Aspartate aminotransferase mutants and their use in the production of l-isoleucine

Abstract

The application discloses an aspartate aminotransferase mutant and application thereof in L-isoleucine production. The application provides an aspartate aminotransferase mutant, which comprises a protein obtained by mutating the 204th amino acid residue of wild-type aspartate aminotransferase from threonine to serine. The application overexpresses the wild-type aspartate aminotransferase gene from corynebacterium glutamicum in a corynebacterium glutamicum receptor aspB or a mutant thereof aspB A610T (including integration into the genome, and also existing in the form of a free plasmid), or directly mutating the endogenous aspB gene to aspB A610T a mutant gene can produce a more cost-effective L-isoleucine fermentation production strain. The application has important significance for improving the fermentation yield of L-isoleucine.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to aspartate aminotransferase mutants and their application in the production of L-isoleucine. Background Technology

[0002] L-Isoleucine is an essential branched-chain amino acid that cannot be synthesized by humans and animals. It plays a central role in regulating protein synthesis, blood glucose balance, and immune signaling. The global annual demand has exceeded 60,000 tons, with 70% used in high-end feed and the remainder used in parenteral nutrition infusions and sports nutrition fortifiers.

[0003] Currently, the industrial production of isoleucine almost entirely employs direct microbial fermentation. However, wild-type strains suffer from multiple feedback inhibitions, and the proportion of byproducts such as valine and leucine is high. Downstream ion exchange separation costs account for over 40% of the total cost. With the advancement of non-grain raw material routes and green manufacturing policies, there is an urgent need to build efficient cell factories.

[0004] The catalytic performance of natural metabolic enzymes in existing wild-type strains has inherent defects, including limited catalytic efficiency and poor substrate adaptability, making it difficult to meet the demands of high-density fermentation and high product accumulation in industrial production. This hinders further improvements in overall fermentation capacity and raw material utilization. To address this technological challenge, developing superior mutant enzymes and applying them to L-isoleucine fermentation production is of significant practical and industrial value for optimizing strain metabolic synthesis capabilities, increasing fermentation yield, reducing byproduct generation, and lowering production costs. Summary of the Invention

[0005] The purpose of this invention is to provide an aspartate aminotransferase mutant and its application in the production of L-isoleucine.

[0006] In a first aspect, the present invention claims protection for an aspartate aminotransferase mutant.

[0007] The aspartate aminotransferase mutants claimed in this invention include or are any of the following: A1) The protein obtained by mutating the 204th amino acid residue of wild-type aspartate aminotransferase from threonine to serine. A2) Proteins derived from Corynebacterium glutamicum with the amino acid sequences shown in A1) having undergone substitution and / or deletion and / or addition of one or more amino acid residues and having the same function. A3) Proteins that have at least 90% identity with the amino acid sequence defined by A1) or A2) and have the same function; A4) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the proteins defined in A1)-A3).

[0008] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using identity 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 program, 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 an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0009] In the aforementioned proteins, the phrase "at least 90% identity" can mean at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0010] Further, in A1), the wild-type aspartate aminotransferase is a wild-type aspartate aminotransferase derived from Corynebacterium glutamicum. Correspondingly, the proteins described in A2) and A3) are proteins derived from Corynebacterium glutamicum.

[0011] Furthermore, the amino acid sequence of the wild-type aspartate aminotransferase (i.e., wild-type aspartate aminotransferase derived from Corynebacterium glutamicum) includes SEQ ID NO:8. In some embodiments of the present invention, the amino acid sequence of the wild-type aspartate aminotransferase (i.e., wild-type aspartate aminotransferase derived from Corynebacterium glutamicum) is SEQ ID NO:8.

[0012] More specifically, the amino acid sequence of the aspartate aminotransferase mutant includes that shown in SEQ ID NO:12. In some embodiments of the present invention, the amino acid sequence of the aspartate aminotransferase mutant is SEQ ID NO:12.

[0013] Secondly, this invention claims protection for biological materials.

[0014] The biological materials claimed in this invention include or are any of the following: B1) The nucleic acid molecule encoding the aspartate aminotransferase mutant described in the first aspect above; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); 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); B5) A whole-cell catalyst containing the nucleic acid molecule described in B1), or a whole-cell catalyst containing the expression cassette described in B2), or a whole-cell catalyst containing the recombinant vector described in B3), or a whole-cell catalyst containing the recombinant microorganism described in B4).

[0015] Furthermore, the nucleic acid molecule described in B1) includes or is as follows: (b11) or (b12) or (b13) b11) Nucleic acid molecules that contain or have the coding sequence shown in SEQ ID NO:11; b12) The nucleic acid molecule described in b11) has more than 80% similarity to the nucleic acid molecule described in the first aspect above and encodes the nucleic acid molecule of the aspartate aminotransferase mutant described in the first aspect above; b13) Hybridizes under stringent conditions with the nucleic acid molecule defined by b11) or b12) and encodes the nucleic acid molecule of the aspartate aminotransferase mutant described in the first aspect above.

[0016] The stringent conditions for the above nucleic acid molecules can be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 2×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 1×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.5×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.1 ...7% SDS, 0.5M Na3PO4, and 1mM EDTA; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.1×SSC and 0.1% SDS. Hybridize in a mixed solution of SDS, 0.5 M Na3PO4 and 1 mM EDTA, and wash at 65 °C with 0.1 × SSC and 0.1% SDS; alternatively, hybridize in a solution of 6 × SSC and 0.5% SDS at 65 °C, and then wash once each with 2 × SSC and 0.1% SDS and 1 × SSC and 0.1% SDS.

[0017] For the aforementioned nucleic acid molecules, the identity of nucleotide sequences can be determined using identity retrieval sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastn as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gapexistence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, the identity of a pair of nucleotide sequences can be retrieved and calculated, and then the identity value (%) can be obtained.

[0018] In the aforementioned nucleic acid molecules, the 80% or more 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.

