IlvC mutant, valine-producing strain and application thereof

Abstract

The application provides an ilvC mutant, a valine-producing strain and application thereof, and in the construction process, first, Corynebacterium glutamicum ATCC 13032 is used as a mutagenesis starting point, key positive mutation sites on an ilvC gene are identified from a high-valine-producing Corynebacterium glutamicum mutant strain screened through ARTP mutagenesis, and the specific mutations are as follows: arginine at the 49th position is mutated into lysine, serine at the 52nd position is mutated into threonine, glutamine at the 87th position is mutated into histidine, and glycine at the 133rd position is mutated into cysteine; subsequently, the gene editing technology is used to integrate the mutant ilvC gene fragment into the genome of Escherichia coli W3110 as a chassis strain, and the valine synthesis capacity can still be effectively enhanced; the strain effectively improves the valine production capacity by introducing the exogenous positive mutation site.

Description

Technical Field

[0001] This invention relates to the fields of metabolic engineering and genetic engineering, and in particular to an ilvC mutant, a valine-producing strain and their applications. Background Technology

[0002] L-valine (chemical name: 2-amino-3-methylbutyric acid) is a branched-chain amino acid that is not only a basic building block of protein synthesis but also plays a crucial role in physiological processes such as muscle metabolism, tissue repair, and energy supply. Due to its unique branched-chain structure, L-valine is widely used in the pharmaceutical, food, feed, and cosmetic industries, and has significant application value in areas such as antibiotic synthesis, nutritional preparations, and functional additives.

[0003] Currently, the main methods for producing L-valine include chemical synthesis, direct extraction, and microbial fermentation. Among these, microbial fermentation has become the mainstream industrial production method due to its advantages such as low raw material costs, mild reaction conditions, environmental friendliness, and ease of large-scale production. Among various production strains, *Escherichia coli* has gradually become a dominant strain in the field of amino acid production due to its clear genetic background, short fermentation cycle, and mature gene manipulation technology.

[0004] In recent years, researchers have enhanced the valine production capacity of *E. coli* through metabolic engineering strategies: increasing the expression of key enzymes such as acetolactate synthase, knocking out competing pathway genes, and optimizing the transport system, effectively increasing the valine synthesis flux. At the fermentation process level, strategies such as two-stage dissolved oxygen control have further improved production intensity. Although studies have reported that engineered *E. coli* strains can achieve high yields in fermenters, current industrial production still faces technical bottlenecks such as uneven carbon flux distribution and imbalances in cofactor supply and demand. Therefore, constructing a high-yielding L-valine-producing *E. coli* strain with a clear genetic background, optimized metabolism, and stable performance remains a core technical challenge that urgently needs to be overcome in this field. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an ilvC mutant.

[0006] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned ilvC mutant.

[0007] The technical problem to be solved by the present invention is to provide a valine-producing strain constructed using the above-mentioned ilvC mutant.

[0008] Another technical problem to be solved by the present invention is to provide a method for constructing the above-mentioned valine-producing strain.

[0009] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned valine-producing strain.

[0010] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: An ilvC mutant is obtained by mutating the wild-type ilvC gene. Specifically, the mutations are as follows: arginine (Arg) at position 49 is mutated to lysine (Lys), serine (Ser) at position 52 is mutated to threonine (Thr), glutamine (Gln) at position 87 is mutated to histidine (His), and glycine (Gly) at position 133 is mutated to cysteine ​​(Cys). The amino acid sequence of the wild-type ilvC gene is shown in SEQ ID NO.4 (the nucleotide sequence is shown in SEQ ID NO.3).

[0011] Preferably, the above-mentioned ilvC mutant has an amino acid sequence as shown in SEQ ID NO.2 (nucleotide sequence as shown in SEQ ID NO.1) or is more than 95% identical to the sequence and originates from the same species.

[0012] The biological material associated with the above-mentioned ilvC mutant is any one of the following (a1) to (a4): (a1) The nucleic acid molecule encoding the above-mentioned ilvC mutant; (a2) An expression cassette containing the nucleic acid molecule described in (a1): (a3) A recombinant vector comprising the nucleic acid molecule described in (a1) or the expression cassette described in (a2); (a4) A recombinant microorganism comprising the nucleic acid molecule of (a1), the expression cassette of (a2), or the recombinant vector of (a3).

