Mutant branched-chain amino acid aminotransferase, l-valine-producing strain, and construction method and application thereof

Abstract

The application provides branched-chain amino acid transaminase mutants, L-valine production strains and construction methods and applications thereof. The branched-chain amino acid transaminase mutant comprises the following amino acid substitutions relative to wild-type branched-chain amino acid transaminase: glycine (Gly) at the 39th position is replaced by serine (Ser); arginine (Arg) at the 98th position is replaced by lysine (Lys); glycine (Gly) at the 197th position is replaced by alanine (Ala); and alanine (Ala) at the 260th position is replaced by valine (Val). A new strain expressing the mutant does not contain plasmids, has no growth defects, does not need to be induced, can stably produce valine, and has the advantages of high fermentation yield and the like.

Description

Technical Field

[0001] This invention belongs to the field of metabolic engineering and genetic engineering technology, and relates to branched-chain amino acid transaminase mutants, as well as L-valine producing strains, their construction methods and applications. Background Technology

[0002] L-valine (2-amino-3-methylbutyric acid) belongs to the branched-chain amino acid family. It serves as a core building block in protein synthesis and is deeply involved in various life activities such as skeletal muscle energy metabolism, tissue damage repair, and energy regulation. Due to its unique branched side chain configuration, L-valine occupies an irreplaceable position in diverse industries including pharmaceuticals, food fortification, livestock feed additives, and daily chemical products. It shows particularly broad application prospects in the preparation of antibacterial drugs, special medical purpose foods, and functional compound additives.

[0003] In terms of preparation processes, current methods for obtaining L-valine can be broadly categorized into three types: chemical directed synthesis, natural product extraction, and microbial transformation and fermentation. Comparatively, fermentation has gradually established its dominant position in industrial production due to its advantages such as convenient raw material acquisition, mild operating conditions, low environmental impact, and suitability for large-scale scaling. Among the many available production hosts, *Escherichia coli*, with its rich genetic manipulation toolbox, short cell doubling time, and thorough understanding of its physiological metabolic network, is widely recognized as a highly competitive candidate cell for the industrial preparation of amino acids.

[0004] In recent years, researchers have systematically enhanced the valine synthesis pathway in *E. coli* through metabolic regulation, effectively increasing the carbon metabolic flux of the target product. Among these enhancements, branched-chain amino acid transaminases (BCATs) have been significantly improved. ilvE) As a key gene at the end of the valine biosynthesis pathway, the gene plays a crucial role in the conversion of the precursor α-ketoisovalerate into the final product L-valine. This step directly determines the accumulation rate and conversion yield of the product. More importantly, ilvE Located at the intersection of multiple branched amino acid synthesis pathways, the regulation and modification of this gene have global regulatory significance for the targeted accumulation of the target product. Simultaneously, reactor-level process control has improved production efficiency, but under current conditions, deep-seated limitations such as the imbalance of carbon metabolic flux distribution still exist in scale-up engineering. Therefore, developing a high-performance L-valine-producing strain with a clear genetic makeup, coordinated physiological functions, and long-term passage stability remains a key challenge that urgently needs to be addressed in this field. Summary of the Invention

[0005] In one aspect, the present invention provides a branched-chain amino acid transaminase (ilvE) mutant, which, relative to wild-type ilvE, includes any one or any combination of the following amino acid substitutions: 1) Replace the glycine (Gly) at position 39 with serine (Ser); 2) Replace the arginine (Arg) at position 98 with lysine (Lys); 3) Replace the glycine (Gly) at position 197 with alanine (Ala); 4) Replace the alanine (Ala) at position 260 with valine (Val).

[0006] In some embodiments, the ilvE mutant, relative to wild-type ilvE, includes the following amino acid substitutions: 1) Replace the glycine (Gly) at position 39 with serine (Ser); 2) Replace the arginine (Arg) at position 98 with lysine (Lys); 3) Replace the glycine (Gly) at position 197 with alanine (Ala); 4) Replace the alanine (Ala) at position 260 with valine (Val).

