGCACCGTGGGCGCAAAACCAGTGGTGGTGCACGGTGGTGGACCTCAGATTTCTGAGATGCTAAACCGTGTGGGTCTCCAGGGCGAGTTCAAGGGTGGTTTCCGTGTGACCACTCCTGAGGTCATGGACATTGTGCGCATGGTGCTCTTTGGTCAGGTCGGTCGCGATTTAGTTGGTTTGATCAACTCTCATGGCCCTTACGCTGTGGGAACCTCCGGTGAGGATGCCGGCCTGTTTACCGCGCAGAAGCGCATGGTCAACATCGATGGCGTACCCACTGATATTGGTTTGGTCGGAGACATCATTAATGTCGATGCCTCTTCCTTGATGGATATCATCGAGGCCGGTCGCATTCCTGTGGTCTCTACGATTGCTCCAGGCGAAGACGGCCAGATTTACAACATTAACGCCGATACCGCAGCAGGTGCTTTGGCTGCAGCGATTGGTGCAGAACGCCTGCTGGTTCTCACCAATGTGGAAGGTCTGTACACCGATTGGCCTGATAAGAGCTCACTGGTGTCCAAGATCAAGGCCACCGAGCTGGAGGCCATTCTTCCGGGACTTGATTCCGGCATGATTCCAAAGATGGAGTCTTGCTTGAACGCGGTGCGTGGGGGAGTAAGCGCTGCTCATGTCATTGACGGCCGCATCGCGCACTCGGTGTTGCTGGAGCTTTTGACCATGGGTGGAATTGGCACGATGGTGCTGCCGGATGTTTTTGATCGGGAGAATTATCCTGAAGGCACCGTTTTTAGAAAAGACGACAAGGATGGGGAACTGTAA。

[0092] The amino acid sequence corresponding to the original gene of the acetylglutamate kinase CGL_RS06975 is shown in SEQ ID NO. 38. SEQ ID NO.38 MNDLIKDLGSEVRANVLAEALPWLQHFRDKIVVVKYGGNAMVDDDLKAAFAADMVFLRTVGAKPVVVHGGGPQISEMLNRVGLQGEFKGGFRVTTPEVMDIVRMVLFGQVGRDLVGLINSHGPYAVGTSGEDAGLFTAQKRMVNIDGVPTDIGLVGDII NVDASSLMDIIEAGRIPVVSTIAPGEDGQIYNINADTAAGALAAAIGAERLLVLTNVEGLYTDWPDKSSLVSKIKATELEAILPGLDSGMIPKMESCLNAVRGGVSAAHVIDGRIAHSVLLELLTMGGIGTMVLPDVFDRENYPEGTVFRKDDKDGEL.

[0093] Mutant 5E16 is derived by mutating G to A at position 133 of the acetylglutamate kinase CGL_RS06975 gene sequence of the originating strain Corynebacterium glutamicum ATCC 13032, as shown in SEQ ID NO. 39. SEQ ID NO.39 ATGAATGACTTGATCAAAGATTTAGGCTCTGAGGTGCGCGCAAATGTCCTCGCTGAGGCGTTGCCATGGTTGCAGCACTTCCGCGACAAGATTGTTGTCGTGAAATATGGCGGAAACGCCATGGTGGATGATAATCTCAAGGCTGCTTTTGCTGCCGACATGGTCTTCTTGCGCACCGTGGGCGCAAAACCAGTGGTGGTGCACGGTGGTGGACCTCAGATTTCTGAGATGCTAAACCGTGTGGGTCTCCAGGGCGAGTTCAAGGGTGGTTTCCGTGTGACCACTCCTGAGGTCATGGACATTGTGCGCATGGTGCTCTTTGGTCAGGTCGGTCGCGATTTAGTTGGTTTGATCAACTCTCATGGCCCTTACGCTGTGGGAACCTCCGGTGAGGATGCCGGCCTGTTTACCGCGCAGAAGCGCATGGTCAACATCGATGGCGTACCCACTGATATTGGTTTGGTCGGAGACATCATTAATGTCGATGCCTCTTCCTTGATGGATATCATCGAGGCCGGTCGCATTCCTGTGGTCTCTACGATTGCTCCAGGCGAAGACGGCCAGATTTACAACATTAACGCCGATACCGCAGCAGGTGCTTTGGCTGCAGCGATTGGTGCAGAACGCCTGCTGGTTCTCACCAATGTGGAAGGTCTGTACACCGATTGGCCTGATAAGAGCTCACTGGTGTCCAAGATCAAGGCCACCGAGCTGGAGGCCATTCTTCCGGGACTTGATTCCGGCATGATTCCAAAGATGGAGTCTTGCTTGAACGCGGTGCGTGGGGGAGTAAGCGCTGCTCATGTCATTGACGGCCGCATCGCGCACTCGGTGTTGCTGGAGCTTTTGACCATGGGTGGAATTGGCACGATGGTGCTGCCGGATGTTTTTGATCGGGAGAATTATCCTGAAGGCACCGTTTTTAGAAAAGACGACAAGGATGGGGAACTGTAA。