[0013] Preferably, in the above-mentioned biological material, the nucleotide sequence of the nucleic acid molecule encoding the ilvC mutant in (a1) is as shown in the sequence listing SEQ ID NO.1 or is more than 95% identical to the sequence and originates from the same species.

[0014] Preferably, in the above-mentioned biomaterials, the vector skeleton of the recombinant vector in (a3) ​​includes pGRB, pK18, pK19, pK18mobsacB, pNV18, or pNV19, etc.

[0015] Preferably, in the above-mentioned biological material, the recombinant microorganisms in (a4) include Escherichia coli, Corynebacterium glutamicum, or Bacillus.

[0016] The above-mentioned ilvC mutants or biological materials are used in the construction of valine-producing strains or in the fermentation production of valine.

[0017] A valine-producing strain, obtained by modifying a starting strain, wherein the modification includes: introducing multiple mutation sites into the wild-type ilvC gene, and the nucleotide sequence of the mutant ilvC gene after the introduction of multiple mutation sites is as shown in SEQ ID NO.1 of the sequence listing or is more than 95% identical to the sequence and originates from the same species.

[0018] Preferably, the valine-producing strain mentioned above is *Escherichia coli* (E. coli). Escherichia coli ).

[0019] Preferably, the valine-producing strain mentioned above is Escherichia coli W3110.

[0020] Preferably, in the above-mentioned valine-producing strain, the nucleotide sequence of the wild-type ilvC gene is as shown in SEQ ID NO.3 of the sequence listing, or is more than 95% identical to the sequence and originates from the same species.

[0021] The specific steps for constructing the above-mentioned valine-producing strain are as follows: using gene editing technology, the above-mentioned gene mutant (mutant ilvC gene) is integrated into the genome of the starting strain Escherichia coli through homologous recombination to construct a recombinant strain.

[0022] Application of the above-mentioned valine-producing strains in the fermentation production of valine.

[0023] Preferably, in the above application, valine is produced using shake-flask fermentation, and the specific steps are as follows: (1) Seed activation and culture: After the bacterial solution is evenly spread on the slant of the activation medium for culture, it is transferred to the slant of the activation medium for further culture, and then transferred to the shake tube containing the seed medium for seed culture. (2) Fermentation culture: Inoculate the seed liquid into the fermentation medium, shake and culture, and maintain the pH at 7.0-7.2 during the fermentation process.

[0024] Preferably, in the above application, valine is produced using shake-flask fermentation, and the specific steps are as follows: (1) Seed activation and culture: The bacterial solution was evenly spread on the slant of the activation medium and cultured at 32℃ for 12h. It was then transferred to the slant of the activation medium and cultured for another 10h. Finally, it was transferred to a shaker containing seed medium for seed culture. (2) Fermentation culture: Inoculate the seed liquid into the Erlenmeyer flask containing the fermentation medium at a rate of 15%, seal the flask with gauze, and culture at 32℃ and 220r / min with shaking. During the fermentation process, the pH is maintained at 7.0-7.2 by adding urea (using phenol red as an indicator, when the fermentation liquid turns yellow, it is considered to have become acidic, and urea is added).

[0025] No antibiotics or inducing agents are added during the above fermentation process.

[0026] Preferably, in the above application, the activation culture medium used is: glucose 1.0-3.0 g / L, peptone 8.0-12.0 g / L, yeast extract 4.0-6.0 g / L, sodium chloride 2.0-3.0 g / L, KH2PO4 0.5-1.5 g / L, MgSO4 0.1-0.3 g / L, agar powder 23-27 g / L, with the remainder being water, and pH 7.0-7.2.

[0027] Preferably, in the above application, the activation culture medium used is: glucose 2.0 g / L, peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 25 g / L, with the remainder being water, pH 7.0–7.2.

[0028] Preferably, in the above application, the seed culture medium used is: yeast extract 7.0-9.0 g / L, peptone 2.0-4.0 g / L, KH2PO4 2.0-4.0 g / L, V B1 V B2 V B3 V B5 V B12 1-3 mg / L each, V H 0.5-1.5 mg / L, MgSO4·7H2O 0.4-0.6 g / L, the remainder is water.