[0007] In some embodiments, the ilvE mutant has at least 80%, 85%, 90%, or 95% amino acid sequence identity with the wild-type ilvE.

[0008] In some embodiments, the amino acid sequence of the wild-type ilvE is shown in SEQ ID NO: 4.

[0009] In some embodiments, the ilvE mutant comprises the amino acid sequence shown in SEQ ID NO: 2 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: 2.

[0010] On the other hand, the present invention provides biomaterials related to the above-mentioned ilvE mutant, which are any of the following: (1) The nucleic acid molecule encoded by the ilvE 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; (4) A host cell comprising (1) the encoding nucleic acid molecule, (2) the expression cassette or (3) the recombinant vector.

[0011] In some embodiments, the encoding nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO: 1.

[0012] In some implementations, the host cell is a prokaryote or a eukaryote.

[0013] In some implementations, the host cell includes Escherichia coli, Corynebacterium glutamicum, or Bacillus.

[0014] On the other hand, the present invention provides the application of the above-mentioned ilvE mutant or biological material in the construction of valine-producing strains.

[0015] On the other hand, the present invention provides a valine-producing strain comprising the above-mentioned biological material or expressing the above-mentioned ilvE mutant.

[0016] In some embodiments, the valine-producing strain includes Escherichia coli, Corynebacterium glutamicum, or Bacillus.

[0017] In some embodiments, the valine-producing strain is Escherichia coli W3110.

[0018] On the other hand, the present invention provides a method for preparing L-valine-producing bacteria, comprising: 1) Construct random mutants of the ilvE-encoded nucleic acid molecule; 2) Introduce the random mutant into the starting strain; and 3) Cultivate the strains with introduced random mutants and screen for strains that produce high levels of L-valine.

[0019] In some implementations, the starting strain is a bacterium.

[0020] In some embodiments, the high-L-valine-producing strain produces at least 1, 1.5, 2, or more L-valine-enhancing amounts compared to strains without the introduction of the random mutant.

[0021] In some embodiments, the method includes integrating the encoding nucleic acid molecule of the ilvE mutant into the Escherichia coli genome, wherein the nucleotide sequence encoding the nucleic acid molecule is shown in SEQ ID NO: 1.

[0022] On the other hand, the present invention provides the application of the above-mentioned ilvE mutant, biomaterial, or L-valine-producing strain in the preparation (or production) of L-valine or in increasing the yield of L-valine.

[0023] 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.

[0024] The Escherichia coli prepared by this invention can be stably produced and efficiently synthesizes valine using glucose as a substrate. After 36 hours of shake-flask fermentation, the valine yield can reach up to 1.69 g / L, which is more than 4 times the yield of wild-type Escherichia coli W3110. Detailed Implementation

[0025] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0026] When referring to branched-chain amino acid transaminases (ilvE), the term "mutant" (e.g., ilvE mutant) refers to an amino acid sequence modified compared to wild-type ilvE, such as the substitution, deletion, and / or insertion of one or more amino acids; when referring to a strain, the term "mutant" (e.g., mutant strain) refers to a strain that can express the aforementioned ilvE mutant and / or encoding nucleic acid molecules including the ilvE mutant.

[0027] The wild-type leucine dehydrogenase used in this article can be derived from Escherichia coli, and its amino acid sequence is shown in SEQ ID NO: 4. When describing specific amino acid mutation sites, this wild-type ilvE amino acid sequence is used as a reference.

[0028] 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.

[0029] 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.

[0030] As used herein, "recombinant vector" refers to a linear or circular nucleic acid molecule construct containing a polynucleotide encoding a mutant such as the ilvE mutant 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.

[0031] 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.

[0032] 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).

[0033] 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.

[0034] 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.

[0035] 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.

