Leucine dehydrogenase mutant, valine production strain including the same, and application thereof

Abstract

This invention provides a leucine dehydrogenase mutant, a valine-producing strain comprising this leucine dehydrogenase mutant, and its applications. The invention designs a leucine dehydrogenase mutant with the following mutations: tyrosine (Tyr) at position 48 is mutated to serine (Ser), histidine (His) at position 127 is mutated to arginine (Arg), tyrosine (Tyr) at position 186 is mutated to phenylalanine (Phe), isoleucine (Ile) at position 256 is mutated to valine (Val), and lysine (Lys) at position 308 is mutated to arginine (Arg). These mutations are used to construct a valine-producing strain. The new strain is plasmid-free, has no growth defects, requires no induction, and exhibits advantages such as good genetic stability and high fermentation yield. It is an excellent strain capable of stably producing valine. This strain efficiently synthesizes valine de novo using glucose as a substrate, significantly increasing valine yield.

Description

Technical Field

[0001] This invention relates to the fields of metabolic engineering and genetic engineering, and in particular to a leucine dehydrogenase mutant derived from Bacillus subtilis, a valine-producing strain, its construction method, and its application. Background Technology

[0002] L-valine, chemically known as 2-amino-3-methylbutyric acid, is an important branched-chain amino acid (BCAA) that plays a crucial role in protein synthesis, muscle metabolism, tissue repair, and energy supply. As an essential component of human and animal nutrition, L-valine is widely used in food fortification, feed additives, pharmaceuticals, and cosmetics. Furthermore, L-valine is also a vital pharmaceutical and chemical intermediate for the synthesis of antibiotics, antihypertensive drugs, and herbicides, with continuously growing market demand. In recent years, with the rapid development of metabolic engineering and synthetic biology technologies, constructing efficient, green, and economical L-valine production systems has become a research hotspot in the industry.

[0003] Currently, the main production methods for L-valine include chemical synthesis, direct extraction, and microbial fermentation. Among these, chemical synthesis and direct extraction suffer from significant drawbacks such as limited raw materials, harsh reaction conditions, low product optical purity, and environmental pollution, and have been gradually phased out. In contrast, microbial fermentation, with its advantages of low raw material costs, mild reaction conditions, high product purity, environmental friendliness, and ease of large-scale production, has become the mainstream solution for L-valine industrial production. Corynebacterium glutamicum, due to its clear genetic background, well-defined metabolic pathway, high food safety, and strong tolerance, is widely used in the industrial production of L-valine. However, in the L-valine synthesis pathway of Corynebacterium glutamicum, the key step in the conversion of L-valine from the keto acid precursor (2-ketoisovaleric acid) is usually catalyzed by endogenous transaminase B (encoded by the ilvE gene). This reaction uses L-glutamate as the amino donor, simultaneously consuming NADPH and generating NADH, leading to an imbalance in intracellular redox states, thus limiting the yield of L-valine.

[0004] Leucine dehydrogenase is a type of NAD+. + L-valine-dependent oxidoreductases are widely found in microorganisms such as Bacillus. These enzymes catalyze the reversible oxidative deamination of branched-chain L-amino acids (L-leucine, L-valine, L-isoleucine, etc.) to produce the corresponding α-keto acids, ammonia, and NADH. Studies have shown that leucine dehydrogenase has a good substrate affinity for L-valine and exhibits high catalytic activity in the oxidative deamination of L-valine. However, there are few reports on constructing L-valine-producing strains using leucine dehydrogenase mutants. Summary of the Invention

[0005] In one aspect, the present invention provides a leucine dehydrogenase mutant that comprises any one or any combination of the following amino-terminal substitutions relative to Bacillus subtilis leucine dehydrogenase: 1) Replace the tyrosine (Tyr) at position 48 with serine (Ser); 2) Replace histidine (His) at position 127 with arginine (Arg); 3) Replace the tyrosine (Tyr) at position 186 with phenylalanine (Phe); 4) Replace isoleucine (Ile) at position 256 with valine (Val); 5) Replace the lysine (Lys) at position 308 with arginine (Arg).

