Construction and application of escherichia coli for producing L-valine
By constructing the E. coli mutant ilvHM and integrating the expression of related genes, and optimizing fermentation conditions, multiple bottleneck problems in the production of L-valine in existing technologies have been solved, achieving high-efficiency production and significantly improving yield and conversion rate, making it suitable for industrial applications.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2026-01-26
- Publication Date
- 2026-04-24
AI Technical Summary
Existing technologies can increase L-valine production through partial genetic modification, but they have failed to systematically and synergistically address multiple bottlenecks such as precursor supply, elimination of competing pathways, cofactor regeneration, and product efflux, resulting in significant room for improvement in yield and conversion rate.
A strain of Escherichia coli was constructed, and through the mutant ilvH (ilvHM), genes such as acetylhydroxy acid synthase, acetylhydroxy acid reductase, and leucine dehydrogenase were integrated and expressed. Fermentation conditions were optimized to form a highly efficient L-valine producing strain, E. coli MMEC-R08.
Fermentation in a 5 L fermenter for 40 h yielded an L-valine yield of over 100 g/L and a glucose conversion rate of 50.5%, demonstrating good genetic stability and suitability for industrial production.
Smart Images
Figure CN121915005A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the construction and application of an L-valine-producing Escherichia coli strain, belonging to the field of bioengineering technology. Background Technology
[0002] L-valine, chemically known as 2-amino-3-methylbutyric acid, has a molecular weight of 117.146 and the chemical formula C5H. 11 NO2 is one of the three branched-chain amino acids and is an essential amino acid for the human body. It cannot be synthesized by the body itself and must be obtained from external sources. Due to its important functions in regulating blood sugar, controlling protein synthesis and lipid metabolism, and providing energy needed by the body, it is widely used in the food, feed, cosmetics, and pharmaceutical industries.
[0003] Microbial fermentation has become the mainstream method for the industrial production of L-valine due to its advantages such as low raw material costs, environmental friendliness, and high product purity. Although microbial cells can directly synthesize L... Valine is a crucial amino acid, but the intracellular feedback inhibition and other regulatory networks severely limit the cell's production capacity. For example, in the synthesis of L-valine, acetylhydroxy acid synthase (AHAS) is the first rate-limiting enzyme in its synthetic pathway, catalyzing the decarboxylation of 2 mol of pyruvate to produce 1 mol of acetylhydroxy acid. Its enzyme activity is subject to feedback inhibition by the final product valine, severely impacting the efficient synthesis of L-valine. In *E. coli*, three isoenzymes of AHAS exist: AHAS I, AHAS II, and AHAS III. Each isoenzyme consists of two subunits, a large subunit responsible for catalysis and a small subunit responsible for regulation. The large and small subunits must combine to form a holoenzyme to maintain activity: AHAS I consists of... ilvBN Manipulator coding, AHAS II by ilvGM Manipulator coding, AHAS III by ilvIH Manipulator code.
[0004] With the development and application of systems metabolic engineering, the production of L-valine has been improved through partial genetic modification. However, it often fails to systematically and synergistically solve multiple bottlenecks such as precursor supply, elimination of competitive pathways, cofactor regeneration, and product efflux, resulting in significant room for improvement in yield and conversion rate. Summary of the Invention
[0005] To address the shortcomings of the existing technologies, this invention provides the construction and application of an L-valine-producing Escherichia coli strain. The aim is to solve the technical problem that although existing technologies have improved yield through partial genetic modification, they often fail to systematically and synergistically solve multiple bottlenecks such as precursor supply, elimination of competing pathways, cofactor regeneration, and product efflux, resulting in significant room for improvement in yield and conversion rate.
[0006] The first technical solution provided by this invention is a ilvH mutant, the ilvH mutant (i.e.) ilvH M The method involves mutating glutamic acid at position 10 to alanine and asparagine at position 29 to histidine, based on the parental amino acid sequence shown in SEQ ID NO.7.