[0094] The amino acid sequence corresponding to the above-mentioned mutated gene (Asp at position 45 is mutated to Asn) is shown in SEQ ID NO.40. SEQ ID NO.40 MNDLIKDLGSEVRANVLAEALPWLQHFRDKIVVVKYGGNAMVDDNLKAAFAADMVFLRTVGAKPVVVHGGGPQISEMLNRVGLQGEFKGGFRVTTPEVMDIVRMVLFGQVGRDLVGLINSHGPYAVGTSGEDAGLFTAQKRMVNIDGVPTDIGLVGDII NVDASSLMDIIEAGRIPVVSTIAPGEDGQIYNINADTAAGALAAAIGAERLLVLTNVEGLYTDWPDKSSLVSKIKATELEAILPGLDSGMIPKMESCLNAVRGGVSAAHVIDGRIAHSVLLELLTMGGIGTMVLPDVFDRENYPEGTVFRKDDKDGEL.

[0095] The original gene sequence of the NADP-dependent phosphoglucose dehydrogenase CGL_RS07265 of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO. 41. SEQ ID NO.41

[0096] The amino acid sequence corresponding to the original gene of the NADP-dependent phosphoglucose dehydrogenase CGL_RS07265 is shown in SEQ ID NO. 42. SEQ ID NO.42 MPSSTINNMTNGDNLAQIGVVGLAVMGSNLARNFARNGNTVAVYNRSTDKTDKLIADHGSEGNFIPSATVEEFVASLEKPRRAIIMVQAGNATDAVINQLADAMDEGDIIIDGGNALYTDTIR REKEISARGLHFVGAGISGGEEGALNGPSIMPGGPAKSYESLGPLLESIAANVDGTPCVTHIGPDGAGHFVKMVHNGIEYADMQVIGEAYHLLRYAAGMQPAEIAEVFKEWNAGDLDSYLIEI TAEVLSQVDAETGKPLIDVIVDAAGQKGTGRWTVKAALDLGIATTGIGEAVFARALSGATSQRAAAQGNLPAGVLTDLEALGVDKAQFVEDVRRALYASKLVAYAQGFDEIKAGSDENNWDVD PRDLATIWRGGCIIRAKFLNRIVEAYDANAELESLLLDPYFKSELGDLIDSWRRVIVTATQLGLPIPVFASSLSYYDSLRAERLPAALIQGQRDFFGAHTYKRIDKDGSFHTEWSGDRSEVEA.

