Lrp mutants and uses thereof

Abstract

The application discloses an Lrp mutant and application thereof. The application relates to an Lrp mutant and application thereof in the field of biotechnology. The leucine-responsive transcriptional regulator mutant provided by the application is any one of the following: A1) a protein obtained by mutating a histidine at the 107th position of a protein with an amino acid sequence of SEQ ID No: 1 into tyrosine; A2) a protein obtained by substituting, deleting and / or adding amino acid residues of the protein of A1) and having 75% or more identity with the protein shown in A1) and the same function; and A3) a fusion protein obtained by connecting a protein tag to the N terminal or / and C terminal of A1) or A2). The yield of L-valine of the engineering strain is improved through site-directed mutation, and the application has industrial application value.

Description

Technical Field

[0001] This invention relates to an Lrp mutant and its applications in the field of biotechnology. Background Technology

[0002] With the rapid development of synthetic biology and metabolic engineering, the green and efficient synthesis of bulk chemicals, fine chemicals, natural products, and key pharmaceutical components through microbial fermentation has become a new trend in industrial production, providing new ideas and approaches to solve problems such as energy crisis, environmental pollution, and resource scarcity. L-valine, one of the three branched-chain amino acids (BCAAs), is an essential amino acid and is currently widely used in food, pharmaceuticals, cosmetics, and feed additives. Microbial fermentation is the main method for valine production, and several engineered valine-producing bacteria have been reported. For example, Chen Ning et al. used *Bacillus flavus* as the starting strain and employed a strategy of protoplast ultraviolet mutagenesis combined with DES chemical mutagenesis to screen and obtain a high-yield L-valine bacterium, TV2564, with an L-valine yield as high as 29.39 g / L. The research group of Sang Yup Lee in South Korea, starting with Escherichia coli W3110, combined rational metabolic engineering, transcriptome analysis and genetic modification, and gene knockout techniques to obtain L-valine engineered bacteria that can produce 7.55 g / L of valine under aerobic conditions, with a sugar-acid conversion rate of 0.378 g / g. The creation of these engineered strains has laid a solid foundation for the industrial development of valine.

[0003] Iterative upgrades of engineered strains are crucial for improving the efficiency and reducing the cost of L-valine biosynthesis. Besides optimizing and improving synthetic pathways and key protein expression, the creation and upgrading of engineered strains also rely heavily on technologies such as artificial intelligence, big data analysis, and omics screening to obtain mutations in key proteins. This optimization of the production performance, physiological characteristics, and fermentation capacity of engineered strains significantly impacts the iterative upgrades of core strains. For example, mutations in key enzymes of the synthetic pathway can significantly improve their catalytic properties, and mutations in core regulatory factors can achieve global regulation of the engineered strain. These are all essential components for the iterative upgrades of existing engineered strains. Summary of the Invention

[0004] The main problem this invention aims to solve is how to improve the biosynthesis efficiency of L-valine and reduce its cost.

[0005] To address the aforementioned problems, this invention provides a leucine-responsive regulatory protein (Lrp) mutant and its application in the creation and fermentation production of branched-chain amino acids, particularly high-yield L-valine engineered strains.

[0006] This invention first provides a leucine-responsive transcriptional regulator mutant, which may be any of the following:

[0007] A1) The protein obtained by mutating histidine at position 107 of the protein with the amino acid sequence SEQ ID No:1 to tyrosine;

[0008] A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in A1) that has more than 75% identity with the protein shown in A1) and has the same function.

[0009] A3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0010] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0011] The leucine-responsive transcriptional regulator mutant can be Lrp M .

[0012] The tag proteins include, but are not limited to: GST (glutathione thiotransferase) tag protein, His6 tag protein (His-tag), MBP (maltose-binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tag protein.

[0013] Those skilled in the art can readily employ known methods, such as directed evolution or point mutation, to modify the Lrp protein encoded by this invention. M The nucleotide sequence was mutated. Those artificially modified proteins with the same characteristics as the Lrp protein obtained in this invention... M Nucleotides with 75% or more nucleotide sequence identity are required to encode the protein Lrp. M And it contains protein Lrp M All functions are derived from and are equivalent to the nucleotide sequences of this invention.

[0014] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid or nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid sequences or nucleotide sequences, then the identity value (%) can be obtained.

[0015] In this article, the 75% or more identity can be 80%, 85%, 90% or 95% or more identity.