[0029] Preferably, in the above application, the seed culture medium used is: yeast extract 8.0 g / L, peptone 3.0 g / L, KH2PO4 3.0 g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, the remainder is water.

[0030] Preferably, in the above application, the fermentation medium used is: glucose 75.0-85.0 g / L, corn flour 13.0-17.0 g / L, glutamic acid 1.0-3.0 g / L, KH2PO4 2.0-2.5 g / L, MgSO4·7H2O 1.3-1.7 g / L, FeSO4·7H2O 8-12 mg / L, V B1 V B2 V B3 V B5 V B12 Each 0.7-1.3 mg / L, V H 0.08-0.12 mg / L, phenol red 1-3%, the remainder is water.

[0031] Preferably, in the above application, the fermentation medium used is: glucose 80.0 g / L, corn flour 15.0 g / L, glutamic acid 2.0 g / L, KH2PO4 2.3 g / L, MgSO4·7H2O 1.5 g / L, FeSO4·7H2O 10 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H 0.1 mg / L, phenol red 2%, the remainder is water.

[0032] All of the above-mentioned culture media can be prepared using standard methods.

[0033] Beneficial effects: The above-mentioned valine-producing strain was constructed by first using *Corynebacterium glutamicum* ATCC 13032 as the mutagenesis starting point, identifying a key positive mutation site on the ilvC gene from a high-valine-producing *Corynebacterium glutamicum* mutant strain obtained through ARTP mutagenesis screening; subsequently, using gene editing technology with *Escherichia coli* W3110 as the chassis strain, the mutant ilvC gene fragment was integrated into the *E. coli* W3110 genome; this was the first confirmation that *Corynebacterium glutamicum*... ilvC After the key mutation site of the gene was applied to heterologous expression in Escherichia coli, it could still effectively enhance the valine synthesis capacity, providing an important heterologous gene target and engineering strategy for constructing a high-efficiency valine-producing strain. The strain does not contain plasmids, has no growth defects, does not require induction, and has good genetic stability. By introducing exogenous positive mutation sites, its valine production capacity was effectively improved. After 36 hours of shake-flask fermentation with glucose as substrate, the valine yield of this strain reached 1.79 g / L, which was significantly higher than that of the original E. coli chassis strain. Detailed Implementation

[0034] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0035] The starting strain used in the examples was wild-type Escherichia coli (E. coli). Escherichia coli W3110.

[0036] The gene editing method used is referenced in the literature (Li Y, Lin Z, Huang C, et al. Metabolic engineering of Escherichia coli using CRISPR-Cas9 meditated genome editing. Metabolic Engineering, 2015, 31: 13-21.). The technical terms such as gene integration and homologous recombination used in the following examples are explained in this article.

[0037] Example 1 Artificial mutagenesis was performed using ARTP, and positive mutant strains were screened. To screen for mutant strains with enhanced valine production capacity, *Corynebacterium glutamicum* (Gamma) was used. Corynebacterium glutamicum ATCC 13032 (commercially available, purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd., catalog number ml-CC1275) was used as the starting strain. It was inoculated into BHI liquid medium and cultured with shaking at 32℃ and 220r / min.

[0038] (1) Preparation of bacterial suspension: Collect the bacterial culture that has reached the logarithmic growth phase, centrifuge and discard the supernatant, wash the bacterial cells twice with physiological saline, and dilute to adjust the OD value to between 0.6 and 0.8 to obtain the bacterial suspension. In a clean bench, place the metal slide in the outer flame of an alcohol lamp for 30 seconds to sterilize, cool and place it on a sterile plate, and take 10 μL of bacterial suspension to spread evenly on the surface of the slide.

[0039] (2) Transfer the slide containing the sample to the operating chamber of the ARTP mutagenesis system. Use sterile forceps to place the slide in the corresponding well position. Adjust the knob under the stage so that the surface of the slide is 2 mm below the airflow port. Close the chamber door.