[0097] Mutant 5E16 is a mutation in the gene sequence of the NADP-dependent phosphoglucose dehydrogenase CGL_RS07265 from the originating strain Corynebacterium glutamicum ATCC 13032, where the G gene is changed to the A gene. The gene sequence is shown in SEQ ID NO. 43. SEQ ID NO.43

[0098] The amino acid sequence corresponding to the above-mentioned mutated gene (Ser is mutated to Phe at position 361) is shown in SEQ ID NO.44. SEQ ID NO.44 MPSSTINNMTNGDNLAQIGVVGLAVMGSNLARNFARNGNTVAVYNRSTDKTDKLIADHGSEGNFIPSATVEEFVASLEKPRRAIIMVQAGNATDAVINQLADAMDEGDIIIDGGNALYTDTIR REKEISARGLHFVGAGISGGEEGALNGPSIMPGGPAKSYESLGPLLESIAANVDGTPCVTHIGPDGAGHFVKMVHNGIEYADMQVIGEAYHLLRYAAGMQPAEIAEVFKEWNAGDLDSYLIEI TAEVLSQVDAETGKPLIDVIVDAAGQKGTGRWTVKAALDLGIATTGIGEAVFARALSGATSQRAAAQGNLPAGVLTDLEALGVDKAQFVEDVRRALYASKLVAYAQGFDEIKAGFDENNWDVD PRDLATIWRGGCIIRAKFLNRIVEAYDANAELESLLLDPYFKSELGDLIDSWRRVIVTATQLGLPIPVFASSLSYYDSLRAERLPAALIQGQRDFFGAHTYKRIDKDGSFHTEWSGDRSEVEA.

[0099] The original gene sequence of phosphoenolpyruvate carboxylase CGL_RS07925 of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO. 45. SEQ ID NO.45

[0100] The amino acid sequence corresponding to the original gene of the phosphoenolpyruvate carboxylase CGL_RS07925 is shown in SEQ ID NO. 46. SEQ ID NO.46 。

[0101] Mutant 5E16 is a mutation in the gene sequence of phosphoenolpyruvate carboxylase CGL_RS07925 from the originating strain Corynebacterium glutamicum ATCC 13032, where G is mutated to A at position 1316. The gene sequence is shown in SEQ ID NO. 47. SEQ ID NO.47

[0102] The amino acid sequence corresponding to the above-mentioned mutated gene (Ala is mutated to Val at position 482) is shown in SEQ ID NO. 48. SEQ ID NO.48 。

[0103] The original gene sequence of malate dehydrogenase (quinone) CGL_RS09955 of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO. 49. SEQ ID NO.49

[0104] The amino acid sequence corresponding to the original gene of the malate dehydrogenase (quinone) CGL_RS09955 is shown in SEQ ID NO. 50. SEQ ID NO.50 MANTCIRVLAIIYEMTPCVGVRQRTRGDKRKLNMSDSPKNAPRITDEADVVLIGAGIMSSTLGAMLRQLEPSWTQIVFERLDGPAQESSSPWNNAGTGHSALCELNYTPEVKGKVEIAKAVGINEKFQVSRQF WSHLVEEGVLSDPKEFINPVPHVSFGQGADQVAYIKARYEALKDHPLFQGMTYADDEATFTEKLPLMAKGRDFSDPVAISWIDEGTDINYGAQTKQYLDAAEVEGTEIRYGHEVKSIKADGAKWIVTVKNVHT GDTKTIKANFVFVGAGGYALDLLRSAGIPQVKGFAGFPVSGLWLRCTNEELIEQHAAKVYGKASVGAPPMSVPHLDTRVIEGEKGLLFGPYGGWTPKFLKEGSYLDLFKSIRPDNIPSYLGVAAQEFDLTKYL VTEVLKDQDKRMDALREYMPEAQNGDWETIVAGQRVQVIKPAGFPKFGSLEFGTTLINNSEGTIAGLGASPGASIAPSAMIELLERCFGDRMIEWGDKLKDMIPSYGKKLASEPALFEQQWARTQKTLKLEEA .