[0007] The second technical solution provided by this invention is to encode the first technical solution. ilvH The mutant gene.
[0008] The third technical solution provided by the present invention is a recombinant vector carrying the gene described in the second technical solution.
[0009] The fourth technical solution provided by this invention is an expression of the first technical solution. ilvH The mutant, or contains the gene described in the second technical solution, or is a recombinant cell transformed with the recombinant vector described in the third technical solution.
[0010] In one embodiment, the recombinant cells use Escherichia coli as a host.
[0011] The fifth technical solution provided by this invention is a genetically engineered bacterium that produces L-valine, wherein the genetically engineered bacterium is formed by knocking out the gene encoding lactose repressor protein in Escherichia coli. lacI Gene encoding formate acetyltransferase pflB Genes encoding phosphorylated acetyltransferase pta Gene encoding malate dehydrogenase mdh Gene encoding lactate dehydrogenase ldhA and the gene encoding valine-pyruvate transaminase avtA and integrates expression of acetylhydroxyl synthase ilvBN M Acetylhydroxy acid reductase ilvC SM Dihydroxy acid dehydratase gene ilvD Leucine dehydrogenase leuDH 3-Phosphoglyceraldehyde dehydrogenase gapA Genes responsible for glucose uptake ptsG L-valine transporter brnFE and acetylhydroxy acid synthase mutant ilvIH M The acetylhydroxy acid synthase mutant is composed of ilvI And the first technical solution described ilvH Mutant composition.
[0012] In one implementation, lacI ,pflB , pta , mdh , ldhA , avtA and yjiT Seven sites each integrate one copy of the tandemly expressed acetylhydroxyl synthase and acetylhydroxyl reductase genes P. trc - ilvBN M C SM (From Corynebacterium glutamicum), 1 copy of the dihydroxy acid dehydratase gene P trc - ilvD 1 copy of the leucine dehydrogenase gene P trc - leuDH (From *Bacillus spheroidae*), 1 copy of the 3-phosphate-glyceraldehyde dehydrogenase gene P trc - gapA One copy of the gene responsible for glucose uptake ptsG One copy of the L-valine transporter gene brnFE (Source: Corynebacterium glutamicum) and 1 copy of the acetylhydroxyl synthase gene P trc - ilvIH Or one copy of the acetylhydroxyl synthase mutant gene P trc - ilvIH M .
[0013] In one embodiment, the ilvIH M The nucleotide sequence of the gene is shown in SEQ ID NO.1.
[0014] In one embodiment, the ilvBN M Genes and ilvC SM The nucleotide sequence of the gene is shown in SEQ ID NO.3 and SEQ ID NO.4.
[0015] In one embodiment, the gene encoding NADH-dependent leucine dehydrogenase in *Bacillus spheroidae*. leuDH SEQ ID NO.5.
[0016] In one embodiment, the ilvIH The amino acid sequence of the gene is shown in SEQ ID NO.6~7.
[0017] In one embodiment, the L-valine transporter is encoded. brnFE The genes have accession numbers NCgl0258 and NCgl0254 on NCBI.
[0018] In one embodiment, a gene encoding dihydroxy acid dehydratase is used. ilvD Gene encoding glyceraldehyde-3-phosphate dehydrogenase gapA Genes responsible for glucose uptake ptsG The GeneIDs are 948277, 947679, and 945651, respectively.
[0019] In one embodiment, the lacI , pflB , pta , mdh , ldhA , avtA and yjiT The GeneIDs of the genes are 945007, 945514, 946778, 947854, 946315, 948087 and 945056.
[0020] In one embodiment, the recombinant Escherichia coli is the model strain W3110 or MG1655.
[0021] The sixth technical solution provided by this invention is a method for producing L-valine by fermentation. This method utilizes the genetically engineered bacteria described in the fifth technical solution to produce L-valine in a glucose-containing fermentation system. In one embodiment, the fermentation uses 40 g / L glucose as the substrate.