[0016] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0017] In this document, the above 90% identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0018] The present invention also provides biomaterials related to the proteins described above, wherein the biomaterials may be any of the following:

[0019] B1) Nucleic acid molecules that encode the proteins described above;

[0020] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0021] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0022] B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

[0023] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0024] The present invention lrp M Genes can be any gene that encodes the protein Lrp M The nucleotide sequence. Considering codon degeneracy and the codon preferences of different species, those skilled in the art can use codons suitable for the expression of a specific species as needed.

[0025] B1) The nucleic acid molecule may also include a nucleic acid molecule obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID No:2.

[0026] B1) The nucleic acid molecule may also include nucleic acid molecules that have a nucleotide sequence identity of more than 95% with the nucleotide sequence shown in SEQ ID No:2 and originate from the same species.

[0027] In one specific embodiment, the nucleotide sequence of the nucleic acid molecule described in B1) may be as follows:

[0028] d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No:2;

[0029] d2) Hybridizes with the nucleotide sequence defined by d1) under strict conditions and encodes the aforementioned protein in a DNA molecule.

[0030] Furthermore, the recombinant microorganism described in B4) integrates the coding gene for the mutant protein described above.

[0031] The integration can be achieved through homologous recombination.

[0032] The present invention also provides a recombinant Escherichia coli containing the gene encoding the mutant protein described above.

[0033] Furthermore, the recombinant E. coli integrates the coding gene of the mutant protein described above through homologous recombination. Lrp M .

[0034] Furthermore, the recipient bacterium that integrates the gene encoding the mutant protein described above can be the L-valine engineered bacterium Sval064.

[0035] The present invention also provides a method for constructing recombinant Escherichia coli, comprising introducing the coding gene of the protein described above into the recipient Escherichia coli, wherein the recipient bacterium is L-valine engineered bacterium Sval064.

[0036] The present invention also provides a method for high-yield L-valine production, comprising introducing the mutant encoding gene described above into a recipient microorganism to obtain a recombinant microorganism expressing the mutant described above, culturing the recombinant microorganism to obtain a fermentation product, and obtaining L-valine from the fermentation product.

[0037] This invention also provides for the application of the mutant protein described above in any of the following:

[0038] P1. Applications in the production of L-valine;

[0039] P2. Application in increasing L-valine production.

[0040] The present invention also provides the recombinant Escherichia coli described above for any of the following applications:

[0041] P1. Applications in the production of L-valine;

[0042] P2. Application in increasing L-valine production.

[0043] This invention provides a novel lrp amino acid point mutation to increase the yield of L-valine in engineered strains. Detailed Implementation

[0044] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0046] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0047] The L-valine engineered bacteria Sval064 and Sval065 in the following examples are described in the authorized patent: CN113278655B Recombinant Microorganisms for L-valine Production and Construction Methods, and Applications. This biological material is available to the public from the applicant and is intended solely for repeating the experiments of this invention; it may not be used for any other purpose.

[0048] The pXZ-CS plasmid used in the following examples is described in: Tan, et al., Activating phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in combination for improvement of succinate production. Appl Environ Microbiol, 2013, 79:4838-4844. This biological material is available to the public from the applicant and is intended solely for the replication of experiments of this invention and may not be used for any other purpose.

[0049] Example 1: Obtaining the lrp mutant

[0050] In previous research (CN113278655B), an engineered bacterium, Sval065, capable of efficiently producing L-valine was obtained. This strain was acquired through anaerobic domestication of the L-valine engineered bacterium Sval064. Under existing fermentation conditions, the L-valine yield of Sval065 increased to 45 g / L compared to 2 g / L of Sval064.

[0051] The genomes of recombinant Escherichia coli Sval064 and Sval065 were resequencing, including the following steps:

[0052] (1) Sval064 and Sval065 were cultured in LB medium to the logarithmic growth phase, and bacterial cells were collected and bacterial genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (promega). The DNA concentration was quantified by Qubit Fluorometer and agarose gel electrophoresis.

[0053] (2) Resequencing of the genomic DNA from Sval064 and Sval065 was performed by BGI Genomics Co., Ltd. in Shenzhen. The extracted genomic DNA was randomly fragmented, and the required DNA fragment lengths were recovered by electrophoresis. Adapters were added to prepare the clusters, and finally, the sequences were sequenced. Bioinformatics analysis was performed on the obtained sequencing information. The reference genome sequence was the ATCC 8939 genome sequence (https: / / www.ncbi.nlm.nih.gov / nuccore / NC_010468.1).