[0105] Mutant 5E16 is a mutation of C to T at position 832 of the N-malate dehydrogenase (quinone) CGL_RS09955 gene sequence of the originating strain Corynebacterium glutamicum ATCC 13032, as shown in SEQ ID NO. 51. SEQ ID NO.51 TCACTTTGCGCCATCAGCCTTGATGCTCTTGACTTCGTGGCCATAGCGGATTTCAGTGCCTTCAACTTCAGCTGCATCCAGGTACTGCTTGGTCTGAGCACCGTAGTTGATGTCGGTGCCTTCATCGATCCAAGAGATTGCTACTGGATCAGAGAAGTCACGGCCCTTTGCCATCAAAGGCAGCTTCTCGGTGAAGGTAGCTTCATCGTCAGCGTAGGTCATGCCCTGGAAGAGTGGGTGATCCTTCAAAGCTTCGTAGCGAGCCTTGATGTATGCAACCTGATCTGCGCCCTGGCCGAAAGATACGTGAGGAACAGGGTTGATGAATTCCTTAGGATCAGACAGCACTCCCTCTTCAACGAGGTGAGACCAGAACTGACGGGAAACCTGGAACTTCTCGTTGATTCCTACAGCCTTGGCAATTTCAACCTTGCCCTTAACCTCTGGGGTGTAGTTCAGCTCGCATAGAGCAGAGTGGCCGGTTCCTGCATTGTTCCACGGGGAGGACGACTCTTGTGCCGGTCCATCCAAACGCTCGAAGACGATCTGAGTCCAGCTTGGCTCCAGCTGACGCAGCATTGCACCCAGCGTGGAGCTCATGATACCGGCACCAATGAGAACTACATCTGCCTCATCGGTAATCCTCGGTGCGTTCTTCGGGGAATCTGACAT。

[0106] The amino acid sequence corresponding to the above mutant gene (Trp at the 257th position is mutated to a stop codon) is shown in SEQ ID NO.52 SEQ ID NO.5​​​

[0107] The original gene sequence of malate dehydrogenase CGL_RS11800 of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO. 53. SEQ ID NO.53 TTAGAGCAAGTCGCGCACTGCCTCGCGCTCGGCCTGCAATTCCTGAGCATTCGCGTCGATGCGGGCGCGCTGGAAATCGGAAATCTCCAGGCCTTCAACGATTTCCCACTCACCGTTGCGGGATACGGTTGGCAGACCGACAAAAATGCCCTCAGGAATGCCGTATGCACCGGTGGAAGGAATTGCCGCAGAGGACCACGCCTCGGTGCCCTGTACCCAATCGCGCATGTGATCAATCGCAGAGGATGCTGCAGAAGCTGCAGAAGACTTTCCACGGACCTCAATGATTTCAGCGCCACGGTTAGCCACGCGAGGAATGAACTCCTCCACATACCAATCGTGATCAACCAGGTCAGTGACCTTTTCTCCACCAACGGTTGCGTAGGTGATGTCTGGGAACTGGGTTGCGGAGTGATTTCCCCAGACCACAATGTTGTTAAATTCCGCAGATCCACGGCCAAGCTTGGTGGCCAGCTGGGAGATCGCACGGTTGTGATCAAGGCGCATCATTGCGTTGAAGCGGGATGCTGGAACATCTGGGGCCGCAGCTGAAGCAATCAACGCGTTGGTGTTCGCTGGGTTTCCAACAACTAGGACACGAATGTCATCTGCGGCGTTGTCATTGATAGCTTTACCTTGAGGTCCGAAAATCTTGCCGTTGTTAGCCAGCAAATCTGCGCGCTCTTCGCCTTTTCCGCGAGGCTTCGCACCGACCAAAAACGCCGCATTAGCGCCGTCGAATGCCTCATTGGCATCCGCGGTGATGGTGATGTTTCGCAGGAGGGGGAAGGCAGAATCCAGAAGTTCCATAGCCACACCCTCTGCCCCGCCAAGAGCCTGAGGGATCTCCAGAAGTTTCAGTTCTACAGGGGTGTCGGTGCCGAATACTTCACCGTTGGCGATGCGCCACAACAGTGAATAAGAGATTTGACCAGCTGCGCCGGTGACGGTGACCTTCTTGGTGGAGACGTTCTGCGGGGAATTCAT。