[0040] (3) Set the mutagenesis parameters to 120W power, 55s processing time, and 10slm gas flow rate, and start the mutagenesis program. After processing, remove the slide with sterile forceps and place it into an EP tube pre-filled with 1mL of physiological saline.

[0041] (4) Place the EP tube on a shaker and shake for 1 min to fully elute the bacteria attached to the slide surface into the liquid, forming a post-mutated bacterial suspension. After appropriate dilution of the bacterial suspension, take 100 μL and spread it on an activation medium plate. After cultivation, hundreds of randomly mutated colonies were obtained, and random mutations have been introduced into the genomes of these strains. The activation medium used was: glucose 2.0 g / L, peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, and agar powder 25 g / L. After dissolving in deionized water, adjust the pH to 7.0–7.2 with sodium hydroxide, and make up to 500 mL. Dispense into test tubes (9 mL / tube) and flasks (45 mL / flask), and autoclave at 121 °C for 20 min.

[0042] 1.2 Evaluation of fermentation potential of mutagenic strains and strain screening To screen for mutant strains with superior valine production capacity compared to the parent strain Corynebacterium glutamicum ATCC 13032, the fermentation performance of colonies obtained through random mutagenesis was evaluated.

[0043] 1.2.1 Culture medium Activation medium: Glucose 2.0 g / L, peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 2.5 g / L, KH₂PO₄ 1.0 g / L, MgSO₄ 0.2 g / L, agar powder 25 g / L. Dissolve in deionized water, adjust pH to 7.0–7.2 with sodium hydroxide, and bring volume to 500 mL.

[0044] Seed culture medium: Yeast powder 8.0g / L, peptone 3.0g / L, KH2PO4 3.0g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, the remainder is water.

[0045] Fermentation medium: Glucose 80.0 g / L, corn flour 15.0 g / L, glutamic acid 2.0 g / L, KH2PO4 2.3 g / L, MgSO4·7H2O 1.5 g / L, FeSO4·7H2O 10 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H0.1 mg / L, phenol red 2%, the remainder is water.

[0046] 1.2.1 Seed activation and culture: The bacterial suspensions of *Corynebacterium glutamicum* ATCC 13032 and *Corynebacterium glutamicum* mutant strain were inoculated into the preservation tube and evenly spread onto the slant of activation medium. The culture was carried out at 32°C for 12 h, then transferred to the activation medium slant and cultured for another 10 h. Finally, the culture was transferred to a shaker containing 5 mL of seed medium for seed culture.

[0047] 1.2.2 Fermentation culture: The seed culture was inoculated at a rate of 15% into 500 mL Erlenmeyer flasks containing fermentation medium (final volume 30 mL). The flasks were sealed with nine layers of gauze and incubated at 32°C with shaking at 220 rpm. During fermentation, the pH was maintained at 7.0-7.2 by supplementing with urea (phenol red was used as an indicator; the fermentation broth turning yellow indicated acidity, at which point urea was added). The fermentation period was 36 hours, and no antibiotics or inducers were added during the fermentation process. After 36 hours of shake-flask fermentation, the concentration of L-valine was analyzed by HPLC, and the analytical concentrations of L-valine are shown in Table 1.

[0048] Table 1

[0049] Based on the results shown in Table 1, strain Vcgb-3, which showed a significant increase in valine production compared to the control strain Corynebacterium glutamicum ATCC 13032, was selected.

[0050] Example 2: Identification of mutations through gene sequencing Genome sequencing was performed on the *Corynebacterium glutamicum* strains obtained through mutagenesis screening, with wild-type *Corynebacterium glutamicum* ATCC 13032 as a control. Sequencing results showed that the Vcgb-3 strain, with significantly enhanced valine production capacity compared to the starting strain, exhibited significantly higher valine production. ilvC A key nucleotide sequence mutation exists in the ORF region of the gene. Specifically, ilvC Base substitutions occurred in the coding regions of all gene subunits, and the corresponding amino acid site changes are shown in Table 2.

[0051] Further gene editing technology can be used to insert the aforementioned genes carrying key mutation sites. ilvC Gene fragments are integrated into the E. coli genome, and unmutated gene fragments are incorporated into the genome. ilvC The gene was integrated into the genome in parallel to verify its regulatory role in valine synthesis.