[0006] In some embodiments, the leucine dehydrogenase mutant, relative to Bacillus subtilis leucine dehydrogenase, includes the following amino acid substitutions: 1) Replace the tyrosine (Tyr) at position 48 with serine (Ser); 2) Replace histidine (His) at position 127 with arginine (Arg); 3) Replace the tyrosine (Tyr) at position 186 with phenylalanine (Phe); 4) Replace isoleucine (Ile) at position 256 with valine (Val); and 5) Replace the lysine (Lys) at position 308 with arginine (Arg).

[0007] In some embodiments, the leucine dehydrogenase mutant has at least 80%, 85%, 90%, or 95% amino acid sequence identity with the Bacillus subtilis leucine dehydrogenase.

[0008] In some embodiments, the Bacillus subtilis leucine dehydrogenase comprises the amino acid sequence shown in SEQ ID NO: 11.

[0009] In some embodiments, the leucine dehydrogenase mutant comprises the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence that has at least 80%, 85%, 90%, or 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 12.

[0010] On the other hand, the present invention provides biological materials related to the above-mentioned leucine dehydrogenase mutant, which are any of the following: (1) The nucleic acid molecule encoded by the leucine dehydrogenase mutant; (2) An expression cassette encoding a nucleic acid molecule as described in (1); (3) A recombinant vector comprising (1) the nucleic acid molecule or (2) the expression cassette; In some embodiments, the encoding nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO: 10.

[0011] On the other hand, the present invention provides a host cell comprising the above-described biological materials.

[0012] In some embodiments, the host cell is Corynebacterium glutamicum.

[0013] On the other hand, the present invention provides a valine-producing strain comprising the above-mentioned biological material or a mutant expressing the above-mentioned leucine dehydrogenase.

[0014] In some embodiments, the valine-producing strain is Corynebacterium glutamicum.

[0015] In some embodiments, the Corynebacterium glutamicum has the encoding nucleic acid molecule recombinantly integrated at its pseudogene site cgl0303.

[0016] On the other hand, the present invention provides a method for preparing L-valine-producing bacteria, comprising: 1) Construct random mutants of nucleic acid molecules encoded by leucine dehydrogenase; 2) Introduce the random mutant into the bacterial strain; and 3) Cultivate the bacterial strains and screen for bacterial strains that produce high levels of L-valine.

[0017] In some embodiments, the high-L-valine-producing bacterial strain produces at least 1, 1.5, 2, or more times more L-valine compared to bacterial strains without the introduction of the random mutant.

[0018] In some embodiments, the method includes integrating a nucleic acid molecule encoding a leucine dehydrogenase mutant into the genome of Corynebacterium glutamicum, wherein the nucleotide sequence encoding the nucleic acid molecule is shown in SEQ ID NO: 10.

[0019] In some implementations, the encoding nucleic acid molecule is integrated into the pseudogene site cgl0303 of the Corynebacterium glutamicum genome.

[0020] On the other hand, the present invention provides the application of the above-mentioned leucine dehydrogenase mutant, biomaterial, or L-valine-producing strain in the preparation of L-valine.

[0021] On the other hand, the present invention provides a method for preparing L-valine, comprising culturing the aforementioned host cells or L-valine-producing strains and isolating the generated L-valine.

[0022] The *Corynebacterium glutamicum* prepared by this invention can be stably produced to efficiently synthesize valine de novo using glucose as a substrate. After 36 hours of shake-flask fermentation, the valine yield can reach up to 2.88 g / L. Detailed Implementation

[0023] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0024] When referring to leucine dehydrogenase, the term "mutant" (e.g., leucine dehydrogenase mutant) refers to a modified amino acid sequence compared to wild-type leucine dehydrogenase, such as the substitution, deletion, and / or insertion of one or more amino acids; when referring to a strain, "mutant" (e.g., mutant strain) refers to a strain that can express the aforementioned leucine dehydrogenase mutant and / or includes the coding nucleic acid molecule of the aforementioned leucine dehydrogenase mutant.

[0025] The wild-type leucine dehydrogenase used in this article can be derived from Bacillus subtilis, and its amino acid sequence is shown in SEQ ID NO: 11. When describing specific amino acid mutation sites, this wild-type leucine dehydrogenase sequence is used as a reference.

