Recombinant strains modified with gene BBD29 14900 and their construction methods and applications

Abstract

A recombinant strain modified with gene BBD29_14900 and its construction method and application are provided. Its specific application is for the production of L-glutamic acid. A method for introducing a point mutation into the BBD29_14900 gene coding sequence or improving its expression in Corynebacterium is also provided. This method can be used to increase the fermentation production of glutamic acid by the mutant strain. The point mutation causes the 1114th base of the BBD29_14900 gene sequence to be mutated from guanine (G) to adenine (A), and the 372nd aspartic acid in the encoded corresponding amino acid sequence is replaced with asparagine.

Description

[Technical Field]

[0001] The present invention relates to the fields of genetic engineering and microbiology, and specifically to a recombinant strain modified with the BBD29_14900 gene, as well as its construction method and application, the specific application of which is the production of L-glutamic acid. [Background technology]

[0002] As is well known, Ajinomoto, a commonly used seasoning, is L-glutamic acid monosodium salt. Since its invention and industrial production in Japan in 1909, Ajinomoto has undergone numerous changes and developed into a global amino acid fermentation industry centered on glutamic acid fermentation. The fermentation that produces Ajinomoto's glutamic acid and other amino acids differs from conventional brewing and antibiotic fermentation in that it is a metabolically controlled fermentation that alters the metabolism of microorganisms.

[0003] Currently, the bacterial strains used by manufacturers for glucose fermentation are mainly several mutant strains, and these producing strains are mainly obtained through microbial breeding, mainly through mutation induction. However, with the existing acid production capacity, it is quite difficult to significantly improve the acid production rate and sugar-to-acid conversion rate by continuing to use classical microbial breeding techniques, such as mutation induction and cell fusion. Therefore, the most effective method currently available is to use modern genetic engineering techniques to modify bacterial strains and improve the acid production rate and sugar-to-acid conversion rate.

[0004] If we can use genetic engineering techniques to modify the bacterial species and produce glutamic acid, there will be a major breakthrough in the acid production rate of glutamic acid and the sugar-acid conversion rate. Summary of the Invention

[0005] The present invention provides a recombinant strain with modified gene BBD29_14900, as well as its construction method and application.

[0006] An object of the present invention is to provide a method for efficiently producing L-glutamic acid by developing a new technology for improving the L-glutamic acid-producing ability of bacteria.

[0007] To achieve the above object, the inventors of the present invention have conducted research into the BBD29_14900 gene or its homologous genes and discovered that by modifying or improving the expression of the gene, bacteria with enhanced L-glutamic acid production ability can be obtained. Based on these findings, the present invention has been completed.

[0008] The present invention provides an L-glutamic acid-producing bacterium, which has improved expression of a polynucleotide encoding the amino acid sequence of SEQ ID NO:3 or a homologous sequence thereof. The present invention also provides a method for producing L-glutamic acid using the microorganism.

[0009] According to a first aspect of the present invention, there is provided an L-glutamic acid-producing bacterium, wherein the expression of a polynucleotide encoding the amino acid sequence of SEQ ID NO:3 or a homologous sequence thereof is improved. According to the present invention, the improved expression means that the expression of the polynucleotide is enhanced, or that the polynucleotide encoding the amino acid sequence of SEQ ID NO:3 or a homologous sequence thereof has a point mutation, or that the polynucleotide encoding the amino acid sequence of SEQ ID NO:3 or a homologous sequence thereof has a point mutation and enhanced expression.

[0010] The amino acid sequence of SEQ ID NO:3 or a homologous sequence thereof is a protein encoded by the gene BBD29_14900 or a homologous gene thereof.

[0011] The bacterium has an enhanced ability to produce L-glutamic acid compared to an unmodified strain.

[0012] In the present invention, the term "bacterium capable of producing L-glutamic acid" refers to a bacterium that has the ability to produce and accumulate the target L-glutamic acid in a medium and / or bacterial cells to the following extent so that L-glutamic acid can be collected when the bacterium is cultured in the medium. The bacterium capable of producing L-glutamic acid may be a bacterium that can accumulate the target L-glutamic acid in a medium and / or bacterial cells in an amount greater than that obtainable by an unmodified strain.

[0013] The term "unmodified strain" refers to a control strain that has not been modified to have a particular characteristic. Thus, examples of unmodified strains include wild-type strains and parental strains.

[0014] The bacterium capable of producing L-glutamic acid may be a bacterium capable of accumulating the target L-glutamic acid in a medium at an amount of preferably 0.5 g / L or more, more preferably 1.0 g / L or more.

[0015] In the present invention, unless otherwise specified, the term "L-glutamic acid" refers to L-glutamic acid in free form, a salt thereof or a mixture thereof.

[0016] The polynucleotide may encode an amino acid sequence having about 90% or more, about 92% or more, about 95% or more, about 97% or more, about 98% or more, or about 99% or more sequence identity with the amino acid sequence of SEQ ID NO:3. As used herein, the term "homology" refers to the percentage identity between two polynucleotide or two polypeptide modules. Sequence homology between one module and another can be measured by methods known in the art. For example, such sequence homology can be measured by the BLAST algorithm.

[0017] Expression of a polynucleotide can be enhanced by substitution or mutation of an expression regulatory sequence, introduction of a mutation into the polynucleotide sequence, increasing the copy number of the polynucleotide introduced via a chromosomal insertion or vector, or a combination thereof.

[0018] The expression control sequence of a polynucleotide may be modified. The expression control sequence controls the expression of a polynucleotide operably linked thereto and may include, for example, a promoter, a terminator, an enhancer, a silencer, etc. The polynucleotide may have a change in the start codon. The copy number may be increased by incorporating the polynucleotide into a specific site on a chromosome. As used herein, the specific site may include, for example, a transposon site or an intergenic site. Alternatively, the copy number may be increased by incorporating the polynucleotide into an expression vector and introducing the expression vector into a host cell.

[0019] In one embodiment of the present invention, the copy number is increased by incorporating a polynucleotide or a polynucleotide with a point mutation into a specific site in the chromosome of a microorganism.

[0020] In one embodiment of the present invention, a polynucleotide with a promoter sequence or a polynucleotide with a promoter sequence having a point mutation is incorporated into a specific site in the chromosome of a microorganism to overexpress the nucleic acid sequence.

[0021] In one embodiment of the present invention, the copy number is increased by incorporating the polynucleotide or the polynucleotide having a point mutation into an expression vector and introducing the expression vector into a host cell.

[0022] In one embodiment of the present invention, a polynucleotide with a promoter sequence or a polynucleotide with a promoter sequence having a point mutation is incorporated into an expression vector, and the expression vector is introduced into a host cell to overexpress the amino acid sequence.