[0022] In one embodiment, the fermentation system contains 7.0 g / L yeast extract, 1.5 g / L magnesium sulfate heptahydrate, 2.5 g / L ammonium sulfate, 3.2 g / L citric acid, 4.5 g / L dipotassium hydrogen phosphate, 1.2 mg / L manganese sulfate, 2.8 mg / L ferrous sulfate, 1.5 mg / L vitamin B1, 1 mg / L vitamin B3, 1.5 mg / L vitamin B5, 0.5 mg / L vitamin B12, 0.5 mg / L biotin, and 0.2 g / L defoamer.
[0023] In one embodiment, the catalytic control conditions are a two-stage fermentation: Aerobic fermentation stage: temperature 35℃, initial tank pressure 0.03 MPa, rotation speed 400 r / min, air volume 2.0 L / min; pH is controlled in stages: OD 600 When the value is higher than 30, control the pH to around 6.3, OD 600 When the value is below 30, control the pH to around 7.0; when the dissolved oxygen is below 30%, slowly increase the rotation speed to 800 r / min, without adjusting the air volume and tank pressure.
[0024] Anaerobic fermentation stage: when OD 600When the pH reaches 18-25, start micro-anaerobic fermentation and reduce the fermentation speed to 300-400 r / min. Add an antifoaming agent in the later stage of fermentation to prevent foam overflow.
[0025] The seventh technical solution provided by this invention is the same as that described in the first technical solution. ilvH The use of mutants, or the gene described in the second technical solution, or the recombinant vector described in the third technical solution, or the recombinant cell described in the fourth technical solution, or the genetically engineered bacteria described in the fifth technical solution, or the method described in the sixth technical solution in the preparation of L-valine or products containing L-valine.
[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is based on E.coli Using MG1655 as the starting strain, a recombinant strain capable of efficiently fermenting and producing L-valine was obtained through metabolic engineering. This strain was named... E.coli MMEC-R08. Fermentation in a 5 L fermenter for 40 h yields L-valine production exceeding 100 g / L and glucose conversion exceeding 50.5%, exhibiting good genetic stability and suitability for industrial production. Attached Figure Description
[0027] Figure 1 This is a diagram showing the fermentation results of the valine-producing strain of this invention. Detailed Implementation
[0028] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.
[0029] In the following embodiments, the technical solutions used are all based on conventional technical means in the field, and the materials used are all commercially available.
[0030] In the following examples, Escherichia coli (Escherichia coli) Escherichia coli The type strain is either W3110 or MG1655.
[0031] Amino acid detection method: Conventional high performance liquid chromatography was used.
[0032] Glucose determination method: The analysis was performed using an SBA-40 biosensor analyzer (Shandong Academy of Sciences Institute of Biology).
[0033] Calculation of sugar-acid conversion rate: Sugar-acid conversion rate = Total accumulation of L-valine in fermentation broth (g) / Total glucose consumption during fermentation (g).
[0034] Activation medium components: yeast extract 2~6 g / L, tryptone 5~10 g / L, NaCl 5 g / L; Seed culture medium composition: base sugar 30 g / L, yeast extract powder 5 g / L, dipotassium hydrogen phosphate trihydrate 2.5 g / L, ammonium sulfate 2.5 g / L, citric acid 2.5 g / L, magnesium sulfate heptahydrate 1.5 g / L, ferrous sulfate 3.0 mg / L, manganese sulfate 2.0 mg / L, biotin 0.5 mg / L, vitamin B1 1.5 mg / L, vitamin B3 1 mg / L, vitamin B5 1.5 mg / L, vitamin B12 0.5 mg / L; Fermentation medium composition: ammonium sulfate 2.5 g / L, dipotassium hydrogen phosphate 4.5 g / L, magnesium sulfate heptahydrate 1.5 g / L, yeast extract powder 7.0 g / L, citric acid 3.2 g / L; ferrous sulfate 2.8 mg / L, manganese sulfate 1.2 mg / L, biotin 0.5 mg / L, vitamin B1 1.5 mg / L, vitamin B3 1 mg / L, vitamin B5 1.5 mg / L, vitamin B12 0.5 mg / L, defoamer 0.2 g / L.