[0019] In some embodiments of the present invention, the coding sequence of the nucleic acid molecule described in B1) is SEQ ID NO:11.

[0020] In the aforementioned biological materials, the microorganisms may be bacteria.

[0021] Furthermore, the bacteria are bacteria capable of producing L-isoleucine.

[0022] "Bacteria capable of producing L-isoleucine" refers to bacteria that possess the following abilities: the ability to produce and accumulate L-isoleucine within the bacteria using external substances (such as culture medium), and may further include the ability to secrete L-isoleucine into the culture system. Thus, L-isoleucine can be collected when bacteria are cultured in a culture medium.

[0023] The bacteria can be naturally collected wild-type bacteria or modified bacteria.

[0024] "Modified bacteria" refers to bacteria that have been artificially mutated and / or induced to grow from naturally collected wild-type bacteria.

[0025] Furthermore, the bacteria may be members of the genus *Corynebacterium*. In some embodiments of the present invention, the bacteria are *Corynebacterium glutamicum*.

[0026] In B5), the whole-cell catalyst uses the recombinant microorganism as its functional core. The aspartate aminotransferase mutant expressed heterologously in the cell can directly catalyze the target reaction without the need for enzyme separation and purification. The catalytic stability of the enzyme can be maintained by the natural microenvironment within the cell. At the same time, the microorganism's own metabolism can achieve self-sufficiency and regeneration of coenzymes, simplifying the catalytic reaction process and making it suitable for large-scale industrial applications.

[0027] Thirdly, the present invention claims protection for the use of the aspartate aminotransferase mutant described in the first aspect above or the biological material described in the second aspect above in any of the following: P1. Production of L-isoleucine; P2. Increase the production of L-isoleucine in recipient bacteria; P3. Construct engineered bacteria that produce L-isoleucine; P4. Prepare products containing L-isoleucine.

[0028] Both the recipient bacteria in P2 and the engineered bacteria in P3 can be bacteria.

[0029] Furthermore, the bacteria are bacteria capable of producing L-isoleucine.

[0030] "Bacteria capable of producing L-isoleucine" refers to bacteria that possess the following abilities: the ability to produce and accumulate L-isoleucine within the bacteria using external substances (such as culture medium), and may further include the ability to secrete L-isoleucine into the culture system. Thus, L-isoleucine can be collected when bacteria are cultured in a culture medium.

[0031] The bacteria can be naturally collected wild-type bacteria or modified bacteria.

[0032] "Modified bacteria" refers to bacteria that have been artificially mutated and / or induced to grow from naturally collected wild-type bacteria.

[0033] Furthermore, the bacteria may be members of the genus *Corynebacterium*. In some embodiments of the present invention, the bacteria are *Corynebacterium glutamicum*.

[0034] Fourthly, the present invention claims protection for a recombinant Corynebacterium glutamicum.

[0035] The recombinant Corynebacterium glutamicum claimed in this invention comprises or is any of the following: C1) Recombinant Corynebacterium glutamicum containing the encoding gene of the aspartate aminotransferase mutant described in the first aspect above; C2) Recombinant Corynebacterium glutamicum obtained by enhancing the expression of the aspartate aminotransferase encoding gene in the recipient Corynebacterium glutamicum.

[0036] The aspartate aminotransferase is either the wild-type aspartate aminotransferase derived from Corynebacterium glutamicum mentioned in the first aspect above, or a mutant of the aspartate aminotransferase mentioned in the first aspect above.

[0037] Furthermore, in C2), the expression of the gene encoding the aspartate aminotransferase can be enhanced in any of the following ways: c1) Replace the coding region of the poxB gene in the genome of the recipient Corynebacterium glutamicum with the expression cassette of the coding gene for wild-type aspartate aminotransferase described in the first aspect above. c2) A point mutation is made in the gene encoding the wild-type aspartate aminotransferase described in the first aspect above in the genome of the recipient Corynebacterium glutamicum, the point mutation causing the 204th amino acid residue of the wild-type aspartate aminotransferase to change from threonine (T) to serine (S). c3) Replace the coding region of the poxB gene in the genome of the recipient Corynebacterium glutamicum with the coding gene expression cassette of the aspartate aminotransferase mutant described in the first aspect above; c4) Introduce into the genome of the recipient Corynebacterium glutamate a recombinant vector (in the form of a free plasmid) carrying the encoding gene of the wild-type aspartate aminotransferase described in the first aspect above. c5) Introduce into the genome of the recipient Corynebacterium glutamicum a recombinant vector (in the form of a free plasmid) carrying the coding gene of the aspartate aminotransferase mutant described in the first aspect above.

[0038] Wherein, the recombinant Corynebacterium glutamicum obtained in c1) is designated as recombinant Corynebacterium glutamicum 1, the recombinant Corynebacterium glutamicum obtained in c2) is designated as recombinant Corynebacterium glutamicum 2, the recombinant Corynebacterium glutamicum obtained in c3) is designated as recombinant Corynebacterium glutamicum 3, the recombinant Corynebacterium glutamicum obtained in c4) is designated as recombinant Corynebacterium glutamicum 4, and the recombinant Corynebacterium glutamicum obtained in c5) is designated as recombinant Corynebacterium glutamicum 5.

[0039] In c1)-c5), the recipient Corynebacterium glutamicum normally expresses its own aspartate aminotransferase.

[0040] In some embodiments of the present invention, the recombinant Corynebacterium glutamicum 1 is a recombinant bacterium obtained by replacing the segment located between SEQ ID NO:1 and SEQ ID NO:3 (i.e., the coding region of the poxB gene) in the genome of the recipient Corynebacterium glutamicum with the segment shown in SEQ ID NO:2 (the coding gene for wild-type aspartate aminotransferase derived from the Corynebacterium glutamicum and its promoter fragment). Further, other sequences remain unchanged.