[0054] The results showed that, compared to the original strain Sval064, the evolved strain Sval065 had multiple mutations in its genome. Among them, histidine at position 107 of the amino acid sequence (SEQ ID No:1) of the leucine-responsive transcriptional regulator was mutated to tyrosine, and the c at position 319 of the corresponding encoding gene lrp was mutated to t. The mutant lrp was labeled as lrp. M Mutant lrp M The nucleotide sequence encoding the gene is SEQ ID No:2.

[0055] The Lrp wild-type amino acid sequence is as follows:

[0056] MVDSKKRPGKDLDRIDRNILNELQKDGRISNVELSKRVGLSPTPCLERVRRLERQGFIQGYTALLNPHYLDASLLVFVEITLNRGAPDVFEQFNTAVQKLEEIQECHLVSGDFDYLLKTRVPDMSAYRKLLGETLLRLPGVNDTRTYVVMEEVKQSNRLVIKTR (SEQ ID No: 1).

[0057] Lrp M The mutant nucleotide sequence is as follows:

[0058] 5'-atggtagatagcaagaagcgccctggcaaagatctcgaccgtatcgatcgtaacattcttaatgagttgcaaaaggatgggcgtatttctaacgtcgagctttctaaacgtgtgggactttcc ccaacgccgtgccttgagcgtgtgcgtcggctggaaagacaagggtttattcagggctatacggcgctgcttaacccccattatctggatgcatcacttctggtattcgttgagattactctgaat cgtggcgcaccggatgtgtttgaacaattcaataccgctgtacaaaaacttgaagaaattcaggagtgtTatttagtatccggtgatttcgactacctgttgaaaacacgcgtgccggatatgtca gcctaccgtaagttgctgggggaaaccctgctgcgtctgcctggcgtcaatgacacacggacatacgttgttatggaagaagtcaagcagagtaatcgtctggttattaagacgcgctaa-3' (seq ID No: 2).

[0059]

[0060] Example 2: Functional verification of the lrp mutant

[0061] To verify the function of the lrp mutant, it was integrated into an L-valine engineered strain and its effects on L-valine production and cell performance in the engineered strain were examined.

[0062] First, lrp from the domesticated strain Sval065 was... M The lrp gene was integrated into the originating strain Sval064 to replace the wild-type lrp gene, and lrp was identified. M The impact on L-valine production in engineered strains with low yields before domestication.

[0063] Replace lrp in strain Sval064 with a mutant lrp, i.e., lrp M The specific steps include:

[0064] In the first step, using pXZ-CS plasmid DNA as a template, a 2720 bp DNA fragment I (nucleotide sequence SEQ ID No: 11) was amplified using primers lrp-CS-up / lrp-CS-down (see Table 1 for details), which was used for homologous recombination in the first step.

[0065] The amplification system consisted of: 10 μl New England Biolabs Phusion 5× buffer, 1 μl dNTPs (10 mM each), 20 ng DNA template, 2 μl primers (10 μM each), 0.5 μl Phusion High-Fidelity DNA polymerase (2.5 U / μl), and 33.5 μl distilled water, for a total volume of 50 μl.

[0066] The amplification conditions were: 98℃ pre-denaturation for 2 minutes (1 cycle); 98℃ denaturation for 10 seconds, 56℃ annealing for 10 seconds, and 72℃ extension for 2 minutes (30 cycles); 72℃ extension for 10 minutes (1 cycle).

[0067] The above DNA fragment I was used for the first homologous recombination: First, the pKD46 plasmid (Datsenko and Wanner2000, Proc Natl Acad Sci USA 97:6640-6645; the plasmid was purchased from the CGSC E. coli Collection Center at Yale University, CGSC#7739) was transformed into the engineered strain Sval064 by electroporation, and then DNA fragment I was electroporated into Sval064 containing pKD46.

[0068] The electroporation conditions were as follows: First, prepare competent cells of the engineered strain Sval064 carrying the pKD46 plasmid (Dower et al., High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res, 1988, Jul 11; 16:6127-6145); place 50 μl of competent cells on ice, add 50 ng of DNA fragment I, incubate on ice for 2 minutes, and then transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5 kV. After electroporation, quickly transfer 1 ml of LB medium to the electroporation cuvette, pipette five times, and then transfer to a test tube. Incubate at 75 rpm and 30°C for 2 hours.

[0069] Take 200 μl of bacterial culture and spread it on an LB agar plate containing ampicillin (final concentration 100 μg / ml) and chloramphenicol (final concentration 34 μg / ml). After incubating overnight at 30°C, select single colonies for PCR verification. The primers used are lrp-CSYZ-up / lrp-CSYZ-down (see Table 1 for details). The correct colony amplification product is a 3700bp fragment (nucleotide sequence is SEQ ID No:12). Select a correct single colony and name it Sval103-CS.