[0035] Example 1: Construction of L-valine-producing strain With Escherichia coli E.coli MG1655 was used as the modified chassis strain to construct recombinant bacteria. E.coli MMEC-R01 E.coli MMEC-R02 E.coli MMEC-R03 E.coli MMEC-R04 E.coli MMEC-R05 E.coli MMEC-R06 E.coli MMEC-R07 E.coli MMEC-R08 and E.coli The genotypes of MMEC-R09 strains are shown in Table 1.
[0036] Table 1 Genotypes of Recombinant Strains
[0037] The recombinant bacteria described in Table 1 are constructed as follows: 1. Ptrc-cg ilvBN M C SM Integration lacI Site (constructing E. coli MMEC-R01): According to E. coli data from NCBI E.coli The MG1655 genome sequence, in lacIPrimers for the upper and lower homologous arms of the gene (GeneID: 945007) were designed with a length of 500 bp (primer sequences are shown in Table 2). The upper and lower homologous arm fragments (UH-cg) were amplified by PCR. ilvBN M C SM ( lacI )-F / R, DH-cg ilvBN M C SM ( lacI -F / R), according to ilvBN M C SM Primers for gene (SEQ ID NO.3, SEQ ID NO.4) amplification were designed, and primers downstream of the Ptrc promoter homologous arm were designed. ilvBN M C SM The upstream primer was obtained by PCR. ilvBN M C SM Gene fragments. Homologous arm fragments and... ilvBN M C SM Gene fragments were obtained into integration frames via fusion PCR. This was achieved using primer N20- lacI -F and N20- lacI -R was used for circular PCR to obtain pTargetF sgRNA- lacI plasmid, and ilvBN M C SM The integrated frame was electroporated into E. coli MG1655 (containing pCas9). Single colonies were selected on kanamycin and spectinomycin double antibody LB agar plates for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, strain E. coli MMEC-R01 was obtained.
[0038] Ptrc- ilvD Integration pflB Site (constructing E. coli MMEC-R02): According to E. coli data from NCBI E.coli The MG1655 genome sequence, in pflB Primers for the upper and lower homologous arms of the gene (GeneID: 945514) were designed with a length of 500 bp upstream and downstream (primer sequences are shown in Table 2). PCR amplification was performed on the upper and lower homologous arm fragments (UH-). ilvD ( pflB-F / R, DH- ilvD ( pflB -F / R), according to ilvD Design amplification primers for gene (GeneID: 948277), and design downstream primers for the Ptrc promoter on the upper homologous arm. ilvD In the upstream primer, the upstream and downstream homologous arm fragments and ilvD Gene fragments were obtained into integration frames via fusion PCR. This was achieved using primer N20- pflB -F and N20- pflB -R was used for circular PCR to obtain pTargetF sgRNA- pflB plasmid, and ilvD The integrated frame was electroporated into E. coli MMEC-R01 (containing pCas9). Single colonies were selected on kanamycin and spectinomycin double antibody LB agar plates for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, strain E. coli MMEC-R02 was obtained.