[0036] This study used Escherichia coli W3110 as the chassis strain, combining strains from the same species and carrying positive mutations. ilvE Genes were introduced into chassis strains to enhance the flow of the valine biosynthetic pathway. Based on this, we first performed ARTP mutagenesis on *E. coli*, obtaining a mutant strain with a significantly increased valine accumulation level. Subsequent sequencing data confirmed that this mutant... ilvE A key amino acid mutation site exists in the coding region of the gene. To assess the actual contribution of this mutation to acid production capacity, this study used CRISPR / Cas9-mediated gene editing technology to insert the mutated amino acid into the gene. ilvEThe gene was precisely integrated into the genome of the chassis strain, and the recombinant strain constructed was named valE-1. Shake-flask fermentation results showed that the final valine concentration of this strain was 1.69 g / L, significantly higher than that of the chassis strain. These results indicate that, through shake-flask fermentation alone... ilvE The discovery that a single point mutation in the gene itself can effectively activate the potential of the intrinsic valine synthesis network in E. coli without the need for exogenous gene elements opens up new entry points and practical pathways for gene manipulation in the cultivation of high-yield industrial strains.

[0037] This invention utilizes endogenous Escherichia coli ilvE By performing site-directed positive mutations on the gene, the overall efficiency of its valine biosynthesis pathway was effectively activated. This discovery provides a simple, efficient, safe, and reliable novel genetic engineering strategy for constructing high-performance E. coli strains for industrial-scale valine production.

[0038] The technical solution of the present invention will be further described below with reference to embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are merely exemplary and do not constitute a limitation on the scope of the present invention. It should be noted that, unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art.

[0039] The starting strain used in the examples was wild-type Escherichia coli W3110.

[0040] The gene editing method used was 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 that article.

[0041] Example 1: Directed Mutagenesis of Escherichia coli Based on ARTP Mutagenesis Technology To screen for mutant strains with enhanced valine production capacity, Escherichia coli W3110 was used as the starting strain and inoculated into LB liquid medium, and cultured with shaking at 37℃ and 220 r / min.

[0042] (1) Preparation of bacterial suspension: Collect bacterial culture to 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 bacterial suspension. In a clean bench, place the metal slide in the outer flame of an alcohol lamp for 30 s for sterilization, cool and place it on a sterile plate, and take 10 μL of bacterial suspension to spread evenly on the surface of the slide.

[0043] (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, and close the chamber door.

[0044] (3) Set the mutagenesis parameters to 120 W power, 55 s treatment time, and 10 slm gas flow rate, and start the mutagenesis program. After treatment, remove the slide with sterile forceps and place it into an EP tube pre-filled with 1 mL of physiological saline.

[0045] (4) Place the EP tube on a shaker and shake for 1 min to fully wash 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 culturing, hundreds of randomly mutated colonies were obtained, and random mutations have been introduced into the genome of these strains.

[0046] 1.1 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, bring volume to 500 mL, dispense into test tubes (9 mL / tube) and flasks (45 mL / flask), and autoclave at 121 °C for 20 min.

[0047] 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 Escherichia coli W3110, the fermentation performance of colonies obtained through random mutagenesis was evaluated.

[0048] 1.2.1 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 H1 mg / L, MgSO4·7H2O 0.5 g / L, the remainder is water.

[0049] 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.

[0050] 1.2.2 Seed activation and culture: Inoculate Escherichia coli W3110 and Escherichia coli mutant strains into the preservation tube, spread them evenly on the activation slant, and incubate at 37°C for 12 h. Transfer to the activation slant and continue incubation for 10 h. Then transfer to a shaker containing 5 mL of seed culture medium for seed culture.

[0051] 1.2.3 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 37°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 concentrations of some strains are shown in Table 1.

[0052] Table 1. L-valine concentration in fermentation broth Based on the results shown in Table 1, the Val-5 strain, which showed a significant increase in valine production compared to the control strain W3110 of Escherichia coli, was selected for subsequent mutation identification.

[0053] Example 2: Identification of Escherichia coli valine mutant strain using whole-genome sequencing technology Whole-genome resequencing was performed on Val-5, a high-yielding mutant strain of *E. coli* obtained through ARTP mutagenesis screening, and the genome of wild-type *E. coli* W3110 was used as a reference for sequence alignment analysis. Sequencing data showed that, compared to the original strain, the mutant strain Val-5, with significantly enhanced valine accumulation capacity, exhibited… ilvECharacteristic nucleotide variations were found within the open reading frame of the gene. Further analysis revealed multiple base substitutions in the coding region of the gene, resulting in the replacement of corresponding amino acid residues. Specific mutation sites and corresponding amino acid changes are detailed in Table 2.