[0023] In one specific embodiment of the present invention, the polynucleotide before mutation may comprise the nucleotide sequence of SEQ ID NO:1.

[0024] In one embodiment of the present invention, the polynucleotide encoding the amino acid sequence of SEQ ID NO:3 has a point mutation such that aspartic acid (D) at position 372 of the amino acid sequence of SEQ ID NO:3 is replaced with a different amino acid.

[0025] According to the present invention, preferably, aspartic acid at position 372 is substituted with asparagine.

[0026] According to the present invention, the amino acid sequence shown in SEQ ID NO:3 after aspartic acid at position 372 has been replaced with asparagine is shown in SEQ ID NO:4.

[0027] In one embodiment of the present invention, the polynucleotide sequence having a point mutation is formed by a mutation at base 1114 of the polynucleotide sequence shown in SEQ ID NO:1.

[0028] According to the present invention, the mutation comprises a mutation from guanine (G) to adenine (A) at base 1114 of the polynucleotide sequence shown in SEQ ID NO:1.

[0029] In one embodiment of the invention, the polynucleotide sequence having the point mutation comprises the polynucleotide sequence shown in SEQ ID NO:2.

[0030] As used herein, the term "operable linkage" refers to a functional linkage between a regulatory sequence and a polynucleotide sequence, whereby the regulatory sequence controls the transcription and / or translation of the polynucleotide sequence. The regulatory sequence may be a strong promoter capable of increasing the expression level of the polynucleotide. The regulatory sequence may be a promoter derived from a microorganism belonging to the genus Corynebacterium, or a promoter derived from another microorganism. For example, the promoter may be a trc promoter, a gap promoter, a tac promoter, a T7 promoter, a lac promoter, a trp promoter, an araBAD promoter, or a cj7 promoter.

[0031] In one specific embodiment of the present invention, the promoter is a promoter of a polynucleotide encoding the amino acid sequence of SEQ ID NO:3 (BBD29_14900 gene).

[0032] As used herein, the term "vector" refers to a polynucleotide construct containing a gene regulatory sequence and a gene sequence and configured to express a target gene in a suitable host cell. Alternatively, a vector may further refer to a polynucleotide construct containing a sequence that can be used for homologous recombination, whereby the vector introduced into a host cell can modify the regulatory sequence of an endogenous gene in the genome of the host cell or insert an expressible target gene into a specific site in the host's genome. In this regard, vectors used in the present invention may further contain a selectable marker that determines the introduction of the vector into a host cell or the insertion of the vector into a chromosome of the host cell. The selectable marker may include a marker that confers a selectable phenotype, such as drug resistance, nutritional deficiency, resistance to cytotoxic agents, or expression of a surface protein. In an environment treated with such a selection agent, only cells that express the selectable marker can survive or exhibit a different phenotypic trait, and thus transformed cells may be selected.

[0033] In some specific embodiments of the present invention, the vectors used are pK18mobsacB plasmid, pXMJ19 plasmid.

[0034] As used herein, the term "transformation" refers to the introduction of a polynucleotide into a host cell so that the polynucleotide can replicate as an extragenomic element or as an inserted part of the genome of the host cell. The method for transforming a vector used in the present invention may include a method for introducing a nucleic acid into a cell. Alternatively, an electric pulse method may be performed depending on the host cell, as disclosed in the related art.

[0035] According to the present invention, the bacterium is a microorganism belonging to the genus Corynebacterium, such as Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium callunae, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Corynebacterium ammoniagenes, Corynebacterium pekinense, Brevibacterium saccharolyticum, and the like. saccharolyticum, Brevibacterium roseum, Brevibacterium thiogenitalis, and the like.

[0036] In one embodiment of the present invention, the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum ATCC 13869.

[0037] In one embodiment of the present invention, the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum YPGLU001, which is a high-yielding glutamic acid producing bacterium, and its conservation information is as follows: Species name: Corynebacterium glutamicum, Latin name: Corynebacterium glutamicum, Strain number: YPGLU001, Conservation organization: Center of Ordinary Microorganisms of the China Committee for the Conservation of Microorganisms, Conservation abbreviation: CGMCC, Address: No. 3, Hall No. 1, Beichen West Road, Chaoyang District, Beijing, Conservation date: November 23, 2020, Conservation center registration number: CGMCC No. 21220.

[0038] According to the present invention, the bacterium may further have other improvements related to increased L-glutamic acid production, such as increased or decreased expression of enzyme activity or genes, such as glutamate dehydrogenase, glutamine synthetase, or glutamine-ketoglutarate aminotransferase, or the genes may be replaced with exogenous genes.

[0039] According to a second aspect of the present invention, there are provided polynucleotide sequences, amino acid sequences encoded by the polynucleotide sequences, recombinant vectors containing the polynucleotide sequences, and recombinant strains containing the polynucleotide sequences.

[0040] According to the present invention, the polynucleotide sequence comprises a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:3, wherein the aspartic acid at position 372 of said sequence is substituted with a different amino acid.

[0041] According to the present invention, preferably, aspartic acid at position 372 is substituted with asparagine.

[0042] According to the present invention, the amino acid sequence shown in SEQ ID NO:3 after aspartic acid at position 372 has been replaced with asparagine is shown in SEQ ID NO:4.

[0043] According to the present invention, preferably, the polynucleotide sequence encoding the polypeptide comprising the amino acid sequence shown in SEQ ID NO:3 comprises the polynucleotide sequence shown in SEQ ID NO:1.

[0044] In one embodiment of the invention, the polynucleotide sequence is formed by mutation of base 1114 of the polynucleotide sequence shown in SEQ ID NO:1.

[0045] According to the present invention, the mutation refers to a change in the base / nucleotide at the site, and the mutation method may be at least one selected from mutagenesis, PCR-directed point mutation, and / or homologous recombination, etc. In the present invention, PCR-directed point mutation and / or homologous recombination are preferably used.

[0046] According to the present invention, the mutation comprises a mutation from guanine (G) to adenine (A) at base 1114 of the polynucleotide sequence shown in SEQ ID NO:1.

[0047] In one embodiment of the invention, the polynucleotide sequence comprises the polynucleotide sequence shown in SEQ ID NO:2.

[0048] According to the present invention, said amino acid sequence comprises the amino acid sequence shown in SEQ ID NO:4.

[0049] According to the present invention, the recombinant vector is constructed by introducing the polynucleotide sequence into a plasmid.

[0050] In one embodiment of the invention, the plasmid is the pK18mobsacB plasmid.

[0051] In another embodiment of the invention, the plasmid is the pXMJ19 plasmid.