[0108] The amino acid sequence corresponding to the original gene of the malate dehydrogenase CGL_RS11800 is shown in SEQ ID NO. 54. SEQ ID NO.54 MNSPQNVSTKKVTVTGAAGQISYSLLWRIANGEVFGTDTPVELKLLEIPQALGGAEGVAMELLDSAFPLLRNITITADANEAFDGANAAFLVGAKPRGKGEERADLLANNGKIFGPQGKAINDNAADDIRVLVVGNPANTNALIASAAAPDVPASRFNAMMRLD HNRAISQLATKLGRGSAEFNNIVVWGNHSATQFPDITYATVGGEKVTDLVDHDWYVEEFIPRVANRGAEIIEVRGKSSAASAASSAIDHMRDWVQGTEAWSSAAIPSTGAYGIPEGIFVGLPTVSRNGEWEIVEGLEISDFQRARIDANAQELQAEREAVRDLL.

[0109] Mutant 5E16 is derived by mutating G to A at position 173 of the malate dehydrogenase CGL_RS11800 gene sequence of the originating strain Corynebacterium glutamicum ATCC 13032, as shown in SEQ ID NO. 55. SEQ ID NO.55 TTAGAGCAAGTCGCGCACTGCCTCGCGCTCGGCCTGCAATTCCTGAGCATTCGCGTCGATGCGGGCGCGCTGGAAATCGGAAATCTCCAGGCCTTCAACGATTTCCCACTCACCGTTGCGGGATACGGTTGGCAGACCGACAAAAATGCCCTCAGGAATGCCGTATGCACCGATGGAAGGAATTGCCGCAGAGGACCACGCCTCGGTGCCCTGTACCCAATCGCGCATGTGATCAATCGCAGAGGATGCTGCAGAAGCTGCAGAAGACTTTCCACGGACCTCAATGATTTCAGCGCCACGGTTAGCCACGCGAGGAATGAACTCCTCCACATACCAATCGTGATCAACCAGGTCAGTGACCTTTTCTCCACCAACGGTTGCGTAGGTGATGTCTGGGAACTGGGTTGCGGAGTGATTTCCCCAGACCACAATGTTGTTAAATTCCGCAGATCCACGGCCAAGCTTGGTGGCCAGCTGGGAGATCGCACGGTTGTGATCAAGGCGCATCATTGCGTTGAAGCGGGATGCTGGAACATCTGGGGCCGCAGCTGAAGCAATCAACGCGTTGGTGTTCGCTGGGTTTCCAACAACTAGGACACGAATGTCATCTGCGGCGTTGTCATTGATAGCTTTACCTTGAGGTCCGAAAATCTTGCCGTTGTTAGCCAGCAAATCTGCGCGCTCTTCGCCTTTTCCGCGAGGCTTCGCACCGACCAAAAACGCCGCATTAGCGCCGTCGAATGCCTCATTGGCATCCGCGGTGATGGTGATGTTTCGCAGGAGGGGGAAGGCAGAATCCAGAAGTTCCATAGCCACACCCTCTGCCCCGCCAAGAGCCTGAGGGATCTCCAGAAGTTTCAGTTCTACAGGGGTGTCGGTGCCGAATACTTCACCGTTGGCGATGCGCCACAACAGTGAATAAGAGATTTGACCAGCTGCGCCGGTGACGGTGACCTTCTTGGTGGAGACGTTCTGCGGGGAATTCAT。