[0026] As used in this article, "encoding nucleic acid molecule" refers to a nucleic acid molecule that follows the rules of the genetic code, directs the synthesis of a specific protein or polypeptide chain, and determines its amino acid sequence. Encoding nucleic acid molecules can be DNA, cDNA, or RNA. For DNA-encoding nucleic acid molecules, their boundaries are typically determined by open reading frames (ORFs), which usually begin with the ATG start codon or alternative start codons such as GTG and TTG, and end with a stop codon such as TAA, TAG, and TGA.

[0027] The term "expression cassette" as used in this article refers to a DNA molecule or fragment thereof, containing the essential elements required for gene expression. In other words, it is the smallest functional unit capable of driving the transcription and translation of a target gene (such as a nucleic acid-coding molecule). Typically, an expression cassette includes at least a promoter, a target gene, and a termination signal sequence.

[0028] As used herein, "recombinant vector" refers to a linear or circular nucleic acid molecule construct containing a polynucleotide encoding a leucine dehydrogenase mutant, as provided herein, and said polynucleotide being operatively linked to additional nucleotides provided for its expression, thus forming an expression cassette. The expression vector also includes host replication capability, typically conferred by the origin of replication, and / or carries selection genes that aid in the identification of transformants. Typically, recombinant vectors used in recombinant DNA technology are in the form of "plasmids," i.e., circular double-stranded DNA loops.

[0029] In this article, "host cell" refers to a cell that can be introduced with the aforementioned coding nucleic acid molecules, expression cassettes, or recombinant vectors, such as *Corynebacterium glutamicum*. After the introduction of these coding nucleic acid molecules, expression cassettes, or recombinant vectors (which may be located outside the host cell genome or integrated into the host cell genome), the host cell can express the protein or polypeptide encoded by that coding nucleic acid molecule. Host cells include the progeny of the host cell, and the progeny may not necessarily be completely identical to the original parent cell due to natural, accidental, or intentional mutations.

[0030] The term “amino acid sequence” as used in this article is synonymous with the terms “polypeptide,” “protein,” and “peptide” and is used interchangeably. A standard single-letter or three-letter code for the amino acid residues is used, where the amino acid sequence is presented with a standard amino-to-carboxyl terminus orientation (i.e., N→C).

[0031] When referring to amino acid or nucleotide sequences, the term "sequence identity" (also known as "sequence uniformity") refers to the degree of similarity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), typically expressed as a percentage. Commonly used sequence alignment algorithms or software include DANMAN, CLUSTALW, MAFFT, BLAST, MUSCLE, etc. For the purposes of this invention, the publicly available alignment software BLAST (available at https: / / www.ncbi.nlm.nih.gov / ) can be used to obtain the optimal sequence alignment and calculate the sequence identity between the two amino acid or nucleotide sequences using default settings.

[0032] As used in this article, “including,” “contains,” and “has” mean “including but not limited to,” but also refers to situations consisting only of the listed elements.

[0033] The term "or" refers to a single element among the listed optional elements, unless the context explicitly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more, or all of the listed optional elements.

[0034] The valine-producing strain provided by this invention uses leucine dehydrogenase (bcd) from Bacillus subtilis as the original sequence, and optimizes the gene sequence based on the codon bias of Corynebacterium glutamicum to obtain a new sequence. bcd- cgl In order to obtain bcd-cgl Advantageous mutants, with bcd-cgl Using the gene as a template, a random mutant library was constructed by error-prone PCR using universal primers pE-F and pE-R. The mutants from this library were integrated into the genome of *Corynebacterium glutamicum* ATCC 13032 to obtain a mutant strain library. Fermentation performance screening revealed that the strain integrating the specific advantageous mutant had significantly higher valine yields than the non-mutated strain and the wild-type strain. The new strain is plasmid-free, has no growth defects, requires no induction, and exhibits advantages such as good genetic stability and high fermentation yield, making it an excellent strain for stable valine production. This strain efficiently synthesizes valine de novo using glucose as a substrate; after 36 hours of shake-flask fermentation, the valine yield can reach as high as 2.88 g / L.

[0035] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described in detail below with reference to specific embodiments.