[0039] Ptrc- ls leuDH Integration pta Site (constructing E. coli MMEC-R03): According to E. coli data from NCBI E.coli The MG1655 genome sequence, in pta Primers for the upper and lower homologous arms of the gene (GeneID: 946778) were designed with a 500 bp primer sequence (see Table 2). The upper and lower homologous arm fragments (UH-) were amplified by PCR. ls leuDH ( pta -F / R, DH- ls leuDH ( pta -F / R), according to ls leuDH Design amplification primers for gene (SEQ ID NO.5), and design downstream primers for the Ptrc promoter on the upper homologous arm. ls leuDH The upstream primer was obtained by PCR. ls leuDH Gene fragments. Homologous arm fragments and... ls leuDH Gene fragments were obtained into integration frames via fusion PCR. This was achieved using primer N20- pta -F and N20- pta -R was used for circular PCR to obtain pTargetF sgRNA- pta plasmid, and ls leuDH The integrated frame was electroporated into E. coli MMEC-R02 (containing pCas9). Single colonies were selected on kanamycin and spectinomycin double antibody LB agar plates for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, strain E. coli MMEC-R03 was obtained.
[0040] Ptrc- gapA Integration mdh Site (constructing E. coli MMEC-R04): According to E. coli data from NCBI E.coli The MG1655 genome sequence, in mdh Primers for the upper and lower homologous arms of the gene (GeneID: 947854) were designed with a length of 500 bp upstream and downstream (primer sequences are shown in Table 2). PCR amplification was performed on the upper and lower homologous arm fragments (UH-). gapA ( mdh -F / R, DH- gapA ( mdh -F / R), according to gapA Design amplification primers for gene (GeneID: 947679), and design downstream primers for the Ptrc promoter on the upper homologous arm. gapA The upstream primer was obtained by PCR. gapA Gene fragments. Homologous arm fragments and... gapA Gene fragments were obtained into integration frames via fusion PCR. This was achieved using primer N20- mdh -F and N20- mdh -R was used for circular PCR to obtain pTargetF sgRNA- mdh plasmid, and gapA The integrated frame was electroporated into E. coli MMEC-R03 (containing pCas9). Single colonies were selected on kanamycin and spectinomycin double antibody LB agar plates for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, strain E. coli MMEC-R04 was obtained.
[0041] Ptrc- ptsG Integration ldhA Site (constructing E. coli MMEC-R05): According to E. coli data from NCBI E.coli The MG1655 genome sequence, in ldhA Primers for the upper and lower homologous arms of the gene (GeneID: 946315) were designed with a length of 500 bp (primer sequences are shown in Table 2). PCR amplification was performed on the upper and lower homologous arm fragments (UH-). ptsG ( ldhA -F / R, DH- ptsG ( ldhA -F / R), according to ptsG Design amplification primers for gene (GeneID: 945651), and design downstream primers for the Ptrc promoter on the upper homologous arm. ptsG The upstream primer was obtained by PCR.ptsG Gene fragments. Homologous arm fragments and... ptsG Gene fragments were obtained into integration frames via fusion PCR. This was achieved using primer N20- ldhA -F and N20- ldhA -R was used for circular PCR to obtain pTargetF sgRNA- ldhA plasmid, and ptsG The integrated frame was electroporated into E. coli MMEC-R04 (containing pCas9). Single colonies were selected on kanamycin and spectinomycin double antibody LB agar plates for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, strain E. coli MMEC-R05 was obtained.
[0042] Ptrc- cg brnFE Integration avtA Site (constructing E. coli MMEC-R06): According to E. coli data from NCBI E.coli The MG1655 genome sequence, in avtA Primers for the upper and lower homologous arms of the gene (GeneID: 948087) were designed with a length of 500 bp (primer sequences are shown in Table 2). PCR amplification was performed on the upper and lower homologous arm fragments (UH-). cg brnFE ( avtA -F / R, DH- cg brnFE ( avtA ) -F / R), according to Corynebacterium glutamicum brnFE Primers were designed for amplification of genes (NCgl0258 and NCgl0254), and primers were designed downstream of the upper homologous arm of the Ptrc promoter. cg brnFE The upstream primer was obtained by PCR. cg brnFE Gene fragments. Homologous arm fragments and... cg brnFE Gene fragments were obtained into integration frames via fusion PCR. This was achieved using primer N20- yjiT -F and N20- yjiT -R was used for circular PCR to obtain pTargetF sgRNA- yjiT plasmid, and cg brnFE The integrated frame was electroporated into E. coli MMEC-R05 (containing pCas9). Single colonies were selected on kanamycin and spectinomycin double antibody LB agar plates for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, strain E. coli MMEC-R06 was obtained.