[0054] Further gene editing technology can be used to insert the aforementioned genes carrying key mutation sites. ilvE Gene fragments are integrated into the E. coli genome, and unmutated gene fragments are incorporated into the genome. ilvE The gene was integrated into the genome in parallel to verify its regulatory role in valine synthesis.

[0055] Table 2. Amino acid changes at mutation sites Functional analysis of the aforementioned mutation sites revealed that these amino acid substitutions have a significant positive regulatory effect on valine synthesis efficiency. This may be because site-directed mutations in the ilvE gene alter the spatial conformation of the branched-chain amino acid transaminase it encodes, thereby increasing the enzyme's catalytic activity or alleviating the rate-limiting step in the metabolic pathway, ultimately leading to a significant increase in the valine accumulation capacity of *E. coli*.

[0056] In the following examples, gene editing technology is used to modify mutants. ilvE Genes and non-mutated ilvE The gene was integrated in parallel into the genome of Escherichia coli W3110, and its fermentation performance was evaluated.

[0057] Example 3 Construction of Escherichia coli W3110 engineered strain based on ilvE gene and verification of valine production 3.1 Integration ilvE Construction of mutant genes and wild-type genetically engineered strains To introduce the point mutation into *E. coli*, genomic DNA was first extracted from *E. coli* W3110 using a bacterial genome extraction kit. The desired ilvE site-directed mutant gene was obtained using GenScript Biotech's gene synthesis service. Using this gene as a template, a 1053 bp fragment containing the target mutation was amplified using primers ilvE-F and ilvE-R. After gel extraction and purification, this fragment was designated as the "introduced mutation fragment ilvE". Using the *E. coli* W3110 genome as a template, a 1053 bp wild-type target fragment was amplified using primers ilvE-F and ilvE-R. After gel extraction and purification, this fragment was designated as "introduced wild-type target fragment ilvE". Using a usable concentration of the *E. coli* W3110 genome as a template, primers Up- yjgX -F / R and Down- yjgX -F / R was used for PCR amplification to obtain the upper homologous arm yjgX -UP, lower homologous armyjgX -DW. With the recovered upstream and downstream homologous arms and the "introduced mutant fragment ilvE" Using "or 'introducing the wild-type order fragment ilvE' as a template, and using primer Up- yjgX -F and Down- yjgX -R is integrated into the target fragment via overlap PCR. yjgX -Ptrc-ilvE and yjgX -Ptrc-ilvE. PCR reaction conditions were: 95℃ pre-denaturation for 5 minutes; followed by 29 cycles, each cycle consisting of 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, and 72℃ extension for 150 seconds; a final extension at 72℃ for 5 minutes. Afterwards, primer pGRB- yjgX -F and pGRB- yjgX The DNA fragment obtained by -R annealing was ligated onto plasmid pGRB to construct pGRB- yjgX Plasmid. Finally, the purified plasmid... yjgX -Ptrc-ilvE or yjgX -Ptrc-ilvE integration fragment and plasmid pGRB- yjgX Simultaneously transferred via electroconversion E.coli In W110 competent cells, primer Up- yjgX -F and Down- yjgX -R is the primer for screening positive transformants. The PCR products were sequenced, and the sequences matched the corresponding sequences in the *E. coli* mutant and wild-type strains, ultimately yielding strains valE-1 and valE-2. The primers used in the strain construction process are shown in Table 3. Table 3 Primers used in strain construction. 3.2 Identification of Valine Production Capacity 3.2.1 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.

[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, valE-1 and valE-2 bacterial suspensions from the preservation tube and spread them evenly on the activation medium slant. Incubate at 37°C for 12 h, transfer to the activation slant and continue incubation for 10 h, then transfer to a shaker containing 5 mL of seed medium for seed culture at 37°C and 220 r / min for 12 h.