[0110] The amino acid sequence corresponding to the above-mentioned mutated gene (Th at position 272 is mutated to Ile) is shown in SEQ ID NO. 56. SEQ ID NO.56 MNSPQNVSTKKVTVTGAAGQISYSLLWRIANGEVFGTDTPVELKLLEIPQALGGAEGVAMELLDSAFPLLRNITITADANEAFDGANAAFLVGAKPRGKGEERADLLANNGKIFGPQGKAINDNAADDIRVLVVGNPANTNALIASAAAPDVPASRFNAMMRLD HNRAISQLATKLGRGSAEFNNIVVWGNHSATQFPDITYATVGGEKVTDLVDHDWYVEEFIPRVANRGAEIIEVRGKSSAASAASSAIDHMRDWVQGTEAWSSAAIPSIGAYGIPEGIFVGLPTVSRNGEWEIVEGLEISDFQRARIDANAQELQAEREAVRDLL.

[0111] The original gene sequence of the LysE family transporter CGL_RS13250 of the originating strain Corynebacterium glutamicum ATCC 13032 is shown in SEQ ID NO. 57. SEQ ID NO.57 CTAAGTGATAAGCCCATAAGCAGTATTAACCAGTAATCCGGCACCAATGAGAAACAGTGCGATGCCACCAACCAGGGTGATAATGCGATCCGCAGACGGTAACTTTTGCAGACCAATACCCCTGACCAATGCGATTGTTCCCAACCACCACGCTCCTGAGCCAAGCACGATGGTGGCACACAATGCCAAGGTCCTCATTTTCATATCTTCCCCATTCCCAATGAACTGAGGAAGAATCGCTGCAAAGAACAACAGCGCTTTCGGGTTCGTGGCATTGGTGATAAATCCTCGAAAATAACCTGCACTCGATTGACTAGCAGCAGTTTCAGATTCCCCGGTATTTTGGGAAGCACGAAACATGTTCGTGCCCATCCACAAAAGCACTCCCGCACCAAGAAGTTGAATAGCGCTCAATACTCCCGGAGCTGATAGCAATAATGCAGTTGCTCCGGCTATCGCAAGACTCGCATGTAACATCAGTCCCGTCATGATTCCTGCCGCAGTCATGACCCCCGTGCGGATCCCGCGGGTTGCAGAATGTAGAACAAGAACAAGGTCAGGTCCGGGCACCGCTAATGCCACCAATAATGCGAGTGCGAATGCGACCCATGATGCTGCGTCCAC。

[0112] The amino acid sequence corresponding to the original gene of the LysE family transporter CGL_RS13250 is shown in SEQ ID NO.58 SEQ ID NO.58 MTAAGIMTGLMLHASLAIAGATALLLSAPGVLSAIQLLGAGVLLWMGTNMFRASQNTGESETAASQSSAGYFRGFITNATNPKALLFFAAILPQFIGNGEDMKMRTLALCATIVLGSGAWWLGTIALVRGIGLQKLPSADRIITLVGGIALFLIGAGLLVNTAYGLIT。

[0113] Mutant 5E16 is a mutation from T to C at position 48 of the LysE family transporter CGL_RS13250 gene sequence of the originating strain Corynebacterium glutamicum ATCC 13032, as shown in SEQ ID NO. 59. SEQ ID NO.59 .

[0114] The amino acid sequence corresponding to the above-mentioned mutated gene (Ile is mutated to Val at position 154) is shown in SEQ ID NO. 60. SEQ ID NO.60 MTAAGIMTGLMLHASLAIAGATALLLSAPGVLSAIQLLGAGVLLWMGTNMFRASQNTGESETAASQSSAGYFRGFITNATNPKALLFFAAILPQFIGNGEDMKMRTLALCATIVLGSGAWWLGTIALVRGIGLQKLPSADRIITLVGGIALFLVGAGLLVNTAYGLIT.

[0115] Example 3: Operation of integrated process for obtaining crude product from syngas fermentation and lysine fermentation Figure 5 An integrated process for obtaining crude products from syngas fermentation and lysine fermentation.