[0052] Specifically, the polynucleotide sequence and the plasmid may be constructed as a recombinant vector via the NEBuider recombination system.

[0053] According to the present invention, said recombinant strain comprises said polynucleotide sequence.

[0054] In one embodiment of the present invention, the starting strain of the recombinant strain is Corynebacterium glutamicum CGMCC No. 21220.

[0055] In one embodiment of the present invention, the starting strain of the recombinant strain is ATCC 13869.

[0056] According to a third aspect of the present invention, there is further provided a method for constructing a recombinant strain that produces L-glutamic acid.

[0057] According to the present invention, the construction method comprises: The method includes modifying the polynucleotide sequence of the wild-type BBD29_14900 gene shown in SEQ ID NO: 1 in a host strain to generate a mutation at base 1114 thereof, thereby obtaining a recombinant strain containing a mutated BBD29_14900-encoding gene.

[0058] According to the construction method of the present invention, the modification includes at least one of the following methods: mutagenesis, PCR fixed point mutation, and / or homologous recombination.

[0059] According to the construction method of the present invention, the mutation refers to a change in the 1114th base in SEQ ID NO:1 from guanine (G) to adenine (A). Specifically, the polynucleotide sequence containing the mutant BBD29_14900-encoding gene is shown in SEQ ID NO:2.

[0060] Furthermore, the construction method includes: (1) modifying the nucleotide sequence of the wild-type BBD29_14900 gene shown in SEQ ID NO: 1 to generate a mutation at base 1114 thereof to obtain a mutant BBD29_14900 gene polynucleotide sequence; (2) constructing a recombinant vector by ligating the mutated polynucleotide sequence with a plasmid; and (3) introducing the recombinant vector into a host strain to obtain a recombinant strain containing the mutant BBD29_14900-encoding gene.

[0061] According to the construction method of the present invention, step (1) includes constructing a point-mutated BBD29_14900 gene. That is, based on the genome sequence of the unmodified strain, two pairs of primers, P1 and P2, and P3 and P4, for amplifying the BBD29_14900 gene fragment are synthesized, and point mutations are introduced into the wild-type BBD29_14900 gene SEQ ID NO:1 by PCR-fixed point mutation method to obtain the point-mutated BBD29_14900 gene nucleotide sequence SEQ ID NO:2, which is then used to construct the BBD29_14900 gene. G1114A Let's say.

[0062] In one embodiment of the present invention, the unmodified strain genome may be derived from the ATCC 13869 strain, the genome sequence of which may be obtained from the NCBI site.

[0063] In one embodiment of the present invention, in step (1), the primers are as follows: P1 (SEQ ID NO: 5): 5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCGCTGTGGTTATCCTCGCTG-3' P2(SEQ ID N0:6):5'-CTGGGGCGACGCGGGGATTCAAGGCGGTCG-3' P3(SEQ ID N0:7):5'-CGACCGCCTTGAATCCCCGCGTCGCCCCAG-3' P4 (SEQ ID NO: 8): 5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGAGTGTCACTAGGCTAGTC-3' In one embodiment of the present invention, the PCR amplification is performed in the following manner: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 40 seconds, 30 cycles, and over-extension at 72°C for 10 minutes.

[0064] In one embodiment of the present invention, the overlap PCR amplification is performed in the following manner: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 90 seconds, 30 cycles, and over-extension at 72°C for 10 minutes.

[0065] According to the construction method of the present invention, the step (2) includes the construction of a recombinant plasmid, and the isolated and purified BBD29_14900 G1114A and pK18mobsacB plasmids were assembled using the NEBuider recombination system to obtain recombinant plasmids.

[0066] According to the construction method of the present invention, the step (3) includes constructing a recombinant strain, that is, transforming the recombinant plasmid into a host strain to obtain a recombinant strain.

[0067] In one embodiment of the present invention, the transformation in step (3) is an electrotransformation method.

[0068] In one embodiment of the invention, the host strain is ATCC 13869.

[0069] In one embodiment of the present invention, the host strain is Corynebacterium glutamicum CGMCC No. 21220.

[0070] In one embodiment of the invention, the recombination is achieved by homologous recombination.

[0071] According to a fourth aspect of the present invention, there is further provided a method for constructing a recombinant strain that produces L-glutamic acid.

[0072] According to the present invention, the construction method comprises: The upstream and downstream homologous arm fragments of BBD29_14900, the BBD29_14900 gene coding region and its promoter region sequence are amplified, and then BBD29_14900 or BBD29_14900 is inserted into the genome of the host strain by homologous recombination. G1114A The gene is introduced, and the strain is BBD29_14900 or BBD29_14900 G1114A The method includes a step of achieving overexpression of the gene.

[0073] In one embodiment of the present invention, the primers for amplifying the upstream homology arm fragment are as follows: P7 (SEQ ID NO: 11): 5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGACCCGCTTGCCATACGAAG-3' P8(SEQ ID NO:12):5'-AGTGGGCTGAATTTGGGCTGATCTACTCATCTGAAGAATC-3' In one embodiment of the invention, the primers for amplifying the downstream homology arm fragment are as follows: P11(SEQ ID NO:15):5'-CCGAAGCGCAAAACGCTTAGTTCGTGGGCACTCTGGTTTG-3' P12 (SEQ ID NO: 16): 5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCATAAGAAACAACCACTTCC-3' In one embodiment of the present invention, the primers for amplifying the gene coding region and its promoter region sequence are as follows: P9(SEQ ID NO:13):5'-GATTCTTCAGATGAGTAGATCAGCCCAAATTCAGCCCACT-3' P10(SEQ ID NO:14):5'-CAAACCAGAGTGCCCACGAACTAAGCGTTTTGCGCTTCGG-3' In one embodiment of the present invention, the P7 / P12 is further used as a primer to amplify a mixture of the amplified upstream homologous arm fragment, downstream homologous arm fragment, gene coding region and its promoter region sequence fragment as a template to obtain an integrated homologous arm fragment.

[0074] In one embodiment of the present invention, the PCR system used comprises 5 μL of 10×Ex Taq Buffer, 4 μL of dNTP Mixture (2.5 mM each), Mg 2+ The PCR mixture consisted of 4 μL of 25 mM ATP, 2 μL of each primer (10 pM), and 0.25 μL of ExTaq (5 U / μL), in a total volume of 50 μL. PCR amplification was performed as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 120 seconds (30 cycles), and over-extension at 72°C for 10 minutes.

[0075] In one embodiment of the present invention, the NEBuider recombination system is employed to assemble the shuttle plasmid PK18mobsacB and the integrated homology arm fragment to obtain the integrated plasmid.