[0036] The starting strain used in the examples was wild-type Corynebacterium glutamicum ( Corynebacterium glutamicum ATCC 13032.

[0037] Leucine dehydrogenase ( bcd The original sequence was derived from Bacillus subtilis ( ) Bacillus subtilis By entrusting GenScript Biotech Co., Ltd. with the initial template, and based on the codon preference of Corynebacterium glutamicum, the gene sequence was optimized to obtain a new gene sequence. bcd-cgl .

[0038] The gene editing method used was based on the literature (Wang T, Li Y, Li J, et al . An update of thesuicide plasmid-mediated genome editing system in Corynebacterium glutamicum. Microbial Biotechnology (2019, 12: 907-919.). The technical terms such as gene integration and homologous recombination used in the following examples are explained in this article.

[0039] Example 1: Expression and purification of leucine dehydrogenase The target gene derived from Bacillus subtilis bcd-cgl After ligation into plasmid pK18, the protein was expressed as a recombinant protein with a C-terminus His-tag in Corynebacterium glutamicum ATCC 13032. The protein was purified using a Ni-His binding column, and the purified protein was collected for SDS-PAGE. Electrophoresis results showed that bcd was efficiently expressed in Corynebacterium glutamicum ATCC 13032, and the purified band was distinct (40 kDa), consistent with the theoretical molecular weight. This indicates that the target gene can be expressed in Corynebacterium glutamicum ATCC 13032 and undergo self-cleavage to generate active subunits.

[0040] Example 2 Construction of mutant libraries and preparation of bacterial strains 2.1 Construction of random mutant libraries In order to obtain a dominant mutant of leucine dehydrogenase, bcd-cgl The gene was used as a mutation template, and a random mutant library was constructed using error-prone PCR with universal primers E-bcd-F and E-bcd-R. The sequence of universal primer pE-F is TGCATGCCTGCAGGTCGAATGGAGCTTTTCAAGTACATGGA (SEQ ID NO: 1), and the sequence of pE-R is GCTATTGACCATGATTACGTTACCTACGGCTCAAGACAGAG (SEQ ID NO: 2). The random mutation reaction system is shown in Table 1. When constructing the random mutant library, the mutation rate was controlled at around 1% by changing the amount of template added, annealing temperature, and the amounts of other components in the PCR system. Agarose gel electrophoresis results showed that the error-prone PCR bands were of the correct size, and sequencing results showed that the mutation rate met the requirements. The PCR products were purified using a DNA purification kit (TIANGEN, China) to obtain... bcd-cgl A library of random mutant genes.

[0041] Table 1 Random Mutation Response System 2.2 Preparation of strains containing random mutants In order to bcd-cgl The gene mutant was inserted into *Corynebacterium glutamicum* strain ATCC 13032, and a vector containing the target mutant fragment was constructed. Specifically, primers Up-bcd-F / Up-bcd-R were designed to amplify the upstream homologous arm and primers Down-bcd-F / Down-bcd-R were designed to amplify the downstream homologous arm, targeting the pseudogene site cgl0303 in *Corynebacterium glutamicum* strain ATCC 13032. The PCR reaction conditions were as follows: denaturation at 95℃ for 5 minutes; then 29 cycles, each cycle including denaturation at 95℃ for 30 seconds, annealing at 58℃ for 30 seconds, and extension at 72℃ for 150 seconds; finally, extension at 72℃ for 5 minutes. (The primers used are shown in Table 2).

[0042] The mutant fragment was ligated to the upstream and downstream homologous arms using a seamless cloning enzyme in a pk18 linear vector (pK18mobSacB, purchased from Miaoling Plasmid Platform, catalog number P0100), and then transformed into *E. coli* Top10 (purchased from Shanghai Angyu Biotechnology Co., Ltd., catalog number: G6015). After transforming the prepared vector into *E. coli* Top10, the transformed strains were selected in LB medium containing kanamycin. Plasmid DNA was obtained from the correct Top10 strains using a plasmid extraction kit, yielding the pk18-bcd-cgl vector containing the mutant.