[0116] Syngas storage tanks are primarily used for syngas storage, gas ratio adjustment, and gas supply. If the upstream gas source is pre-mixed syngas, it can be directly stored and supplied in the syngas tank. If the upstream gas source is CO2, H2, or other proportions of syngas supplied independently, H2, CO2, and syngas can be introduced into the syngas tank separately, and the ratios can be adjusted before storage and supply. The effective component ratio in the syngas is H2:CO2 = 7:3 (volume ratio), and the gas pressure is 0.5 MPa.

[0117] The anaerobic fermenter was a syngas fermentation reactor for acetate (salt). Syngas from the syngas tank was introduced according to the method described in Example 1, with a flow rate controlled at 1000 mL / min. *Clostridium yongdarii* was inoculated at a rate of 10 wt%. The culture medium was PETC. The temperature was controlled at 36°C. The stirring speed was controlled at 500 rpm. During fermentation, the pH was adjusted with ammonia and maintained at 6.0. The fermentation broth was collected after approximately 130 h of fermentation (ammonium acetate concentration greater than 50 g / L).

[0118] Membrane retention device 1 is used to separate the bacterial broth from the anaerobic fermenter (i.e., the fermentation broth collected after 130 h of fermentation) into concentrated broth and clear broth using the device's interception effect. Part of the concentrated broth is returned to the fermenter, and part enters the cell recovery device. The clear broth enters the crude acetate solution storage tank and the concentration and purification system. The membrane retention device is a ceramic membrane with a pore size of 50 nm.

[0119] The crude acetate solution storage tank stores the supernatant from the anaerobic syngas fermentation in the anaerobic fermenter used in the above process. The supernatant is mainly ammonium acetate solution with an ammonium acetate concentration of 60 g / L and contains less than 5 g / L of ethanol.

[0120] Then, using the microorganisms obtained above, the supernatant obtained from the fermentation of syngas was used to prepare a fermentation medium for the production of lysine under aerobic conditions.

[0121] The fermentation medium consisted of the following components added per liter of the supernatant obtained from the fermentation of the microorganisms using syngas as a substrate: glucose 40 g / L; soybean meal hydrolysate 20 g / L; molasses 10 g / L; potassium dihydrogen phosphate 1 g / L; dipotassium hydrogen phosphate 1 g / L; magnesium sulfate 1 g / L; ferrous sulfate 100 mg / L; manganese sulfate 100 mg / L; zinc sulfate 0.5 mg / L; anhydrous copper sulfate 0.5 mg / L; biotin 0.5 mg / L; and vitamin B3 40 mg / L.

[0122] An aerobic fermenter was used for the acetate-based fermentation of lysine. The prepared fermentation medium was added, and then *Corynebacterium glutamicum* 5E16, selected through screening, was inoculated at a rate of 10 wt%. The temperature was controlled at 34°C, and the stirring speed was controlled at 800 rpm. The pH was maintained at approximately 7.2 during fermentation and monitored in real time to obtain lysine.

[0123] The obtained fermentation broth is further purified to obtain pure lysine. Specifically, the bacterial solution in the aerobic fermenter is introduced into membrane retention device 2. Utilizing the flow-blocking effect of this device, the bacterial solution is separated into concentrated bacterial solution and clear liquid. The concentrated bacterial solution enters the cell recovery device. The clear liquid enters the concentration and purification system. The membrane retention device is a ceramic membrane with a pore size of 50 nm.

[0124] The clear liquid obtained from the anaerobic fermenter after the above separation is first passed through an electrodialysis device, and then further processed through a multi-effect continuous evaporation crystallization device to produce lysine hydrochloride or lysine sulfate products.

[0125] Example 4 Strain: Corynebacterium glutamicum 5E16; Reactor: 5L aerobic stirred tank; Culture medium: For each liter of the supernatant obtained by fermentation of the microorganisms described in Example 3 above using syngas as a substrate, glucose 40 g / L; soybean meal hydrolysate 20 g / L; molasses 10 g / L; potassium dihydrogen phosphate 1 g / L; dipotassium hydrogen phosphate 1 g / L; magnesium sulfate 1 g / L; ferrous sulfate 100 mg / L; manganese sulfate 100 mg / L; zinc sulfate 0.5 mg / L; anhydrous copper sulfate 0.5 mg / L; biotin 0.5 mg / L; vitamin B3 40 mg / L.