[0041] In some embodiments of the present invention, the recombinant Corynebacterium glutamicum 2 is a recombinant bacterium obtained by mutating the nucleotide sequence at position 610 (A) of the recipient Corynebacterium glutamicum genome shown in SEQ ID NO:7 to T (this mutation causes the 204th amino acid residue of the wild-type aspartate aminotransferase derived from Corynebacterium glutamicum to change from T to S). Further, other sequences remain unchanged.

[0042] In some embodiments of the present invention, the recombinant Corynebacterium glutamicum 3 is a recombinant bacterium obtained by replacing the segment located between SEQ ID NO:1 and SEQ ID NO:3 (i.e., the coding region of the poxB gene) in the genome of the recipient Corynebacterium glutamicum with the segment shown in SEQ ID NO:13 (the coding gene of the aspartate aminotransferase mutant and its promoter fragment). Further, other sequences remain unchanged.

[0043] In some embodiments of the present invention, the recombinant Corynebacterium glutamicum 4 is a recombinant bacterium obtained by introducing a recombinant vector carrying SEQ ID NO:7 (i.e., the encoding gene for wild-type aspartate aminotransferase from the Corynebacterium glutamicum) into the recipient Corynebacterium glutamicum. Specifically, the recombinant vector is obtained by cloning SEQ ID NO:7 into the pXMJ19 plasmid.

[0044] In some embodiments of the present invention, the recombinant Corynebacterium glutamicum 5 is a recombinant bacterium obtained by introducing a recombinant vector carrying SEQ ID NO:11 (i.e., the encoding gene of the aspartate aminotransferase mutant) into the recipient Corynebacterium glutamicum. Specifically, the recombinant vector is obtained by cloning SEQ ID NO:11 into the pXMJ19 plasmid.

[0045] In the foregoing related aspects, in some embodiments of the present invention, the *Corynebacterium glutamicum* (or the *Corynebacterium glutamicum* receptor) is *Corynebacterium glutamicum* (… Corynebacterium glutamicum CGMCC No. 20437. In some other embodiments of the present invention, the Corynebacterium glutamicum (or the recipient Corynebacterium glutamicum) is Corynebacterium glutamicum ATCC13032.

[0046] Fifthly, the present invention claims protection for the use of the recombinant Corynebacterium glutamicum described in the fourth aspect above in the production of L-isoleucine.

[0047] Sixthly, the present invention claims a method for producing L-isoleucine.

[0048] The method for producing L-isoleucine claimed in this invention may include the following steps: fermenting and culturing the recombinant Corynebacterium glutamicum described in the fifth aspect above, and obtaining L-isoleucine from the fermentation product.

[0049] The cultivation of the recombinant Corynebacterium glutamicum can be carried out by methods known to those skilled in the art using existing techniques, and the fermentation method can also be optimized and improved through routine experiments. The fermentation can be carried out under fermentation conditions known in the art in a suitable culture medium. The culture medium may contain: a carbon source, a nitrogen source, trace elements, and combinations thereof. During cultivation, the pH of the culture can be adjusted. During cultivation, the temperature of the culture can be controlled. During cultivation, the rotation speed can be controlled. During cultivation, the fermentation time can be controlled. During cultivation, dissolved oxygen can be controlled.

[0050] In some embodiments of the present invention, the specific culture medium formulation and fermentation process used are described in the relevant section of Example 5.

[0051] Experiments have demonstrated that this invention enables the overexpression of the wild-type aspartate aminotransferase gene derived from Corynebacterium glutamicum in the recipient Corynebacterium glutamicum. aspB or its mutants aspB A610T (Including integration into the genome, as well as existence as free plasmids), or directly using endogenous... aspB Gene mutation aspB A610T All of these strains can produce more cost-effective L-isoleucine fermentation production strains. This invention is of great significance for improving the fermentation yield of L-isoleucine.

[0052] explanation Classification and nomenclature: Corynebacterium glutamicum ( Corynebacterium glutamicum ); Biological material used for ginseng: YPILE001 Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; The abbreviation for the depository institution is CGMCC. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Date of deposit: August 17, 2020; Registered with the China National Collection Center (CGMCC) No. 20437. Detailed Implementation

[0053] 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.

[0054] 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.

[0055] Corynebacterium glutamicum ATCC13032, also known as ATCC13032, is a product of the American Type Culture Collection (ATCC), with strain number 13032.

[0056] Corynebacterium glutamicum ( Corynebacterium glutamicum CGMCC No. 20437, also known as Corynebacterium glutamicum CGMCC20437, is a strain that has been deposited through a patent procedure with the accession number CGMCC No. 20437.

[0057] Escherichia coli MG1655 was obtained from Beijing Beina Innovation Biotechnology Research Institute (BNCC), strain number: BNCC363342.

[0058] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0059] Example 1: Construction of Corynebacterium glutamicum overexpressing on the genome aspB Genes and E. coli aspC engineered strains of genes Vectors were constructed using NEBuilder recombination technology, based on the *Corynebacterium glutamicum* strain published by NCBI. Corynebacterium glutamicum The genome sequence of ATCC 13032 (GenBank:BA000036.3, 2016-10-07) and Escherichia coli ( Escherichia coli The genome sequence of MG1655 (GenBank: CP032667.1, 2018-10-08) was used to design and synthesize amplified Corynebacterium glutamicum. aspB Gene coding regions and their promoter regions, Escherichia coli aspC Primers for the gene coding region and promoter region were used to overexpress the above gene in *Corynebacterium glutamicum* CGMCC No. 20437 and ATCC 13032 via homologous recombination. Note: *Escherichia coli* does not have this feature. aspB Genes, their aspC The gene is the aspartate aminotransferase encoding gene of Escherichia coli, which belongs to the core metabolic genes.