[0076] In one embodiment of the present invention, the integrative plasmid is transfected into a host strain and BBD29_14900 or BBD29_14900 is integrated into the genome of the host strain by homologous recombination. G1114A Introducing a gene.

[0077] In one embodiment of the present invention, the host strain is Corynebacterium glutamicum CGMCC No. 21220.

[0078] In one embodiment of the invention, the host strain is ATCC 13869.

[0079] In one embodiment of the invention, the host strain is a strain having the polynucleotide sequence shown in SEQ ID NO:2.

[0080] According to a fifth aspect of the present invention, there is further provided a method for constructing a recombinant strain that produces L-glutamic acid.

[0081] According to the present invention, the construction method comprises: BBD29_14900 gene coding region and promoter region sequence, or BBD29_14900 G1114A The gene coding region and promoter region sequences are amplified to construct an overexpression plasmid vector, and the vector is introduced into a host strain, and the strain is BBD29_14900 or BBD29_14900 G1114A The method includes a step of achieving overexpression of the gene.

[0082] In one embodiment of the present invention, the primers for amplifying the gene coding region and its promoter region sequence are as follows: P17(SEQ ID N0:21): 5'-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCAGCCCAAATTCAGCCCACT-3' P18 (SEQ ID NO: 22): 5'-ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACCTAAGCGTTTTGCGCTTCGG-3' In one embodiment of the present invention, the PCR system comprises 5 μL of 10×Ex Taq Buffer, 4 μL of dNTP Mixture (2.5 mM each), Mg 2+ The PCR amplification was carried out in the following cycles: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, extension at 72°C for 90 seconds, and over-extension at 72°C for 10 minutes.

[0083] In one embodiment of the present invention, the NEBuider recombination system is used to transfect the shuttle plasmid pXMJ19 with the self-promoted BBD29_14900 or BBD29_14900 G1114A The fragments are assembled to obtain the overexpression plasmid.

[0084] In one embodiment of the present invention, the host strain is Corynebacterium glutamicum CGMCC No. 21220.

[0085] In one embodiment of the invention, the host strain is ATCC 13869.

[0086] In one embodiment of the invention, the host strain is a strain having the polynucleotide sequence shown in SEQ ID NO:2.

[0087] The recombinant strain obtained in the present invention may be used alone for the fermentative production of L-glutamic acid, or may be mixed with other bacteria that produce L-glutamic acid to produce L-glutamic acid by fermentation.

[0088] According to another aspect of the present invention, there is provided a method for producing L-glutamic acid, the method comprising culturing the bacterium described above and obtaining L-glutamic acid from the culture.

[0089] The bacteria can be cultured in an appropriate medium under culture conditions known in the art. The medium may contain a carbon source, a nitrogen source, trace elements, and combinations thereof. The pH of the culture may be adjusted during the culture. The culture may also include preventing the generation of bubbles, for example, by using an antifoaming agent. The culture may also include injecting gas into the culture. The gas may include any gas capable of maintaining aerobic conditions in the culture. The culture temperature during the culture may be 20 to 45°C. The produced L-glutamic acid can be recovered from the culture by treating the culture with sulfuric acid or hydrochloric acid, and then using a combination of methods, such as anion exchange chromatography, concentration, crystallization, and isoelectric precipitation.

[0090] The present invention relates to a protein (mutated protein BBD29_14900 D372N The protein was obtained by mutating the 372nd amino acid residue of the BBD29_14900 protein from aspartic acid to asparagine, The BBD29_14900 protein is (a1) the protein shown in SEQ ID NO:3, or (a2) a protein derived from a bacterium and having 95% or more identity with (a1) and related to bacterial glutamic acid production; or (a3) A protein derived from (a1) obtained by substituting and / or deleting and / or adding one or more amino acid residues in the protein shown in (a1) and related to bacterial glutamic acid production.

[0091] As used herein, the term "identity" refers to sequence similarity to a naturally occurring amino acid sequence. Identity can be assessed by eye or by computer software. Using computer software, the identity between two or more sequences can be displayed as a percentage (%) and may be used to assess identity between related sequences.

[0092] The 95% or more identity may specifically be 96% or more identity, 97% or more identity, 98% or more identity, or 99% or more identity.

[0093] Specifically, the above BBD29_14900 D372N The protein is shown in SEQ ID NO:4. The BBD29_14900 gene is a gene that encodes the BBD29_14900 protein.

[0094] Specifically, the BBD29_14900 gene is (b1) the coding region is the DNA molecule set forth in SEQ ID NO:1; or (b2) a DNA molecule derived from a bacterium and having 95% or more identity with (b1) and encoding the protein; or (b3) A DNA molecule that hybridizes with (b1) under stringent conditions and encodes the protein.

[0095] As used herein, the term "identity" refers to sequence similarity to naturally occurring nucleic acid sequences. Identity can be assessed by eye or by computer software. Computer software can be used to display the identity between two or more sequences as a percentage (%) and can be used to assess identity between related sequences.

[0096] The 95% or more identity may specifically be 96% or more identity, 97% or more identity, 98% or more identity, or 99% or more identity.

[0097] The stringent conditions may be hybridization and membrane washing in a solution of 0.1x SSPE (or 0.1x SSC) and 0.1% SDS at 65°C.

[0098] BBD29_14900 D372N Protein-encoding gene (BBD29_14900 G1114A The present invention also covers the protection scope of the present invention.

[0099] Specifically, BBD29_14900 G1114A Genes are (c1) the coding region is the DNA molecule set forth in SEQ ID NO:2; or (c2) a DNA molecule derived from a bacterium and having 95% or more identity to (c1) and encoding the protein; or (c3) A DNA molecule that hybridizes with (c1) under stringent conditions and encodes the protein.

[0100] As used herein, the term "identity" refers to sequence similarity to naturally occurring nucleic acid sequences. Identity can be assessed by eye or by computer software. Computer software can be used to display the identity between two or more sequences as a percentage (%) and can be used to assess identity between related sequences.

[0101] The 95% or more identity may specifically be 96% or more identity, 97% or more identity, 98% or more identity, or 99% or more identity.

[0102] The stringent conditions may be hybridization and membrane washing in a solution of 0.1x SSPE (or 0.1x SSC) and 0.1% SDS at 65°C.

[0103] BBD29_14900 G1114A DNA molecule carrying a gene, BBD29_14900 G1114A Expression cassette carrying the gene or BBD29_14900 G1114A Recombinant vector carrying the gene or BBD29_14900 G1114A Any recombinant bacteria carrying the gene are within the scope of protection of the present invention.