[0052] Table 2

[0053] Carrying key mutation sites ilvC The amino acid sequence of the gene is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.1. Analysis of the above mutation region revealed that this mutation has a significant impact on valine synthesis, possibly due to the mutation... ilvC The gene causes a change in the structure of the ketool acid reductase it encodes, enhancing the enzyme's catalytic efficiency or removing metabolic bottlenecks, thereby significantly improving the ability of Corynebacterium glutamicum to synthesize valine.

[0054] The following examples identified ilvC Key mutation sites in genes, and use gene editing technology to remove mutants ilvC Genes and non-mutated ilvC The gene was integrated in parallel into the genome of Escherichia coli W3110, and its fermentation performance was evaluated.

[0055] Example 3 introduces ilvC Preparation of genes from Escherichia coli strain W3110 and identification of its valine production capacity. 3.1 Integration ilvC Construction of mutant genes and wild-type genetically engineered strains To introduce the point mutation into *E. coli*, genomic DNA was first extracted from *Corynebacterium glutamicum* using a bacterial genomic extraction kit, and then PCR amplification was performed using this DNA as a template. The PCR reaction conditions were: 95°C pre-denaturation for 5 minutes; followed by 29 cycles, each cycle consisting of 95°C denaturation for 30 seconds, 58°C annealing for 30 seconds, 72°C extension for 150 seconds, and a final extension at 72°C for 5 minutes. Using GenScript Biotech's gene synthesis service, the desired ilvC site-directed mutant gene was obtained. Using this gene as a template, a 1017 bp fragment containing the target mutation was amplified using primers Ccgb-F and Ccgb-R. After gel extraction and purification, this fragment was used as the "introduced mutant fragment ilvC". Using the genome of *Corynebacterium glutamicum* ATCC 13032 as a template, a 1017 bp wild-type target fragment was amplified using primers Ccgb-F and Ccgb-R. After gel extraction and purification, this fragment was used as "introduced wild-type target fragment ilvC". Using a usable concentration of *Escherichia coli* W3110 genome as a template, primers Up- ycjV -F / Up- ycjV -R and Down- ycjV -F / Down- ycjV -R was used for PCR amplification to obtain the upper homologous arm. ycjV -UP, lower homologous arm ycjV -DW. With the recovered upstream and downstream homologous arms and the "introduced mutant fragment ilvC" Using "and the wild-type order fragment ilvC" as a template, primer Up- ycjV -F and Down- ycjV -R is integrated into the target fragment via overlap PCR. ycjV -Ptrc-ilvC and ycjV -Ptrc-ilvC, then, primer pGRB- ycjV -F and pGRB- ycjV The DNA fragment obtained by -R annealing was ligated onto plasmid pGRB to construct pGRB- ycjV Plasmid. Finally, the purified plasmid... ycjV -Ptrc-ilvC Integration fragment and plasmid pGRB- ycjV Simultaneously, it was transferred into competent E. coli W3110 cells via electroporation, and then... (The sentence is incomplete and requires more context to translate accurately.) ycjV -F and Down- ycjV -R is the primer for identification to screen positive transformants. The PCR product was sequenced, and the sequence matched the corresponding sequence in the *Corynebacterium glutamicum* mutant strain, ultimately obtaining strain Vecj-1. The purified product... ycjV -Ptrc-ilvC integration fragment and plasmid pGRB- ycjV Simultaneously, it was transferred into competent E. coli W3110 cells via electroporation, and then... (The sentence is incomplete and requires more context to translate accurately.) ycjV -F and Down- ycjV -R is the primer for screening positive transformants. The PCR product was sequenced, and the sequence was consistent with the corresponding sequence in the wild-type strain of Corynebacterium glutamicum, finally obtaining strain Vecj-2.

[0056] Table 3

[0057] 3.2 Identification of Valine Production Capacity 3.2.1 Culture medium Seed culture medium: Yeast powder 8.0g / L, peptone 3.0g / L, KH2PO4 3.0g / L, V B1 V B2 V B3 V B5 V B12 2 mg / L each, V H 1 mg / L, MgSO4·7H2O 0.5 g / L, the remainder is water.