[0060] The primers were designed as follows (synthesized by Invitrogen Shanghai), and the nucleotide sequences in lowercase format are the sequences on the pK18mobsacB plasmid: P1: 5'-cagtgccaagcttgcatgcctgcaggtcgactctagCATTGCCATCAATCCATGC-3'; P2: 5'-CAAAGCAGCCATGCGTTGCAGCCTGATTGATACACCTGCTGTT-3'; P3: 5'-AACAGCAGGTGTATCAATCAGGCTGCAACGCATGGCTGCTTTG-3'; P4: 5'-GCCATAACGTTGAGGAGTTCAGTTAGTTAGCGTAATGCTCCGCTGC-3'; P5: 5'-GCAGCGGAGCATTACGCTAACTAACTGAACTCCTCAACGTTATGGC-3'; P6: 5'-cagctatgaccatgattacgaattcgagctcggtacccGCACCAAGTGAAGCGATTC-3'; P7: 5'-CTATTACTGATGAAAACGCAGGCTTGATTGATACACCTGCTGTTC-3'; P8: 5'-GAACAGCAGGTGTATCAATCAAGCCTGCGTTTTCATCAGTAATAG-3'; P9: 5'-GCCATAACGTTGAGGAGTTCAGTTACAGCACTGCCACAATCGCTTC-3'; P10: 5'-GAAGCGATTGTGGCAGTGCTGTAACTGAACTCCTCAACGTTATGGC-3'.

[0061] Using genomic DNA from Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed using primers P1 and P2 to obtain... aspB The upstream homologous arm fragment of the gene is 863 bp (sequence shown in SEQ ID NO:1); PCR amplification was performed using primers P3 and P4 to obtain... aspBThe gene and its promoter fragment 1621 bp (sequence shown in SEQ ID NO:2) were amplified by PCR using primers P5 and P6 to obtain... aspB The downstream homologous arm fragment of the gene is 821 bp (sequence shown in SEQ ID NO:3).

[0062] Using genomic DNA from Escherichia coli MG1655 as a template, PCR amplification was performed using primers P1 and P7 to obtain... aspC The upstream homologous arm fragment of the gene is 864 bp (sequence shown in SEQ ID NO:4); PCR amplification was performed using primers P8 and P9 to obtain... aspC The gene and its promoter fragment 1328 bp (sequence shown in SEQ ID NO:5) were amplified by PCR using primers P10 and P6 to obtain... aspC The downstream homologous arm fragment of the gene is 821 bp (sequence shown in SEQ ID NO:6).

[0063] After the PCR reaction, all amplified fragments were recovered by electrophoresis using a column-based DNA gel extraction kit. The two combined fragments (Combination 1: aspB upstream homologous arm fragment of the gene, aspB Genes and their promoter fragments and aspB Downstream homologous arm fragment of the gene; Combination 2: aspC upstream homologous arm fragment of the gene, aspC Genes and their promoter fragments and aspC The downstream homologous arm fragment of the gene) and the sequence of genes that have undergone homologous arm processing are respectively related to the gene that has undergone homologous arm processing. Xbal I / BamH The pK18mobsacB plasmid purified after I enzyme digestion was ligated with NEBuilder enzyme (NEB) at 50℃ for 30 min. The ligation product was transformed into DH5α and the resulting single clones were identified by PCR using M13 primers (M13F: 5'-TGTAAAACGACGGCCAGT-3'; M13R: 5'-CAGGAAACAGCTATGACC-3') to obtain positive integration plasmids (recombinant vectors). The correctly sequenced integration plasmids were named pK18-aspBOE and pK18-aspCOE according to the different exogenous fragments inserted. The positive integration plasmids contained a kanamycin resistance marker, and recombinants integrated into the genome could be obtained by kanamycin screening.

[0064] The PCR reaction system consisted of: 5 μL of 10×Ex Taq Buffer, 4 μL of dNTP Mixture (2.5 mM each), and Mg... 2+4 μL of (25 mM) primers, 2 μL each of (10 pM) primers, 0.25 μL of Ex Taq (5 U / μL), and ddH2O added to a total volume of 50 μL.

[0065] The PCR reaction program was as follows: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 30 s, 52 °C annealing for 30 s, 72 °C extension for 60 s (30 cycles); 72 °C final extension for 10 min.

[0066] The recombinant vector pK18-aspBOE contains "Corynebacterium glutamicum" as shown in SEQ ID NO:2. aspB "Gene coding region and its promoter region", including Corynebacterium glutamicum aspB The nucleotide sequence of the gene coding region is shown in SEQ ID NO:7, which encodes the amino acid sequence shown in SEQ ID NO:8.

[0067] The recombinant vector pK18-aspCOE contains *Escherichia coli* as shown in SEQ ID NO:5. aspC "Gene coding region and its promoter region".

[0068] The recombinant vectors pK18-aspBOE and pK18-aspCOE were electroporated into Corynebacterium glutamicum CGMCC No. 20437 and ATCC 13032, respectively, and cultured in culture medium. The composition and culture conditions of the culture medium are shown in Table 1.

[0069] Table 1. Composition and culture conditions of the culture medium

[0070] Single colonies generated from the culture of pK18-aspBOE recombinant plasmid were identified by PCR using primers P11 / P12. Strains amplified with a 1585 bp fragment were considered positive strains, while those without amplification were considered the original strains. Positive strains were cultured on medium containing 15% sucrose, and single colonies were further identified by PCR using primers P13 / P14. Strains amplified with a 1512 bp fragment were considered positive strains. Corynebacterium glutamicum... aspB Positive strains of Corynebacterium glutamicum CGMCC No. 20437 and ATCC 13032, whose genes were integrated, were named YPI-aspB-001 and YPI-aspB-002, respectively.

[0071] Single colonies generated from the culture of the pK18-aspCOE recombinant plasmid were identified by PCR using primers P11 / P15. Strains amplified with a 1498 bp fragment were considered positive, while those without amplification were considered the original strain. Positive strains were cultured on medium containing 15% sucrose, and single colonies were further identified by PCR using primers P16 / P14. Strains amplifying a 1516 bp fragment were considered positive. Positive strains of *E. coli* with the aspC gene integrated into *Corynebacterium glutamicum* CGMCC No. 20437 and ATCC 13032 were named YPI-aspC-003 and YPI-aspC-004, respectively.