[0043] Ptrc- ilvIH or Ptrc- ilvIH M1 / M2 Integration yjiTSites (constructing E. coli MMEC-R07~R09): According to E. coli data from NCBI E.coli The MG1655 genome sequence, in yjiT Primers for the upstream and downstream homologous arms of the gene locus (GeneID: 945056) were designed with a length of 500 bp (primer sequences are shown in Table 2). The upstream and downstream homologous arm fragments (UH / DH-) were amplified by PCR. ilvIH ( yjiT )-F / R、 ilvIH ( yjiT )-F / R or ilvIH M1 / M2 ( yjiT )-F / R), according to ilvIH Primers were designed to amplify the gene (SEQ ID NO. 6~7), and primers were designed downstream of the Ptrc promoter on the upper homologous arm. ilvIH The upstream primer was obtained by PCR. ilvIH or ilvIH M1 / M2 Gene fragments (SEQ ID NO.1 / SEQ ID NO.2), in which mutants ilvH M1 The mutant involves mutating glutamic acid at position 10 of the parent amino acid sequence shown in SEQ ID NO.7 to alanine, and simultaneously mutating asparagine at position 29 to histidine; ilvH M2 The amino acid sequence of the parent, as shown in SEQ ID NO.7, involves mutating proline at position 41 to alanine. Homologous arm fragments and... ilvIH or ilvIH M1 / M2 Gene fragments were obtained into integration frames via fusion PCR. This was achieved using primer N20. -yjiT -F and N20- yjiT -R was used for circular PCR to obtain pTargetF sgRNA- yjiT plasmid, and ilvIH or ilvIH M1 / M2 The integrated frame was electroporated into E. coli MMEC-R06 (containing pCas9). Single colonies were selected on kanamycin and spectinomycin double antibody LB agar plates for colony PCR identification and sequencing to obtain the correct recombinant bacteria. After removing the manipulation plasmid, strains E. coli MMEC-R07~R09 were obtained.
[0044] Table 2 Primer Sequences
[0045] Example 2: Production of valine by fermentation of recombinant strains The recombinant strain obtained was fermented in a 5 L fermenter, and the specific steps are as follows: (1) Seed activation and culture Plate activation: Inoculate one loopful of glycerol bacteria from a glycerol storage tube onto a solid LB agar plate and incubate at 37°C for 24 h; Primary seed culture: Pick a well-grown single colony from the plate and inoculate it into a 500 mL Erlenmeyer flask containing 50 mL of LB medium. Incubate at 37°C for 10–12 h until the OD of the primary seed culture is reached. 600 The value is between 6 and 8.
[0046] Secondary seed culture: Inoculate the primary seed culture at a rate of 5-10% into a 500mL Erlenmeyer flask containing 50mL of fermentation medium, and incubate at 37℃ for approximately 8-10 hours until the OD of the secondary seed culture reaches the specified level. 600 The value is between 12 and 16.
[0047] (2) Fermentation culture Aerobic fermentation stage: initial tank pressure 0.03 MPa, air volume 2.0 L / min, rotation speed 400 r / min, temperature 35℃; residual sugar controlled at 0.05%-0.1%; pH adjusted according to OD. 600 Value segmentation control: OD 600 When the dissolved oxygen value is below 30, control the pH at 7.0; when the dissolved oxygen value is ≥30, control the pH at 6.3. When the dissolved oxygen value is below 30%, increase the rotation speed to a maximum of 700-800 r / min.