[0104] For example, BBD29_14900 G1114A The DNA molecule having the gene may be the DNA molecule shown in SEQ ID NO:30 or the DNA molecule shown in SEQ ID NO:31.

[0105] For example, BBD29_14900 G1114A The recombinant vector carrying the gene may be a plasmid carrying the DNA molecule shown in SEQ ID NO:30 or a plasmid carrying the DNA molecule shown in SEQ ID NO:31.

[0106] For example, BBD29_14900 G1114A The recombinant vector carrying the gene is the integrative plasmid PK18mobsacB-BBD29_14900 in the examples.G1114A or overexpression plasmid pXMJ19-BBD29_14900 G1114A may be.

[0107] The recombinant microorganism may specifically be a recombinant bacterium.

[0108] For example, BBD29_14900 G1114A The recombinant bacterium having the gene may be a recombinant bacterium having a DNA molecule shown in SEQ ID NO:30, a recombinant bacterium having a DNA molecule shown in SEQ ID NO:31, or a recombinant bacterium having a DNA molecule shown in SEQ ID NO:2.

[0109] BBD29_14900 G1114A The recombinant bacteria containing the gene specifically locate the BBD29_14900 gene in the bacterial genome. G1114A It may also be produced by gene replacement.

[0110] Exemplarily, the BBD29_14900 gene in the bacterial genome is G1114A The gene replacement is achieved by introducing the DNA molecule shown in SEQ ID NO:29 into bacteria.

[0111] Exemplarily, the BBD29_14900 gene in the bacterial genome is G1114A The method of replacing the gene is as follows: G1114A The goal is to introduce the

[0112] In the recombinant bacteria, BBD29_14900 G1114A The gene may be expressed by integrating it into genomic DNA or by expressing it in a plasmid.

[0113] For example, BBD29_14900 G1114AThe recombinant bacterium carrying the gene may be specifically produced by introducing the DNA molecule shown in SEQ ID NO: 30 or the DNA molecule shown in SEQ ID NO: 31 into the bacterium.

[0114] For example, BBD29_14900 G1114A Specifically, recombinant bacteria containing the gene may be prepared by introducing a plasmid containing the DNA molecule shown in SEQ ID NO:30 or a plasmid containing the DNA molecule shown in SEQ ID NO:31 into bacteria.

[0115] For example, BBD29_14900 G1114A The recombinant bacterium carrying the gene is specifically the integrated plasmid PK18mobsacB-BBD29_14900 in the examples. G1114A or overexpression plasmid pXMJ19-BBD29_14900 G1114A It may also be produced by introducing the

[0116] The present invention is BBD29_14900 D372N Protein, BBD29_14900 G1114A gene, BBD29_14900 G1114A Expression cassette carrying the gene or BBD29_14900 G1114A Recombinant vector carrying the gene or BBD29_14900 G1114A Further protect the application of recombinant bacteria containing genes, The application is (I) Application to improving bacterial glutamic acid production, or (II) Application to glutamic acid production.

[0117] The present invention further protects the application of specific materials, The application is (I) Application to improving bacterial glutamic acid production, or (II) Application to glutamic acid production. The specific substance is (d1)BBD29_14900 G1114A Substances for enhancing gene expression, (d2)BBD29_14900 D372N Substances to improve protein abundance, (d3)BBD29_14900 D372N Substances for improving protein activity, (d4) a substance for improving the expression of the BBD29_14900 gene; (d5) a substance for increasing the abundance of BBD29_14900 protein, or (d6) A substance for improving the activity of BBD29_14900 protein.

[0118] Exemplarily, BBD29_14900 for improving the above G1114A The gene expression substance is specifically BBD29_14900 G1114A Gene or BBD29_14900 G1114A The recombinant plasmid may be a recombinant plasmid carrying the gene. Illustratively, the recombinant plasmid is the integrative plasmid PK18mobsacB-BBD29_14900 in the examples. G1114A or overexpression plasmid pXMJ19-BBD29_14900 G1114A may be.

[0119] For example, the material for enhancing the expression of the BBD29_14900 gene may be the BBD29_14900 gene or a recombinant plasmid carrying the BBD29_14900 gene. For example, the recombinant plasmid may be the integration plasmid PK18mobsacB-BBD29_14900 or the overexpression plasmid pXMJ19-BBD29_14900 in the embodiment.

[0120] The present invention further provides a recombinant bacterium, G1114A These were obtained by overexpressing the gene or BBD29_14900.

[0121] Exemplarily, overexpression BBD29_14900 G1114A The gene implementation method is BBD29_14900 in bacteria. G1114AGene or BBD29_14900 G1114A The recombinant plasmid carrying the gene is introduced. Illustratively, the recombinant plasmid is the integrative plasmid PK18mobsacB-BBD29_14900 in the examples. G1114A or overexpression plasmid pXMJ19-BBD29_14900 G1114A may be.

[0122] For example, the BBD29_14900 gene can be overexpressed by introducing the BBD29_14900 gene or a recombinant plasmid carrying the BBD29_14900 gene into bacteria. For example, the recombinant plasmid can be the integrative plasmid PK18mobsacB-BBD29_14900 or the overexpression plasmid pXMJ19-BBD29_14900 in the embodiment.

[0123] The present invention further covers the application of said recombinant bacteria in the production of glutamic acid.

[0124] The present invention relates to the BBD29_14900 gene in the bacterial genome. G1114A Further disclosed is a method for improving the production of glutamic acid in bacteria, comprising the step of replacing the gene with a .

[0125] Exemplarily, the BBD29_14900 gene in the bacterial genome is G1114A The method of replacing the gene is as follows: G1114A The goal is to introduce the

[0126] The present invention relates to the use of BBD29_14900 in bacteria. G1114A Overexpressing the gene or BBD29_14900 in bacteria D372N Enhancing the abundance of BBD29_14900 protein in bacteria D372NFurther provided is a method for increasing glutamic acid production in a bacterium, comprising the step of increasing the activity of a protein or increasing the activity of a BBD29_14900 protein in the bacterium.

[0127] Exemplarily, overexpression BBD29_14900 G1114A The gene implementation method is BBD29_14900 in bacteria. G1114A Gene or BBD29_14900 G1114A The recombinant plasmid carrying the gene is introduced. Illustratively, the recombinant plasmid is the integrative plasmid PK18mobsacB-BBD29_14900 in the examples. G1114A or overexpression plasmid pXMJ19-BBD29_14900 G1114A may be.

[0128] For example, the BBD29_14900 gene can be overexpressed by introducing the BBD29_14900 gene or a recombinant plasmid carrying the BBD29_14900 gene into bacteria. For example, the recombinant plasmid can be the integrative plasmid PK18mobsacB-BBD29_14900 or the overexpression plasmid pXMJ19-BBD29_14900 in the embodiment.