[0070] In the second step, using the genomic DNA of Sval065 as a template, a 784 bp DNA fragment II (nucleotide sequence SEQ ID No: 13) was amplified using primers lrpM-up50 / lrp-CSYZ-down (see Table 1 for details). DNA fragment II was used for the second homologous recombination. DNA fragment II was electroporated into strain Sval103-CS.

[0071] The electroporation conditions were as follows: First, prepare Sval103-CS competent cells carrying the pKD46 plasmid for electroporation; place 50 μl of competent cells on ice, add 50 ng of DNA fragment II, incubate on ice for 2 minutes, and then transfer to a 0.2 cm Bio-Rad electroporation cuvette. Use a MicroPulser (Bio-Rad) electroporator with an electroporation parameter of 2.5 kV. After electroporation, quickly transfer 1 ml of LB medium to the electroporation cuvette, pipette five times, then transfer to a test tube and incubate at 75 rpm at 30°C for 4 hours.

[0072] The bacterial culture was transferred to LB liquid medium (50 ml in a 250 ml flask) containing 10% sucrose and sodium chloride-free, and incubated for 24 hours. Afterward, it was streaked onto LB solid medium (6% sucrose) containing 6% sucrose and sodium chloride-free. PCR verification confirmed that the primers used were lrp-YZ-up / lrp-YZ-down (see Table 1). The correct colony amplification product was a 1677 bp fragment (nucleotide sequence SEQ ID No: 14). Sequencing was performed, and a single correct colony was selected and named Sval103.

[0073] Sequencing analysis confirmed that, compared to the valine-engineered strain Sval064, the engineered strain Sval103 differs in that the wild-type lrp gene in the genomic DNA of the valine-engineered strain Sval064 has been replaced with a mutated lrp gene. M A recombinant bacterium expressing the mutant lrp gene was successfully obtained. In the mutant bacterium, the c at position 319 of the lrp gene nucleotide sequence was mutated to t, and the histidine at position 107 of the corresponding amino acid sequence was mutated to tyrosine.

[0074] Example 3: Production of L-valine using Sval103 fermentation

[0075] 1. Seed culture

[0076] The seed culture medium consists of the following components (solvent is water): glucose 20 g / L, corn steep liquor powder 10 g / L, KH2PO4 8.8 g / L, (NH4)2SO4 2.5 g / L, MgSO4·7H2O 2 g / L.

[0077] Fresh clones from LB plates were inoculated into test tubes containing 4 mL of seed culture medium and cultured overnight at 37°C and 250 rpm with shaking. Then, the inoculum was transferred at a rate of 2% (v / v) to 250 mL Erlenmeyer flasks containing 30 mL of seed culture medium and cultured at 37°C and 250 rpm for 12 hours to obtain the seed culture, which was then used for inoculation of the fermentation medium.

[0078] 2. Fermentation culture

[0079] The fermentation medium is mostly the same as the seed culture medium, the only difference being that the glucose concentration is 50 g / L.

[0080] 250 mL of fermentation medium was added to a 500 mL anaerobic fermenter. The seed culture was inoculated into the fermentation medium at a final concentration OD550 = 0.1. Fermentation was carried out at 37°C and 150 rpm for 96 hours. The resulting fermentation broth comprised all the substances in the fermenter. During fermentation, the pH was maintained at 7.0 by adding 5M ammonia solution. No gas was introduced during the cultivation process.

[0081] 3. Fermentation broth analysis

[0082] The components in the fermentation broth after fermentation were determined using an Agilent-1260 high-performance liquid chromatograph. The concentrations of glucose and organic acids in the fermentation broth were determined using a Biorad Aminex HPX-87H organic acid analytical column. Amino acid analysis was performed using a Sielc Primesp 100250×4.6 mm amino acid analytical column.

[0083] The results showed that the L-valine content in the fermentation broth obtained after 96 hours of anaerobic fermentation by the engineered strain Sval103 was 4.5 g / L. Compared with the starting strain Sval064, the newly constructed engineered strain Sval103 showed a 125% increase in L-valine production; the only difference between the engineered strain Sval103 and Sval064 was the use of a mutant lrp. MThe Lrp gene replaced the wild-type Lrp gene. Therefore, it can be determined that the increased L-valine production in the engineered strain Sval103 compared to the starting strain Sval064 is due to the Lrp mutant.

[0084] Example 4, lrp M Functional identification of mutants in Sval065, a domesticated bacterium that produces high levels of L-valine.