[0048] Anaerobic fermentation stage: When the OD600 value reaches 18-25, microanaerobic fermentation begins, with a tank pressure of 0.03 MPa, an air volume of 2.0 L / min, and a rotation speed of 300-400 r / min. An antifoaming agent is added in the later stages of fermentation to prevent foam overflow. Samples are taken during the process to detect the accumulation of valine and the byproduct succinic acid (Table 3). The optimal bacterial strain is... E. coli When MMEC-R08 is fermented in a 5 L fermenter for 40 h, the yield of L-valine can reach more than 100.3 g / L, and the glucose conversion rate can reach 50.6%.
[0049] Table 3 Fermentation status of recombinant Escherichia coli
[0050] From Table 3 and Figure 1 It can be seen that, compared to the wild type ilvIH ( E. coli(MMEC-R07), mutant M1 can significantly improve the production performance of L-valine producing strains, with yield and conversion rate increased by 12% and 1%, respectively, while mutant M2 does not improve the production performance of the strains.
[0051] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A kind ilvH Mutant, characterized by, The ilvH The mutant is based on the parental amino acid sequence shown in SEQ ID NO.7, with glutamic acid at position 10 mutated to alanine and asparagine at position 29 mutated to histidine.
2. The encoding as described in claim 1 ilvH The mutant gene.
3. A recombinant vector carrying the gene of claim 2.
4. The expression of claim 1 ilvH The mutant, or contains the gene of claim 2, or is a recombinant cell transformed with the recombinant vector of claim 3.
5. The recombinant cell according to claim 4, characterized in that, The recombinant cells used Escherichia coli as the host.
6. A genetically engineered bacterium that produces L-valine, characterized in that, The genetically engineered bacteria are those in which the gene encoding lactose repressor protein in Escherichia coli has been knocked out. lacI Gene encoding formate acetyltransferase pflB Genes encoding phosphorylated acetyltransferase pta Gene encoding malate dehydrogenase mdh Gene encoding lactate dehydrogenase ldhA and the gene encoding valine-pyruvate transaminase avtA and integrates expression of acetylhydroxyl synthase ilvBN M Acetylhydroxy acid reductase ilvC SM Dihydroxy acid dehydratase gene ilvD Leucine dehydrogenase leuDH 3-Phosphoglyceraldehyde dehydrogenase gapA Genes responsible for glucose uptake ptsG L-valine transporter brnFE and acetylhydroxy acid synthase mutant ilvIH M The acetylhydroxy acid synthase mutant is composed of ilvI And as described in claim 1 ilvH Mutant composition.
7. The genetically engineered bacterium according to claim 6, characterized in that, The ilvIH M The nucleotide sequence of the gene is shown in SEQ ID NO.
1. ilvBN M Genes and ilvC SM The nucleotide sequence of the gene is shown in SEQ ID NO.3 and SEQ ID NO.4, which encodes the NADH-dependent leucine dehydrogenase in *Bacillus spheroidae*. leuDH SEQ ID NO.5 encodes the L-valine transporter protein. brnFE The genes have accession numbers NCgl0258 and NCgl0254 on NCBI, encoding dihydroxy acid dehydrase genes. ilvD Gene encoding glyceraldehyde-3-phosphate dehydrogenase gapA Genes responsible for glucose uptake ptsG The GeneIDs are 948277, 947679, and 945651, respectively. lacI , avtA , pflB , pta , mdh , ldhA , yjiT The GeneIDs of the genes are 945007, 948087, 945514, 946778, 947854, 946315, and 945056, respectively.
8. A method for producing L-valine by fermentation, characterized in that, The method involves using the genetically engineered bacteria described in claim 6 or 7 to produce L-valine in a glucose-containing fermentation system.
9. The method according to claim 8, characterized in that, The reaction is a two-stage fermentation, including an aerobic fermentation stage and an anaerobic fermentation stage.
10. The claim 1 ilvH The use of a mutant, or the gene of claim 2, or the recombinant vector of claim 3, or the recombinant cell of claim 4, or the genetically engineered bacteria of claim 6 or 7, or the method of claim 8 in the preparation of L-valine or products containing L-valine.