[0043] The vector pk18-bcd-cgl was electroporated into *Corynebacterium glutamicum* ATCC13032 via homologous recombination on the chromosome. Strains that had inserted the vector into the chromosome via homologous sequence recombination were selected in BHI medium containing kanamycin. PCR was performed using primers Cva-bcd-F and Cva-bcd-R, with *Corynebacterium glutamicum* transformants that had undergone secondary recombination as templates, to identify whether the target gene mutant had been integrated into the genome. All randomly generated mutant strains were inoculated onto activated medium plates to obtain a strain library containing multiple random mutants.

[0044] Table 2 Primer Sequences Preparation of the activation culture medium used: Glucose 2.0 g / L, peptone 10.0 g / L, yeast powder 5.0 g / L, sodium chloride 2.5 g / L, KH2PO4 1.0 g / L, MgSO4 0.2 g / L, agar powder 2.5%, dissolved in water, then the pH was adjusted to 7.0-7.2 with sodium hydroxide, and the volume was brought to 500 mL. The mixture was then sterilized in an autoclave at 121℃ for 20 min.

[0045] Example 3 Evaluation of dominant mutants and screening of strains To select mutant strains with increased L-valine production capacity compared to Corynebacterium glutamicum ATCC 13032, fermentation potential tests were performed on strains from a library of random mutant strains.

[0046] 3.1 Culture medium Seed culture medium: Yeast powder 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.

[0047] 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.

[0048] 3.2 Seed activation and culture: The bacterial suspensions of Corynebacterium glutamicum ATCC 13032 inoculated in the preservation tube and the library strain prepared in Example 2 were evenly spread on the slant of activation medium and cultured at 32°C for 12 h. The culture was then transferred to the slant of activation medium and cultured for another 10 h. Finally, the culture was transferred to a shaker containing 5 mL of seed medium for seed culture.

[0049] 3.3 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 h, and no antibiotics or inducers were added during the fermentation process. After 36 h of shake-flask fermentation, the concentration of L-valine in the fermentation broth was analyzed by HPLC. The L-valine concentration is shown in Table 3.

[0050] Table 3. L-valine concentration in fermentation broth Based on the results shown in Table 3, it was found that *Corynebacterium glutamicum* ATCC 13032 and the Cval-bcd strain with integrated unmutated gene did not significantly increase valine production. The Cval-bcd-E9 strain, which showed the greatest increase in valine production compared to *Corynebacterium glutamicum* ATCC 13032 and the Cval-bcd (unmutated) strain (i.e., the control group), was selected for subsequent experiments. The results of this example confirm that introducing the bcd dominant mutant can effectively improve the valine production of the strain.

[0051] Example 4: Identification of mutations through gene sequencing The mutant gene of a strain with outstanding valine production was sequenced and compared with the starting point. bcd-cgl Gene comparisons were conducted. The results revealed a mutant in the Cval-bcd-E9 strain with enhanced valine production (…). bcd-cgl-E9 )exist bcd A specific location in the ORF region of a gene contains multiple nucleotide sequence mutations.

[0052] Specifically, it was discovered bcd-E9 and bcd-cgl The gene underwent multiple site mutations, and the specific locations of the resulting amino acid mutations and changes are shown in Table 4.

[0053] Table 4 bcd-cgl-E9 The encoded protein is relatively bcd-cgl Mutation location and amino acid changes in encoded proteins In summary, this invention employs random mutation technology to... bcd-cgl Gene sequences were randomly mutagenized to construct random mutant libraries. Mutants from this library were integrated into the genome of *Corynebacterium glutamicum* ATCC 13032 to obtain mutant strain libraries. Fermentation performance screening revealed that strains integrating specific advantageous mutants exhibited significantly higher valine production than both non-mutated and wild-type strains.

[0054] This application utilizes error-prone PCR technology to randomly mutate the leucine dehydrogenase encoding gene, constructs a mutant library, and screens for mutants with significantly enhanced catalytic activity. These mutants are then introduced into wild-type Corynebacterium glutamicum to further improve L-valine yield and production efficiency. This strategy not only promises to address the key bottleneck of redox imbalance in the traditional transaminase pathway but also provides a new approach for constructing highly efficient L-valine industrial production strains, possessing significant practical value and broad application prospects.

[0055] 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.