[0126] Fermentation conditions: temperature 32℃, pH controlled at 7.2 using ammonia and acetic acid solutions; dissolved oxygen level maintained at 40% through stirring and aeration; inoculum size 10%. During fermentation, the stirring speed and gas flow rate were gradually increased as the biomass increased. During fermentation, the fed-batch medium maintained the acetate content in the tank at 10 g / L. Figure 6As can be seen, after 100 hours of cultivation, 2 ml of fermentation broth was taken, centrifuged at 12,000 rpm for 10 minutes to obtain the supernatant, diluted 300 times, and the lysine content was detected by the S-10 biosensor analyzer of Silman Technology Co., Ltd. The average concentration was 0.85 g / L, and the actual concentration was 255 g / L.

Claims

1. A strain that uses syngas fermentation products as substrates to produce lysine, characterized in that: The strain is Corynebacterium glutamicum ( Corynebacterium glutamicum 5E16 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 27, 2025, with accession number GDMCC No:67371.

2. The application of the strain according to claim 1, which uses syngas fermentation products as a substrate to produce lysine, characterized in that: Application of the strain in the fermentation production of lysine.

3. The application of the strain that utilizes syngas fermentation products as a substrate to produce lysine according to claim 2, characterized in that: Application of the strain in the fermentation production of lysine using syngas fermentation products as substrates.

4. The application of the strain that utilizes syngas fermentation products as a substrate to produce lysine according to claim 3, characterized in that: Fermentation temperature is controlled at 32~35℃, pH at 7.0~7.2, and dissolved oxygen at 30%~40%.

5. The application of the strain that utilizes syngas fermentation products as a substrate to produce lysine according to claim 3, characterized in that: Syngas fermentation products contain ammonium acetate or other acetates.

6. The application of the strain that utilizes syngas fermentation products as a substrate to produce lysine according to claim 3 or 5, characterized in that: The fermentation medium consisted of the following ingredients added per liter of supernatant obtained from microbial fermentation using syngas as a substrate: glucose 0-40 g / L, soybean meal hydrolysate 20 g / L, molasses 10 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium sulfate 1 g / L, ferrous sulfate 100 mg / L, manganese sulfate 100 mg / L, zinc sulfate 0.5 mg / L, anhydrous copper sulfate 0.5 mg / L, biotin 0.5 mg / L, and vitamin B3 40 mg / L. The ammonium acetate concentration in the supernatant obtained from microbial fermentation using syngas as a substrate was 40-60 g / L.

7. The application of the strain according to claim 6, which uses syngas fermentation products as substrates to produce lysine, is characterized in that: The process involves microorganisms using syngas as a substrate for fermentation to obtain a supernatant. This process involves anaerobic fermentation of microorganisms in a liquid culture medium in the presence of syngas at 30–60°C, pH 4.5–6.5, and with stirring, followed by separation of the supernatant. The syngas mainly consists of H2 and CO2, with a volume ratio of 9:1 to 5:

5. The microorganisms are one or more types of aerobic microorganisms.

8. The application of the strain that utilizes syngas fermentation products as a substrate to produce lysine according to claim 7, characterized in that: The microorganism is Clostridium difficile, a self-producing alcohol-producing bacterium (… Clostridium autoethanogenum Clostridium yongdarii ( Clostridium ljungdahlii ), Clostridium carbonmonoxide ( Clostridium carboxidivorans ), Acetobacter wuerii ( Acetobacterium woodii ) and hot acetylcholine ( Moorella thermoacetica ).

9. The application of the strain according to claim 7 that uses syngas fermentation products as substrates to produce lysine, characterized in that: The selected synthesis gas flow rate is 1~20 L / min.

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