[0129] The present invention is BBD29_14900 D372N The present invention further protects the application of the BBD29_14900 protein in regulating bacterial glutamate production.

[0130] The control is a positive control, i.e., BBD29_14900 D372N The protein content increases and glutamate production increases.

[0131] The control is a positive control, i.e., BBD29_14900 D372N Protein content decreases and glutamate production decreases.

[0132] The regulation is positive, i.e., the content of BBD29_14900 protein increases and the production of glutamic acid increases.

[0133] The regulation is positive, i.e., the content of BBD29_14900 protein decreases and the production of glutamic acid decreases.

[0134] When using the recombinant bacteria to produce glutamic acid, a specific method includes fermenting the recombinant bacteria.

[0135] Those skilled in the art can carry out fermentation using conventional fermentation methods. Optimization and improvement of fermentation methods can also be achieved through routine testing. Bacterial fermentation can be carried out in an appropriate medium under fermentation conditions known in the art. The medium may contain a carbon source, a nitrogen source, trace elements, and combinations thereof. The pH of the culture may be adjusted during cultivation. The cultivation may also include preventing the generation of bubbles, for example, by using an antifoaming agent. The cultivation may also include injecting gas into the culture. The gas may include any gas capable of maintaining aerobic conditions in the culture. The temperature of the culture may be 20 to 45°C.

[0136] The method may further include obtaining glutamic acid from the culture, which can be achieved in a variety of ways, including, but not limited to, treating the culture with, for example, sulfuric acid or hydrochloric acid, and combining methods such as anion exchange chromatography, concentration, crystallization, and isoelectric precipitation.

[0137] During the fermentation, an exemplary fermentation medium formulation is shown in Table 3, with the balance being water.

[0138] During the fermentation, an exemplary fermentation control process is shown in Table 4.

[0139] For example, in the fermentation, the bacterial concentration in the system is 15 g / L.

[0140] For example, during the fermentation process, the sugar content (residual sugar) of the system is controlled by supplementing the aqueous glucose solution.

[0141] Any one of the above-mentioned bacteria is a bacterium of the genus Corynebacterium, preferably Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium callunae, Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium lactofermentum, Corynebacterium ammoniagenes, Corynebacterium pekinense, Brevibacterium saccharolyticum, or the like. saccharolyticum, Brevibacterium roseum, and Brevibacterium thiogenitalis.

[0142] Any one of the above-mentioned bacteria is a bacterium capable of producing glutamic acid.

[0143] "Bacteria capable of producing glutamic acid" refers to bacteria that have the ability to produce and accumulate glutamic acid in a medium and / or bacterial cells, which allows glutamic acid to be collected when cultured in a bacterial medium.

[0144] The bacteria may be wild-type bacteria collected from nature, or may be modified bacteria.

[0145] "Modified bacteria" refers to modified bacteria obtained by artificially mutating and / or mutating wild-type bacteria collected from nature.

[0146] Specifically, the Corynebacterium glutamicum may be Corynebacterium glutamicum CGMCC21260.

[0147] Corynebacterium glutamicum CGMCC21220 has the strain name YPGLU001 and is abbreviated as Corynebacterium glutamicum CGMCC21220. It was preserved at the Center for Ordinary Microorganisms of the China Committee for the Collection and Management of Microbial Species of China (abbreviated as CGMCC, address: No. 3, Beichen West Road, Chaoyang District, Beijing, China) on November 23, 2020, with the preservation registration number CGMCC 21220.

[0148] The glutamic acid in any one of the above means glutamic acid in the broad sense, and includes glutamic acid in free form, a salt of glutamic acid, or a mixture of both.

[0149] Specifically, the glutamic acid is L-glutamic acid.

[0150] Any of the above methods or applications may also be used in the production of downstream products of glutamic acid.

[0151] The BBD29_14900 protein in Corynebacterium glutamicum is shown in SEQ ID NO:3, and its encoding gene is shown in SEQ ID NO:1. In the present invention, the BBD29_14900 protein shown in SEQ ID NO:4 is isolated by introducing point mutations. D372N Protein obtained, BBD29_14900 D372N The protein-encoding gene is shown in SEQ ID NO:2. BBD29_14900 is the phase of the BBD29_14900 gene. G1114AThe difference from the gene is that position 1114 is changed from guanine deoxyribonucleotide (G) to adenine deoxyribonucleotide (A). Compared to the BBD29_14900 protein, BBD29_14900 D372N The difference in the protein is that the amino acid residue at position 372 is changed from aspartic acid (D) to asparagine (N).

[0152] The present invention has discovered that the BBD29_14900 protein positively regulates the production of glutamate in bacteria, i.e., an increase in the content of BBD29_14900 protein leads to an increase in glutamate production, while a decrease in the content of BBD29_14900 protein leads to a decrease in glutamate production. By inhibiting the expression of the BBD29_14900 gene, the production of glutamate can be reduced, and by overexpressing the BBD29_14900 gene, the production of glutamate can be increased. Furthermore, the present invention has discovered that point mutations in BBD29_14900 D372N The obtained protein has superior function to BBD29_14900 protein. The present invention has important application value for industrial production of glutamic acid. DETAILED DESCRIPTION OF THE INVENTION

[0153] The following examples will facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

[0154] The media used to culture the strains in the following examples are all obtained by adding other components to the basal medium. These other components include sucrose, kanamycin, or chloramphenicol. Solid media contain agarose. The composition of the basal medium is shown in Table 1. Unless otherwise specified, the culture temperature for all strains in the examples is 32°C. [Table 1] The PAGE recipe for sscp electrophoresis is shown in Table 2. The electrophoresis conditions are as follows: the electrophoresis tank is placed on ice, 1x TBE buffer is used, the voltage is 120V, and the electrophoresis time is 10 hours. [Table 2] Corynebacterium glutamicum CGMCC21220 has the strain name YPGLU001 and is abbreviated as Corynebacterium glutamicum CGMCC21220. It was preserved at the Center of Ordinary Microorganisms of the China Commission on Microbial Species Collection (abbreviated as CGMCC, address: No. 3, Hall 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences) on November 23, 2020, with the preservation registration number CGMCC 21220 Corynebacterium glutamicum ATCC 13869, i.e., Corynebacterium glutamicum CICC20216. The full name of CICC is China Center for Industrial Microorganism Collection.