[0060] 3.2.2 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 37°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 concentrations of the fermentation broths for each strain are shown in Table 4.

[0061] Table 4 L-valine concentration in fermentation broth of each strain In this embodiment, gene editing technology was used to integrate the mutant ilvE gene of *E. coli* obtained through ARTP mutagenesis screening and its wild-type control gene into the genome of *E. coli* W3110, thereby constructing recombinant strains valE-1 and valE-2. Subsequent shake-flask fermentation performance evaluation showed that the mutant ilvE gene... ilvE The recombinant strain valE-1, which carries the gene, has a final valine concentration that is higher than that of the control strain and the wild-type strain. ilvE All recombinant strains showed a significant increase in gene expression. This result clearly indicates that... ilvE Point mutations in genes can effectively drive the enhancement of valine synthesis in Escherichia coli.

[0062] This study used *Escherichia coli* as the starting strain and employed gene editing technology to enhance its valine synthesis pathway. First, wild-type *E. coli* strains were targeted using ARTP mutagenesis to screen for a mutant strain with significantly increased valine production. Genome sequencing analysis successfully located the mutant... ilvE Key positive mutation sites in genes. Based on this, using the CRISPR / Cas9-mediated gene editing system, the mutant... ilvE The gene was precisely integrated into the genome of wild-type Escherichia coli W3110. The resulting recombinant strain exhibits advantages such as no need for exogenous plasmids, unaffected cell growth, independence from inducer addition, and excellent passage stability. After 36 hours of fermentation in a shake-flask culture system with glucose as the carbon source, the engineered strain achieved a cumulative valine concentration of 1.69 g / L, demonstrating a significant yield advantage over the original chassis strain.

[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.

[0064] The relevant nucleic acid and amino acid sequences involved in this article are as follows.