[0072] The PCR identification primers are shown below: P11: 5'-GCCAGAAAATGGGTTGCGTGA-3'; P12: 5'-AGGGCAAATCCACTTAACGGTTTC-3'; P13: 5'-CCGGACTTCCGCGTTGTGTG-3'; P14: 5'-TCAAACGTAATGCGGATCAG-3'; P15: 5'-GAACAGCACTACGTCGCCAGCC-3'; P16: 5'-CCATAACCCAACCGGTATCGAC-3'.

[0073] Both recombinant bacteria YPI-aspB-001 and YPI-aspB-002 contain two copies of the enzyme shown in SEQ ID NO:7. aspB Gene. Specifically, the recombinant strain YPI-aspB-001 is derived from Corynebacterium glutamicum CGMCC No. 20437 by replacing the coding region of the poxB gene (Gene ID: 1020557, 12-Dec-2025. Note: Corynebacterium glutamicum CGMCC 20437 is derived from Corynebacterium glutamicum ATCC13032, and the coding region of the poxB gene has not been edited, so the sequence at this site is consistent with ATCC 13032) (i.e., the fragment between SEQ ID NO:1 and SEQ ID NO:3) from wild-type Corynebacterium glutamicum. aspB The recombinant strain YPI-aspB-002 was obtained by modifying the genome of Corynebacterium glutamicum CGMCC No. 20437 with the ORF gene and its promoter fragment (SEQ ID NO: 2) intact, while maintaining other nucleotides in the genome of Corynebacterium glutamicum CGMCC No. 20437. poxBThe gene coding region (i.e., the segment located between SEQ ID NO:1 and SEQ ID NO:3) was replaced with a wild-type Corynebacterium glutamicum. aspB The recombinant bacteria were obtained by preserving the gene ORF and its promoter fragment (SEQ ID NO:2), while maintaining other nucleotides in the genome of Corynebacterium glutamicum ATCC 13032. It contains two copies of the gene ORF and its promoter fragment (SEQ ID NO:2). aspB The recombinant bacteria of the gene can significantly and stably increase the expression level of aspB protein (as shown in SEQ ID NO:8).

[0074] Recombinant bacteria YPI-aspC-003 and YPI-aspC-004 contain Escherichia coli MG1655 as shown in SEQ ID NO:9. aspC Gene. Specifically, the recombinant strain YPI-aspC-003 is formed by replacing the coding region of the poxB gene (GeneID: 1020557, 12-Dec-2025) in the genome of Corynebacterium glutamicum CGMCC No. 20437 (i.e., the segment located between SEQ ID NO:4 and SEQ ID NO:6) with that from Escherichia coli MG1655. aspC The recombinant strain YPI-aspC-004 was obtained by extracting the gene and its promoter fragment (SEQ ID NO:5) from the genome of Corynebacterium glutamicum CGMCC No. 20437, while keeping other nucleotides unchanged. poxB The gene coding region (i.e., the segment located between SEQ ID NO:4 and SEQ ID NO:6) was replaced with a gene from E. coli MG1655. aspC The recombinant bacteria were obtained by preserving the gene and its promoter fragment (SEQ ID NO:5) of *Corynebacterium glutamicum* ATCC 13032 while maintaining other nucleotides in the genome. These two recombinant bacteria can express the gene derived from *Escherichia coli* MG1655. aspC The gene-encoded aspC protein (as shown in SEQ ID NO:10).

[0075] Example 2: Construction of Corynebacterium glutamicum overexpressing on the genome aspB A610T engineered strains of genes Obtaining high-yielding mutant strains: *Corynebacterium glutamicum* CGMCC No. 20437 was mutagenized using ambient temperature and pressure plasma (ARTP) mutagenesis. The mutagenized bacterial culture was spread and isolated, and single colonies were picked and inoculated into Erlenmeyer flasks for shake-flask fermentation for 48 hours. After fermentation, the concentration of L-isoleucine in the fermentation broth was determined by high-performance liquid chromatography (HPLC) (as shown in Table 2). The L-isoleucine production capacity of mutant strain 1 was significantly higher than that of the original mutant strain (P < 0.05).

[0076] Table 2. Results of L-amino acid analysis by high performance liquid chromatography (HPLC) of the mutant strain *Corynebacterium glutamicum* CGMCC No. 20437.

[0077] Whole-genome sequencing was performed on mutant strain 1 obtained through screening. Comparison with the genome sequence of the original strain confirmed that it encodes aspartate transaminase. aspB The gene has point mutations ( aspB A610T ),Right now aspB At position 610 of the coding region nucleotide sequence, adenine (A) is mutated to thymine (T) (sequence shown in SEQ ID NO:11), and at position 204 of the corresponding amino acid sequence, threonine (T) is mutated to serine (S) (sequence shown in SEQ ID NO:12). Except for this site, the rest of the genome sequence of mutant strain 1 is completely identical to that of Corynebacterium glutamicum CGMCC No.20437. aspB This gene mutation site is closely related to the increase in L-isoleucine production and is a key functional mutation site. The mutant strain was named YPI-aspB-005.

[0078] Using the genomic DNA of the high-yielding mutant strain YPI-aspB-005 as a template, PCR amplification was performed using primers P1 and P2 to obtain... aspB A610T The upstream homologous arm fragment of the gene is 863 bp (sequence shown in SEQ ID NO:1); PCR amplification was performed using primers P3 and P4 to obtain... aspB A610T The gene and its promoter fragment 1621 bp (sequence shown in SEQ ID NO:13) were amplified by PCR using primers P5 and P6 to obtain... aspB A610T The downstream homologous arm fragment of the gene is 821 bp (sequence shown in SEQ ID NO:3).