[0085] By using lrp from Sval065 containing the lrp mutant M Replace with wild-type LRP and identify LRP. M The effect of domesticated engineered strain Sval065 on L-valine production.

[0086] Using the same method as the first step of homologous recombination in Example 2, a 2720 bp DNA fragment I was amplified using pXZ-CS plasmid DNA as a template and primers lrp-CS-up / lrp-CS-down (see Table 1 for details). This fragment was then integrated into the Sval065 strain to obtain the engineered strain Sval104-CS.

[0087] In the second step, using the genomic DNA of Sval064 as a template, a 784 bp DNA fragment III was amplified using primers lrp-up50 / lrp-CSYZ-down. DNA fragment III (nucleotide sequence SEQ ID No:15) was used for the second homologous recombination.

[0088] DNA fragment III was electroporated into strain Sval104-CS using the same method as described in the second step of homologous recombination in Example 2. PCR verification and sequencing were performed using primers lrp-YZ-up / lrp-YZ-down (see Table 1). The correct colony amplification product was a 1677 bp fragment (nucleotide sequence SEQ ID No: 16). A correct single colony was sequenced and selected, and named Sval104.

[0089] Sequencing analysis confirmed that the engineered strain Sval104 differs from the valine-engineered strain Sval065 in that the mutant lrp gene in the genomic DNA of the valine-engineered strain Sval065 is present. M The wild-type lrp gene was replaced, and an engineered bacterium expressing the wild-type lrp gene was successfully obtained.

[0090] Compared to strain Sval065, lrp in the obtained strain Sval104... MThe T at position 319 in the gene nucleotide sequence was restored to C, and the tyrosine at position 107 in the corresponding amino acid sequence was restored to wild-type histidine.

[0091] Example 5: Production of L-valine using Sval104 fermentation

[0092] 1. Seed culture

[0093] The seed culture medium was prepared and the seed solution was obtained using the same method as in Example 3.

[0094] 2. Fermentation culture

[0095] The fermentation medium is mostly the same as the seed culture medium, the only difference being that the glucose concentration is 100 g / L.

[0096] As described in Example 3, the fermentation culture was carried out in a 500 mL anaerobic fermenter containing 250 mL of fermentation medium. The fermentation conditions and culture were the same as those described in Example 2, except that the fermentation time was 48 hours.

[0097] 3. Fermentation broth analysis

[0098] The treatment of the fermentation broth and the detection of compounds in the fermentation broth were the same as described in Example 3.

[0099] The results showed that the L-valine content in the fermentation broth obtained after 48 hours of anaerobic fermentation by the engineered strain Sval104 was 39.2 g / L. Compared with the engineered strain Sval065, the L-valine yield of the newly constructed engineered strain Sval104 was reduced by 12.9%. The only difference between the engineered strain Sval104 and Sval065 was the replacement of the mutant lrp gene with the wild-type lrp gene. M Therefore, it can be determined that the decrease in L-valine production in the engineered strain Sval104 compared to the engineered strain Sval065 is due to changes in the nucleotide sequence of the lrp gene.

[0100] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for high-yield L-valine, comprising introducing the coding gene of a leucine-responsive transcriptional regulator mutant into a recipient microorganism to obtain a recombinant microorganism expressing the mutant, culturing the recombinant microorganism to obtain a fermentation product, and obtaining L-valine from the fermentation product; The mutant is any one of the following: A1) The protein obtained by mutating histidine at position 107 of the protein with the amino acid sequence SEQ ID No:1 to tyrosine; A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1); The microorganism in question is Escherichia coli.

2. The method according to claim 1, characterized in that, The encoding gene is a DNA molecule with a nucleotide sequence shown in SEQ ID No:

2.

3. Leucine-responsive transcriptional regulator mutants in any of the following applications: P1. Application of Escherichia coli in the production of L-valine; P2. Application in increasing L-valine production in Escherichia coli; The mutant is any one of the following: A1) The protein obtained by mutating histidine at position 107 of the protein with the amino acid sequence SEQ ID No:1 to tyrosine; A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1).

4. Recombinant Escherichia coli in any of the following applications: P1. Applications in the production of L-valine; P2. Application in increasing L-valine production; The recombinant Escherichia coli contains the encoding gene of a leucine-responsive transcriptional regulator mutant; The mutant is any one of the following: A1) The protein obtained by mutating histidine at position 107 of the protein with the amino acid sequence SEQ ID No:1 to tyrosine; A2) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1).

5. The application according to claim 4, characterized in that, The encoding gene is a DNA molecule with a nucleotide sequence shown in SEQ ID No:2.

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