[0065] ilvE mutant nucleotide sequence (SEQ ID NO: 1): atgaccacgaagaaagctgattacatttggttcaatggggagatggttcgctgggaagacgcgaaggtgcatgtgatgtcgcacgcgctgcactatggcacttcggtttttgaa a gcatccgttgctacgactcgcacaaaggaccggttgtattccgccatcgtgagcatatgcagcgtctgcatgactccgccaaaatctatcgcttcccggtttcgcagagcattgatgagctgatggaagcttgtcgtgacgtgatccgcaaaaacaatctcaccagcgcctatatc aaaccgctgatcttcgtcggtgatgttggcatgggagtaaacccgccagcgggatactcaaccgacgtgattatcgctgctttcccgtggggagcgtatctgggcgcagaagcgctggagcaggggatcgatgcgatggtttcctcctggaaccgcgcagcaccaaacaccatcccgacggcggcaaaagccggtggtaactacctctcttccctgctggtgggtagcgaagcgcgccgccacggttatcaggaaggtatcgcgctggatgtgaacggttatatctctgaaggcgcag c cgaaaacctgtttgaagtgaaagatggtgtgctgttcaccccaccgttcacctcctccgcgctgccgggtattacccgtgatgccatcatcaaactggcgaaagagctgggaattgaagtacgtgagcaggtgctgtcgcgcgaatccctgtacctggcggatgaagtgtttatgtccggtacggcgg tg gaaatcacgccagtgcgcagcgtagacggtattcaggttggcgaaggccgttgtggcccggttaccaaacgcattcagcaagccttcttcggcctcttcactggcgaaaccgaagataaatggggctggttagatcaagttaatcaataa ilvE Mutant amino acid sequence (SEQ ID NO: 2): MTTKKADYIWFNGEMVRWEDAKVHVMSHALHYGTSVFESIRCYDSHKGPVVFRHREHMQRLHDSAKIYRFPVSQSIDELMEACRDVIRKNNLTSAYIKPLIFVGDVGMGVNPPAGYSDTVIIAAFPWGAYLGAEALEQGIDAMVSSWNRAAPNT IPTAAKAGGNYLSSLLVGSEARRHGYQEGIALDVNGYISEGAAENLFEVKDGVLFTPPFTSSALPGITRDAIIKLAKELGIEVREQVLSRESLYLADEVFMSGTAVEITPVRSVDGIQVGEGRCPVTKRIQQAFFGLFTGETEDKWGWLDQVNQ Wild type ilvE nucleotide SEQ ID NO:3: atgaccacgaagaaagctgattacatttggttcaatggggagatggttcgctgggaagacgcgaaggtgcatgtgatgtcgcacgcgctgcactatggcacttcggtttttgaaggcatccgttgctacgactcgcacaaaggaccggttgtattccgccatcgtgagcatatgcagcgtctgcatgactccgccaaaatctatcgcttcccggtttcgcagagcattgatgagctgatggaagcttgtcgtgacgtgatccgcaaaaacaatctcaccagcgcctatatccgtccgctgatcttcgtcggtgatgttggcatgggagtaaacccgccagcgggatactcaaccgacgtgattatcgctgctttcccgtggggagcgtatctgggcgcagaagcgctggagcaggggatcgatgcgatggtttcctcctggaaccgcgcagcaccaaacaccatcccgacggcggcaaaagccggtggtaactacctctcttccctgctggtgggtagcgaagcgcgccgccacggttatcaggaaggtatcgcgctggatgtgaacggttatatctctgaaggcgcaggcgaaaacctgtttgaagtgaaagatggtgtgctgttcaccccaccgttcacctcctccgcgctgccgggtattacccgtgatgccatcatcaaactggcgaaagagctgggaattgaagtacgtgagcaggtgctgtcgcgcgaatccctgtacctggcggatgaagtgtttatgtccggtacggcggcagaaatcacgccagtgcgcagcgtagacggtattcaggttggcgaaggccgttgtggcccggttaccaaacgcattcagcaagccttcttcggcctcttcactggcgaaaccgaagataaatggggctggttagatcaagttaatcaataa Wild type ilvE Amino acid sequence (SEQ ID NO: 4): MTTKKADYIWFNGEMVRWEDAKVHVMSHALHYGTSVFEGIRCYDSHKGPVVFRHREHMQRLHDSAKIYRFPVSQSIDELMEACRDVIRKNNLTSAYIRPLIFVGDVGMGVNPPAGYSTDVIIAAFPWGAYLGAEALEQGIDAMVSSWNRAAPNTIPTAAKAGGNYLSSLLVGSEARRHGYQEGIALDVNGYISEGAGENLFEVKDGVLFTPPFTSSALPGITRDAIIKLAKELGIEVREQVLSRESLYLADEVFMSGTAAEITPVRSVDGIQVGEGRCGPVTKRIQQAFFGLFTGETEDKWGWLDQVNQ。

Claims

1. A branched-chain amino acid transaminase mutant, characterized in that, The amino acid sequence of the branched-chain amino acid transaminase mutant is shown in SEQ ID NO:

2.

2. A biomaterial related to the branched-chain amino acid transaminase mutant of claim 1, characterized in that, It is any of the following: (1) The nucleic acid molecule encoded by the branched-chain amino acid transaminase 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; (4) A host cell comprising (1) the encoding nucleic acid molecule, (2) the expression cassette or (3) the recombinant vector.

3. The biomaterial as described in claim 2, characterized in that, The encoded nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO:

1.

4. The biomaterial as described in claim 2 or 3, characterized in that, The host cell is Escherichia coli.

5. A valine-producing strain, characterized in that, Includes the biological material according to any one of claims 2-4 or the mutant expressing the branched-chain amino acid transaminase according to claim 1.

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

7. The application of the branched-chain amino acid transaminase mutant of claim 1 or the biomaterials of claims 2-4 in the construction of L-valine-producing strains.

8. The application as described in claim 7, characterized in that, The L-valine-producing strain is Escherichia coli.

9. The use of the branched-chain amino acid transaminase mutant of claim 1, the biomaterial of any one of claims 2-4, or the valine-producing strain of claim 5 or 6 in the production of L-valine or in increasing L-valine yield.

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