[0079] After the PCR reaction, all amplified fragments were recovered by electrophoresis using a column-based DNA gel extraction kit. The three fragments were then compared with the recovered fragments... Xbal I / BamH The pK18mobsacB plasmid purified after I enzyme digestion was ligated with NEBuilder enzyme (NEB product) at 50℃ for 30 min. The ligation product was transformed into DH5α, and the resulting single clones were identified by PCR using M13 primers (M13F: 5'-TGTAAAACGACGGCCAGT-3'; M13R: 5'-CAGGAAACAGCTATGACC-3') to obtain a positive integrative plasmid (recombinant vector). The integrative plasmid, verified by sequencing, was named pK18-aspB. A610T OE, the positive integration plasmid contains a kanamycin resistance marker, and recombinants integrated into the genome can be obtained through kanamycin screening.

[0080] The PCR reaction system consisted of: 5 μL of 10×Ex Taq Buffer, 4 μL of dNTP Mixture (2.5 mM each), and Mg... 2+ 4 μL of (25 mM) primers, 2 μL each of (10 pM) primers, 0.25 μL of Ex Taq (5 U / μL), and ddH2O added to a total volume of 50 μL.

[0081] The PCR reaction program was as follows: 94 °C pre-denaturation for 5 min; 94 °C denaturation for 30 s, 52 °C annealing for 30 s, 72 °C extension for 60 s (30 cycles); 72 °C final extension for 10 min.

[0082] Recombinant vector pK18-aspB A610T OE contains "Corynebacterium glutamicum" as shown in SEQ ID NO:13. aspB A610T "Gene coding region and its promoter region", including Corynebacterium glutamicum aspB A610T The nucleotide sequence of the gene coding region is shown in SEQ ID NO:11, which encodes the amino acid sequence shown in SEQ ID NO:12.

[0083] The recombinant vector pK18-aspB A610T OE was electroporated into Corynebacterium glutamicum CGMCC No.20437 and ATCC13032, respectively, and cultured in culture medium. The composition of the culture medium and the culture conditions are shown in Table 1.

[0084] For the transfer to pK18-aspB A610TSingle colonies generated from OE recombinant plasmid culture were identified by PCR using primers P11 / P12. Strains amplified with a 1585 bp fragment were considered positive strains, while those without amplification were considered the original strains. Positive strains were cultured on 15% sucrose medium, and single colonies were further identified by PCR using primers P13 / P14. Strains amplified with a 1512 bp fragment were considered positive strains. *Corynebacterium glutamicum*... aspB A610T Positive strains of Corynebacterium glutamicum CGMCC No. 20437 and ATCC 13032, whose genes were integrated, were named YPI-aspB-006 and YPI-aspB-007, respectively.

[0085] Both recombinant bacteria YPI-aspB-006 and YPI-aspB-007 contain one copy of the enzyme shown in SEQ ID NO:7. aspB The gene and 1 copy shown in SEQ ID NO:11 aspB A610T Gene. Specifically, the recombinant strain YPI-aspB-006 is formed by replacing the coding region of the poxB gene (GeneID: 1020557, 12-Dec-2025) in the genome of Corynebacterium glutamicum CGMCC No. 20437 (i.e., the segment located between SEQ ID NO:1 and SEQ ID NO:3) with that from the mutant strain YPI-aspB-005. aspB A610T The recombinant bacteria YPI-aspB-007 was obtained by modifying the genome of Corynebacterium glutamicum CGMCC No. 20437 with the ORF gene and its promoter fragment (SEQ ID NO: 13) intact, while maintaining other nucleotides in the genome of Corynebacterium glutamicum CGMCC No. 20437. poxB The coding region of the gene (GeneID: 1020557, 12-Dec-2025) (i.e., the segment located between SEQ ID NO:1 and SEQ ID NO:3) was replaced with a gene from the mutant strain YPI-aspB-005. aspB A610T The recombinant bacteria were obtained by keeping the gene ORF and its promoter fragment (i.e., SEQ ID NO:13) unchanged, while maintaining other nucleotides in the genome of Corynebacterium glutamicum ATCC 13032.

[0086] Example 3: Overexpression on plasmid aspB Genes and aspB A610T engineered strains of genes Vectors were constructed using NEBuilder recombination technology. Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, a pair of amplified vectors were designed and synthesized. aspB Genes and aspB A610T Primers for the gene coding region and its promoter region were designed as follows (synthesized by Invitrogen Shanghai). The lowercase nucleotide sequences are those found on the pXMJ19 plasmid: P17: 5'-gcttgcatgcctgcaggtcgactctagaggatccccGGCTGCAACGCATGGCTGCTTTG-3'; P18: 5'-atcaggctgaaaatcttctctcatccgccaaaacTTAGTTAGCGTAATGCTCCGCTGC-3'.

[0087] Construction method: Using Corynebacterium glutamicum YPI-aspB-001 and YPI-aspB-007 as templates, PCR amplification was performed using primers P17 / P18 to obtain... aspB Gene and its promoter fragment (sequence shown in SEQ ID NO:14) and aspB A610T The gene coding region and its promoter fragment (sequence shown in SEQ ID NO:15). The amplification products were subjected to electrophoresis and purified using a column-based DNA gel extraction kit. The recovered DNA fragments were compared with those obtained from the electrophoresis process. EcoR I / Kpn The shuttle plasmid pXMJ19 (Biovector pXMJ19, containing chloramphenicol resistance as a selection marker), recovered by enzyme digestion, was ligated with NEBuilder enzyme (NEB) at 50°C for 30 min. The ligation product was transformed into DH5α competent cells, and the resulting single clones were identified by PCR using primers M13R(-48)(5'-AGCGGATAACAATTTCACACAGGA-3') / P18 to obtain the positive overexpression plasmid pXMJ19-aspB (containing...). aspB The gene, amplified to a size of 1688 bp (sequence shown in SEQ ID NO:16), is pXMJ19-aspB. A610T (contain aspB A610T The gene, with an amplified product size of 1688 bp, has the sequence (SEQ ID NO:16, position 965, A replaced with T). Both recombinant plasmids were sent for sequencing. Because the plasmids contain a chloramphenicol resistance marker, chloramphenicol can be used to screen whether the plasmids have been transformed into the bacterial strain.