[0058] Fermentation medium: Glucose 80.0 g / L, corn flour 15.0 g / L, glutamic acid 2.0 g / L, KH2PO4 2.3 g / L, MgSO4·7H2O 1.5 g / L, FeSO4·7H2O 10 mg / L, V B1 V B2 V B3 V B5 V B12 1 mg / L each, V H 0.1 mg / L, phenol red 2%, the remainder is water.

[0059] 3.2.1 Seed activation and culture: Inoculate Escherichia coli W3110, Vecj-1 and Vecj-2 bacterial suspensions from the preservation tubes and spread them evenly on the slant of activation medium. Incubate at 32°C for 12 h, transfer to the activation medium slant and continue incubation for 10 h, then transfer to a shaker containing 5 mL of seed medium for seed culture.

[0060] 2.2.2 Fermentation culture: The seed culture was inoculated at a rate of 15% into 500 mL Erlenmeyer flasks containing fermentation medium (final volume 30 mL). The flasks were sealed with nine layers of gauze and incubated at 32°C with shaking at 220 rpm. During fermentation, the pH was maintained at 7.0-7.2 by supplementing with urea (phenol red was used as an indicator; the fermentation broth turning yellow indicated acidity, at which point urea was added). The fermentation period was 36 hours, and no antibiotics or inducers were added during the fermentation process. After 36 hours of shake-flask fermentation, the concentration of L-valine was analyzed by HPLC, and the analytical concentrations of L-valine are shown in Table 4.

[0061] Table 4

[0062] In this embodiment, the mutant and wild-type ilvC genes from *Corynebacterium glutamicum* ATCC 13032 were introduced into *Escherichia coli* W3110 using gene editing technology to construct recombinant strains. Subsequent fermentation potential experiments showed that the valine production of the recombinant strain Vecj-1 containing the mutant ilvC gene was significantly increased compared to the control strain W3110 and the strain Vecj-2 containing the wild-type ilvC gene. This result confirms that point mutations in the ilvC gene can effectively increase the valine production of strains.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention. Improvements and modifications such as strain modification based on the method of the present invention or based on the method are all considered to be within the scope of protection of the present invention.

Claims

1. An ilvC mutant, characterized in that: Its amino acid sequence is shown in the sequence listing SEQ ID NO.

2.

2. A biomaterial relating to the ilvC mutant of claim 1, characterized in that: It can be any one of the following (a1) to (a4): (a1) Nucleic acid molecule encoding the ilvC mutant; (a2) An expression cassette containing the nucleic acid molecule described in (a1): (a3) A recombinant vector comprising the nucleic acid molecule described in (a1) or the expression cassette described in (a2); (a4) A recombinant microorganism comprising the nucleic acid molecule of (a1), the expression cassette of (a2), or the recombinant vector of (a3).

3. The biomaterial according to claim 2, characterized in that: The nucleotide sequence of the nucleic acid molecule encoding the ilvC mutant in (a1) is shown in the sequence listing SEQ ID NO.

1.

4. The use of the ilvC mutant of claim 1 or the biomaterial of claim 2 or 3 in constructing valine-producing strains or in the fermentation production of valine.

5. A valine-producing strain, characterized in that: It was obtained by modifying the original strain, and the modification included: introducing multiple mutation sites into the wild-type ilvC gene, and the nucleotide sequence of the mutant ilvC gene after introducing multiple mutation sites is shown in the sequence listing SEQ ID NO.

1.

6. The valine-producing strain according to claim 5, characterized in that: The starting strain was Escherichia coli.

7. The valine-producing strain according to claim 5 or 6, characterized in that: The starting strain was Escherichia coli W3110.

8. The valine-producing strain according to claim 5, characterized in that: The nucleotide sequence of the wild-type ilvC gene is shown in the sequence listing SEQ ID NO.

3.

9. The method for constructing the valine-producing strain according to any one of claims 5-8, characterized in that: The specific steps are as follows: Using gene editing technology, the mutant ilvC gene is integrated into the genome of the starting strain Escherichia coli through homologous recombination to construct a recombinant strain.

10. The use of the valine-producing strain according to any one of claims 5-8 in the fermentation production of valine.

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