[0155] Example 1: Transformation vector pK18-BBD29_14900 containing point-mutated BBD29_14900 gene coding region G1114A Construction Based on the genome sequence of Corynebacterium glutamicum ATCC 13869 published by NCBI, two pairs of primers were designed and synthesized to amplify the BBD29_14900 gene coding region sequence, and point mutations were introduced into Corynebacterium glutamicum CGMCC21220 by allele substitution (sequencing confirmed that the BBD29_14900 gene coding region on the Corynebacterium glutamicum CGMCC21220 chromosome was identical to the BBD29_14900 gene coding region on the Corynebacterium glutamicum ATCC 13869 chromosome). The amino acid sequence of the corresponding encoded protein before mutation is SEQ ID NO:3. The nucleotide sequence of the BBD29_14900 gene before mutation is SEQ ID NO:1 gene, in which a single point mutation was introduced into the coding region, i.e., the 1114th amino acid residue was changed from guanine deoxyribonucleotide (G) to adenine deoxyribonucleotide (A). Accordingly, a single point mutation was introduced into the protein, i.e., the 372nd amino acid residue was changed from aspartic acid (D) to asparagine (N). The gene after mutation is BBD29_14900. G1114A The gene is referred to as BBD29_14900 and is shown in SEQ ID NO:2. D372N The protein is referred to as SEQ ID NO:4.

[0156] The primers are as follows (synthesized by Shanghai Invitrogen Company): P1 (SEQ ID NO: 5): 5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCGCTGTGGTTATCCTCGCTG-3' P2(SEQ ID NO:6):5'-CTGGGGCGACGCGGGGATTCAAGGCGGTCG-3' P3(SEQ ID NO:7):5'-CGACCGCCTTGAATCCCCGCGTCGCCCCAG-3' P4 (SEQ ID NO: 8): 5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGAGTGTCACTAGGCTAGTC-3'

[0157] 1. Using Corynebacterium glutamicum ATCC 13869 as a template, PCR amplification was performed using a primer set consisting of P1 and P2 or a primer set consisting of P3 and P4. PCR system (50 μL): Template, 10x Ex Taq Buffer 5 μL, dNTP Mixture (2.5 mM each) 4 μL, Mg 2+ (25 mM) 4 μL, primers (10 μM) 2 μL each, Ex Taq (5 U / μL) 0.25 μL, and the remaining amount was water.

[0158] PCR amplification was performed in the following format: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 40 s, 30 cycles, and overextension at 72°C for 10 min.

[0159] 2. Recovery The amplified products of the two PCR systems in step 1 were recovered and PCR amplification was carried out using a primer set consisting of P1 and P4 as a template. PCR system (50 μL): Template, 10x Ex Taq Buffer 5 μL, dNTP Mixture (2.5 mM each) 4 μL, Mg 2+ (25 mM) 4 μL, primers (10 μM) 2 μL each, Ex Taq (5 U / μL) 0.25 μL, and the remaining amount was water. PCR amplification was performed in the following format: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 90 s, 30 cycles, and overextension at 72°C for 10 min.

[0160] 3. The approximately 1253 bp amplification product obtained by PCR amplification in step 2 was recovered, and the recovered DNA fragment was identified as BBD29_14900 G1114A (After sequencing, the DNA fragment is shown in SEQ ID NO:29). BBD29_14900 G1114A The fragment was successfully integrated into Corynebacterium glutamicum CGMCC21220 by homologous recombination, resulting in a mutation of nucleotide 1114 in the BBD29_14900 gene coding region from guanine deoxyribonucleotide (G) to adenine deoxyribonucleotide (A), and ultimately a mutation of amino acid residue 372 in the encoded protein from aspartic acid (D) to asparagine (N).

[0161] 4. The pK18mobsacB plasmid (Addgene) was cleaved using the restriction enzyme Xba I to recover a linear plasmid. 5. BBD29_14900 obtained in step 3 G1114A The fragment and the linear plasmid obtained in step 4 were assembled using the NEBuider recombination system to create the vector pK18-BBD29_14900 G1114A The vector pK18-BBD29_14900 was obtained. G1114A The sequence has been verified, including BBD29_14900, which is shown in SEQ ID NO: 29. G1114A The vector pK18-BBD29_14900 contained the fragment G1114A had a kanamycin resistance marker.

[0162] Example 2, Point Mutation BBD29_14900 G1114A Construction of engineered strains containing Vector pK18-BBD29_14900 constructed in Example 1 G1114AThe gene was introduced into Corynebacterium glutamicum CGMCC21220 by electrophoretic transformation. Single colonies resulting from the culture were analyzed by PCR using a primer pair consisting of primer P1 and universal primer M13R (M13R: 5'-CAGGAAACAGCTATGACC-3'). Strains with a band of approximately 1260 bp were identified as positive. Positive strains were cultured on plates containing 15% sucrose. Single colonies resulting from the culture were then cultured on plates containing and not containing kanamycin. Strains that grew on the kanamycin-free medium but not on the kanamycin-containing medium were further analyzed by PCR using a primer set consisting of P5 and P6 (synthesized by Shanghai Invitrogen). The PCR amplified products were collected, denatured at high temperature, and subjected to ice bath denaturation followed by SSCP electrophoresis (vector pK18-BBD29_14900). G1114A The amplified fragment from Corynebacterium glutamicum ATCC 13869 was used as the positive control, the amplified fragment from Corynebacterium glutamicum ATCC 13869 was used as the negative control, and water was used as the blank control. Because the fragment structures and electrophoretic positions differed, strains whose electrophoretic positions did not match those of the negative control fragment but matched those of the positive control fragment were considered to have successfully undergone isosite substitution. The strains screened for successful isosite substitution were amplified by PCR using a primer set consisting of P5 and P6. The amplified product was recovered, ligated into the PMD19-T vector, and sequenced. The strain with successful isosite substitution was verified by sequence ratio and named Corynebacterium glutamicum YPG-019. P5(SEQ ID NO:9):5'-GCAGCCAAGGCCAAGAAGAT-3' P6(SEQ ID NO:10):5'-TCCCTGTTTAAGACTGCATT-3' The difference between Corynebacterium glutamicum CGMCC21220 and Corynebacterium glutamicum YPG-019 is that the BBD29_14900 gene shown in Sequence 1 of the Sequence Listing in the genome of Corynebacterium glutamicum CGMCC21220 is BBD29_14900 shown in Sequence 2 of the Sequence Listing. G1114AThe only difference between sequence 1 and sequence 2 is the nucleotide at position 1114.

[0163] Example 3, BBD29_14900 or BBD29_14900 in the genome G1114A Construction of engineered strains with overexpressed genes I. Construction of integrative plasmids Based on the Corynebacterium glutamicum ATCC 13869 genome sequence published by NCBI, three pairs of primers were designed and synthesized to target the upstream homologous arm fragment, the BBD29_14900 gene coding region (or BBD29_14900 G1114A The downstream homologous arm fragment was amplified and inserted into the genomic DNA of Corynebacterium glutamicum CGMCC21220 by homologous recombination. G1114A Genes were integrated.