[0088] The PCR reaction system consisted of: 5 μL of 10×Ex Taq Buffer, 4 μL of dNTP Mixture (2.5 mM each), and Mg... 2+ 4 μL of (25 mM) primers, 2 μL each of (10 pM) primers, 0.25 μL of Ex Taq (5 U / μL), for a total volume of 50 μL.

[0089] The PCR reaction program was as follows: 94 °C pre-denaturation for 5 min, 94 °C denaturation for 30 s; 52 °C annealing for 30 s; 72 °C extension for 60 s (30 cycles), and 72 °C final extension for 10 min.

[0090] The recombinant vector pXMJ19-aspB contains the substance shown in SEQ ID NO:7. aspB The gene is capable of expressing the aspB protein shown in SEQ ID NO:8.

[0091] Recombinant vector pXMJ19-aspB A610T Contains the substance shown in SEQ ID NO:11 aspB A610T The gene is capable of expressing aspB as shown in SEQ ID NO:12. T204S protein.

[0092] The correctly sequenced plasmids pXMJ19-aspB and pXMJ19-aspB A610T Electroporation was performed on Corynebacterium glutamicum CGMCC No. 20437 and ATCC 13032, respectively, and the cultures were incubated in culture media. The culture medium composition and conditions are shown in Table 1. Single colonies were identified by PCR using primers M13R(-48)(5'-AGCGGATAAC AATTTCACAC AGGA-3') / P18. Strains amplified by PCR containing a 1688 bp fragment were considered positive. Modified strains obtained from CGMCC No. 20437 were named YPI-aspB-008 and YPI-aspB-009, respectively; modified strains obtained from ATCC 13032 were named YPI-aspB-010 and YPI-aspB-011, respectively.

[0093] Recombinant bacteria YPI-aspB-008 and YPI-aspB-010 contain plasmid pXMJ19 overexpression as shown in SEQ ID NO:7. aspB Gene; recombinant bacteria YPI-aspB-009 and YPI-aspB-011 contain the mutation shown in SEQ ID NO:11 that overexpresses plasmid pXMJ19. aspB A610TGene.

[0094] Example 4: Constructing a genome-deleted... aspB engineered strains of genes Vectors were constructed using NEBuilder recombination technology. Based on the genome sequence of Corynebacterium glutamicum ATCC13032 published by NCBI, two pairs of primers were synthesized to amplify the fragments at both ends of the aspB gene coding region, serving as upstream and downstream homologous arms. The primer design is as follows (synthesized by Invitrogen Shanghai): P19: 5'-cagtgccaagcttgcatgcctgcaggtcgactctagGCCGTTAGTGTGGTGACATCGTC-3'; P20: 5'-CAATGTGCTGTCTCCGCTGTATTCACTTAACTGCGTACCTCCGCATGTGG-3'; P21: 5'-CCACATGCGGAGGTACGCAGTTAAGTGAATACAGCGGAGACAGCACATTG-3'; P22: 5'-cagctatgaccatgattacgaattcgagctcggtacccTACGCATGGTTGATGCGCCCGC-3'.

[0095] Construction method: Using Corynebacterium glutamicum ATCC 13032 as a template, PCR amplification was performed using primers P19 / P20 and P21 / P22, respectively, to obtain... aspB The upstream homologous arm of the gene is 789 bp and aspB The downstream homologous arm of the gene is 826 bp.

[0096] The amplified products were subjected to electrophoresis and purified using a column-based DNA gel extraction kit. The recovered DNA fragments were compared with those obtained through electrophoresis. Xbal I / BamH The pK18mobsacB plasmid purified after I enzyme digestion was ligated with NEBuilder enzyme (NEB product) at 50℃ for 30 min. The single clones grown after transformation were identified by PCR using M13 primers to obtain the positive knockout vector pK18-ΔaspB. This recombinant plasmid pK18-ΔaspB contains 1565 bp of ΔaspB up-down DNA (sequence shown in SEQ ID NO:17).

[0097] The plasmid was sent for sequencing. The correctly sequenced knockout plasmid pK18-ΔaspB was electroporated into Corynebacterium glutamicum CGMCC No. 20437 and cultured in culture medium. The composition and culture conditions of the culture medium are shown in Table 1. Single colonies generated by the culture were identified by PCR using the following primers (synthesized by Invitrogen Shanghai): P23: 5'-GCCGTTAGTGTGGTGACATCGTC-3'; P24: 5'-TACGCATGGTTGATGCGCCCGC-3'.

[0098] The strains that simultaneously amplified bands of 1491 bp and 2772 bp were identified as positive strains by the above PCR, while the strains that amplified only the 2772 bp band were identified as the original strain. Positive strains were screened on 15% sucrose medium and then cultured on media containing and without kanamycin. Strains that grew on the kanamycin-free medium but not on the kanamycin-containing medium were further identified by PCR using primers P23 / P24. Strains amplifying a 1491 bp band were identified as positive strains with the aspB gene coding region knocked out. Positive strains were then amplified again by PCR using primers P23 / P24. aspB Fragmentation and sequencing were performed, and the correctly sequenced strain was named YPI-aspB-012 (from the genome of Corynebacterium glutamicum CGMCC No. 20437). aspB (The gene was knocked out).

[0099] Example 5: L-Isoleucine Fermentation Experiment The strains constructed in the above embodiments, as well as the original strains *Corynebacterium glutamicum* CGMCC No. 20437 and ATCC13032, were fermented in a BLBIO-5GC-4-H fermenter (Shanghai Bailun Biotechnology Co., Ltd.) using fermentation medium and the control process shown in Table 3. Each strain was replicated three times, and the results are shown in Table 4.