[0056] Some of the nucleic acid and amino acid sequences involved in this article: bcd-cgl bcd-cgl-E9 Gene sequence: bcd-cgl Amino acid sequence corresponding to the gene: MELFKYMEKYDYEQLVFCQDEQSGLKAIIAIHDTTLGPALGGTRMWTYENEEAAIEDALRLARGMTYKNAAAGLNLGGGKTVIIGDPRKDKNEEMFRAFGRYIQGLNGRYITAEDVGTTVEDMDIIHDETDYVTGISPAFGSSGNPSPVTAYGVYRGMKAAAKAAFGTDSLEGKTIAVQGVGNVAYNLCRHLHEEGANLIVTDINKQSVQRAVEDFGARAVDPDDIYSQDCDIYAPCALGATINDDTIKQLKAKVIAGAANNQLKETRHGDQIHEMGIVYAPDYVINAGGVINVADELYGYNAERALKKVEGIYGNIERVLEISQRDGIPAYLAADRLAEERIERMRRSRSQFLQNGHSVLSRR (SEQ ID NO: 11) bcd-cgl-E9 Amino acid sequence corresponding to the gene: MELFKYMEKYDYEQLVFCQDEQSGLKAIIAIHDTTLGPALGGTRMWTSENEEAAIEDALRLARGMTYKNAAAGLNLGGGKTVIIGDPRKDKNEEMFRAFGRYIQGLNGRYITAEDVGTTVEDMDIIRDETDYVTGISPAFGSSGNPSPVTAYGVYRGMKAAAKAAFGTDSLEGKTIAVQGVGNVAFNLCRHLHEEGANLIVTDINKQSVQRAVEDFGARAVDPDDIYSQDCDIYAPCALGATINDDTIKQLKAKVVAGAANNQLKETRHGDQIHEMGIVYAPDYVINAGGVINVADELYGYNAERALRKVEGIYGNIERVLEISQRDGIPAYLAADRLAEERIERMRRSRSQFLQNGHSVLSRR (SEQ ID NO: 12).

Claims

1. A leucine dehydrogenase mutant, characterized in that, The amino acid substitutions compared to Bacillus subtilis leucine dehydrogenase include the following: 1) Replace the tyrosine (Tyr) at position 48 with serine (Ser); 2) Replace histidine (His) at position 127 with arginine (Arg); 3) Replace the tyrosine (Tyr) at position 186 with phenylalanine (Phe); 4) Replace isoleucine (Ile) at position 256 with valine (Val); and 5) Replace the lysine (Lys) at position 308 with arginine (Arg).

2. The leucine dehydrogenase mutant as described in claim 1, characterized in that, The leucine dehydrogenase mutant includes the amino acid sequence shown in SEQ ID NO:

12.

3. Biomaterials related to the leucine dehydrogenase mutant of claim 1 or 2, characterized in that, It is any of the following: (1) The nucleic acid molecule encoded by the leucine dehydrogenase mutant; (2) An expression cassette encoding a nucleic acid molecule as described in (1); (3) A recombinant vector comprising the nucleic acid molecule encoded by (1) or the expression cassette by (2).

4. The biomaterial as described in claim 3, characterized in that, The encoded nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO:

10.

5. A valine-producing strain, characterized in that, Includes the biological material as described in claim 3 or 4, or the mutant expressing the leucine dehydrogenase as described in claim 1 or 2.

6. The valine-producing strain according to claim 5, characterized in that, The valine-producing strain is Corynebacterium glutamicum.

7. The valine-producing strain according to claim 6, characterized in that, The *Corynebacterium glutamicum* has the encoded nucleic acid molecule recombined and integrated at its pseudogene site cgl0303.

8. A method for preparing an L-valine-producing strain, characterized in that, This includes integrating a nucleic acid molecule encoding a leucine dehydrogenase mutant into the genome of Corynebacterium glutamicum, wherein the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

10.

9. The method as described in claim 8, characterized in that, The pseudogene site cgl0303, which encodes a nucleic acid molecule, is integrated into the genome of Corynebacterium glutamicum.

10. The use of the leucine dehydrogenase mutant of claim 1 or 2, the biomaterial of claim 3 or 4, or the L-valine-producing strain of any one of claims 5-7 in the preparation of L-valine.

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