[0100] The fermentation medium formula is as follows: glucose 5.0 g / L, phosphate 0.38 g / L, magnesium sulfate 1.85 g / L, potassium chloride 1.6 g / L, biotin 550 μg / L, vitamin biotin B1 300 μg / L, ferrous sulfate 10 mg / L, manganese sulfate 10 g / dL, KH2PO4 2.8 g / L, vitamin C 0.75 mg / L, vitamin B12 2.5 μg / L, para-aminobenzoic acid 0.75 mg / L, antifoaming agent 0.0015 ml / dL, betaine 1.5 g / L, sugarcane molasses 7 ml / L, corn steep liquor 77 ml / L, aspartic acid 1.7 g / L, hair powder 2 g / L, and water as the solvent.

[0101] The results are shown in Table 4. Overexpression of itself in Corynebacterium glutamicum aspB Genes, or the effects of aspB Point mutations in the coding region of a gene aspB A610T Both expression and overexpression contribute to increased L-isoleucine production; overexpression of E. coli-derived L-isoleucine... aspC The gene had no significant effect on isoleucine accumulation; aspB Knocking out or weakening genes is not conducive to the accumulation of L-isoleucine.

[0102] Table 3. Fermentation Control Process

[0103] Table 4. Results of L-Isoleucine Fermentation Experiment

[0104] Note: The P-values ​​of strains YPI-aspB-001, YPI-aspC-003, YPI-aspB-005, YPI-aspB-006, YPI-aspB-008, YPI-aspB-009, and YPI-aspB-012 are compared with CGMCC No. 20437. The P-values ​​of strains YPI-aspB-002, YPI-aspC-004, YPI-aspB-007, YPI-aspB-010, and YPI-aspB-011 are compared with ATCC 13032.

[0105] 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. An aspartate aminotransferase mutant, characterized in that: The aspartate aminotransferase mutant includes any one of the following: A1) The protein obtained by mutating the 204th amino acid residue of wild-type aspartate aminotransferase from threonine to serine. A2) Proteins derived from Corynebacterium glutamicum with the amino acid sequences shown in A1) having undergone substitution and / or deletion and / or addition of one or more amino acid residues and having the same function. A3) Proteins that have at least 90% identity with the amino acid sequence defined by A1) or A2) and have the same function; A4) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of any of the proteins defined in A1)-A3).

2. The aspartate aminotransferase mutant according to claim 1, characterized in that: The wild-type aspartate aminotransferase contains the amino acid sequence shown in SEQ ID NO:

8.

3. The aspartate aminotransferase mutant according to claim 1 or 2, characterized in that: The aspartate aminotransferase mutant contains the amino acid sequence shown in SEQ ID NO:

12.

4. A biomaterial, characterized in that: The biomaterial includes any of the following: B1) A nucleic acid molecule encoding any of the aspartate aminotransferase mutants described in claims 1-3; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); 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); B5) A whole-cell catalyst containing the nucleic acid molecule described in B1), or a whole-cell catalyst containing the expression cassette described in B2), or a whole-cell catalyst containing the recombinant vector described in B3), or a whole-cell catalyst containing the recombinant microorganism described in B4).

5. The biomaterial according to claim 4, characterized in that: The nucleic acid molecules described in B1) include the following: (b11) or (b12) or (b13) b11) A nucleic acid molecule containing the coding sequence shown in SEQ ID NO:11; b12) The nucleic acid molecule described in b11) has more than 80% similarity to the nucleic acid molecule described in b11) and encodes a nucleic acid molecule of any of the aspartate aminotransferase mutants described in claims 1-3; b13) Hybridizes under stringent conditions with the nucleic acid molecule defined in b11) or b12) and encodes a nucleic acid molecule of any of the aspartate aminotransferase mutants of claims 1-3.

6. The use of the aspartate aminotransferase mutant according to any one of claims 1-3 or the biomaterial according to claim 4 or 5 in any of the following: P1. Production of L-isoleucine; P2. Increase the production of L-isoleucine in recipient bacteria; P3. Construct engineered bacteria that produce L-isoleucine; P4. Prepare products containing L-isoleucine.

7. Recombinant Corynebacterium glutamicum, characterized in that: The recombinant Corynebacterium glutamicum comprises any of the following: C1) Recombinant Corynebacterium glutamicum containing the encoding gene of any of the aspartate aminotransferase mutants according to claims 1-3; C2) Recombinant Corynebacterium glutamicum obtained by enhancing the expression of the gene encoding aspartate aminotransferase in the recipient Corynebacterium glutamicum.

8. The recombinant Corynebacterium glutamicum according to claim 7, characterized in that: In C2), the expression of aspartate aminotransferase is enhanced by any of the following methods: c1) Replace the coding region of the poxB gene in the genome of the recipient Corynebacterium glutamicum with the gene expression cassette encoding the wild-type aspartate aminotransferase as described in claim 1 or 2; c2) A point mutation is made in the gene encoding the wild-type aspartate aminotransferase as described in claim 1 or 2 in the genome of the recipient Corynebacterium glutamicum, wherein the point mutation causes the amino acid residue at position 204 of the wild-type aspartate aminotransferase to change from threonine to serine. c3) Replace the coding region of the poxB gene in the genome of the recipient Corynebacterium glutamicum with the coding gene expression cassette of the aspartate aminotransferase mutant of claim 3; c4) Introduce a recombinant vector carrying the encoding gene of the wild-type aspartate aminotransferase as described in claim 1 or 2 into the genome of the recipient Corynebacterium glutamicum; c5) Introduce a recombinant vector carrying the encoding gene of the aspartate aminotransferase mutant of claim 3 into the genome of the recipient Corynebacterium glutamicum.

9. The use of the recombinant Corynebacterium glutamicum according to claim 7 or 8 in the production of L-isoleucine.

10. A method for producing L-isoleucine, comprising the following steps: fermenting and culturing the recombinant Corynebacterium glutamicum as described in claim 7 or 8, and obtaining L-isoleucine from the fermentation product.

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