[0164] The primers are as follows (synthesized by Shanghai Invitrogen Company): P7 (SEQ ID NO: 11): 5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGACCCGCTTGCCATACGAAG-3' P8(SEQ ID NO:12):5'-AGTGGGCTGAATTTGGGCTGATCTACTCATCTGAAGAATC-3' P9(SEQ ID NO:13):5'-GATTCTTCAGATGAGTAGATCAGCCCAAATTCAGCCCACT-3' P10(SEQ ID NO:14):5'-CAAACCAGAGTGCCCACGAACTAAGCGTTTTGCGCTTCGG-3' P11(SEQ ID NO:15):5'-CCGAAGCGCAAAACGCTTAGTTCGTGGGCACTCTGGTTTG-3' P12 (SEQ ID NO: 16): 5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCATAAGAAACAACCACTTCC-3' Using Corynebacterium glutamicum YPG-019 as a template, PCR amplification was performed using the primer set consisting of P7 / P8, the primer set consisting of P9 / P10, and the primer set consisting of P11 / P12. The upstream homologous arm fragment of approximately 806 bp, BBD29_14900, was obtained. G1114A A gene fragment of approximately 1494 bp and a downstream homologous arm fragment of approximately 788 bp were obtained. The resulting mixture of the three fragments was then amplified using the P7 / P12 primer set as a template to obtain an integrated homologous arm fragment of approximately 3008 bp (sequenced as shown in SEQ ID NO: 30). The integrated homologous arm fragment was ligated to the shuttle plasmid PK18mobsacB, which had been recovered by XbaI digestion, using the rice NEBuider recombination system to generate the integrated plasmid PK18mobsacB-BBD29_14900. G1114A The integrated plasmid contains a kanamycin resistance marker, and recombinants with the integrated plasmid can be obtained by kanamycin screening. PCR system (50 μL): Template, 10× Ex Taq Buffer 5 μL, dNTP Mixture (2.5 mM each) 4 μL, Mg 2+ The PCR amplification consisted of 4 μL of 25 mM ATP, 2 μL of each primer (10 μM), 0.25 μL of ExTaq (5 U / μL), and water. The PCR amplification consisted of 30 cycles of pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 120 seconds, followed by over-extension at 72°C for 10 minutes. The above steps were performed using Corynebacterium glutamicum ATCC 13869 instead of Corynebacterium glutamicum YPG-019, and the integrative plasmid PK18mobsacB-BBD29_14900 was obtained.

[0165] II. Production of recombinant bacteria The integrative plasmid was electrotransformed into Corynebacterium glutamicum CGMCC21220, and single colonies grown were identified by PCR using the P13 / P14 primer set. Those containing a PCR-amplified fragment of approximately 1827 bp were positive for the transformation, while those lacking amplified fragment were unsuccessful. After screening with 15% sucrose, the positive strains were cultured on plates containing and without kanamycin. Those that grew on the kanamycin-free medium but not on the kanamycin-containing medium were further identified by PCR using the P15 / P16 primer set. Those containing a PCR-amplified fragment of approximately 1517 bp were identified as strains in which the target gene had been integrated into the Corynebacterium glutamicum CGMCC21220 genome. P13(SEQ ID NO:17):5'-GTCCAAGGTGACGGCCGCAC-3' P14(SEQ ID NO:18):5'-GCAGCCTTAACTGGGGAAAG-3' P15(SEQ ID NO:19):5'-GGAGCGCCGCCTCATCGAGC-3' P16(SEQ ID NO:20):5'-ATATTCGGCCCAGCAGCAGC-3' The above steps were carried out with the PK18mobsacB-BBD29_14900 integrated plasmid, resulting in the recombinant Corynebacterium glutamicum YPG-020. Corynebacterium glutamicum YPG-020 is a recombinant bacterium in which the BBD29_14900 gene is overexpressed in the genomic DNA.

[0166] Integrative plasmid PK18mobsacB-BBD29_14900 G1114A The recombinant bacterium obtained by carrying out the above steps is Corynebacterium glutamicum YPG-021. Corynebacterium glutamicum YPG-021 contains the BBD29_14900 sequence in its genomic DNA. G1114A It is a recombinant bacterium that overexpresses genes.

[0167] Example 4: BBD29_14900 or BBD29_14900 on a plasmid G1114A Construction of engineered strains with overexpressed genes I. Construction of recombinant plasmids Based on the wild-type Corynebacterium glutamicum ATCC 13869 genome sequence published by NCBI, a pair of primers was designed and synthesized to amplify the coding and promoter sequences of the BBD29_14900 gene, designated P17 and P18 (synthesized by Shanghai Invitrogen). P17 (SEQ ID NO: 21): 5'-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCAGCCCAAATTCAGCCCACT-3' P18 (SEQ ID NO: 22): 5'-ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACCTAAGCGTTTTGCGCTTCGG-3' Using Corynebacterium glutamicum YPG-019 as a template, PCR amplification was performed with a primer set consisting of primers P17 / P18 to obtain the 1524 bp BBD29_14900 G1114A Gene fragment (after sequencing, BBD29_14900 G1114A The gene fragment was obtained (shown in SEQ ID NO: 31). The NEBuider recombination system was employed to obtain BBD29_14900 G1114A The gene fragment was ligated with the shuttle plasmid pXMJ19 recovered by digestion with EcoRI enzyme to create the overexpression plasmid pXMJ19-BBD29_14900. G1114 A The overexpression plasmid contained a chloramphenicol resistance marker, and the resulting plasmid could be transformed into a strain by chloramphenicol screening. PCR system (50 μL): template, 5 μL of 10x Ex Taq Buffer, 4 μL of dNTP mixture (2.5 mM each), Mg 2+The PCR amplification consisted of 4 μL of 25 mM ATP, 2 μL of each primer (10 pM), 0.25 μL of ExTaq (5 U / μL), and the remaining volume was water. PCR amplification was performed using the following cycles: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 52°C for 30 seconds, and extension at 72°C for 90 seconds, followed by 30 cycles of overextension at 72°C for 10 minutes. The above steps were performed using Corynebacterium glutamicum YPG-020 instead of Corynebacterium glutamicum YPG-019, and the overexpression plasmid pXMJ19-BBD29_14900 was obtained.

[0168] II. Production of recombinant bacteria The overexpression plasmid was electrotransformed into Corynebacterium glutamicum CGMCC21220, and single colonies grown were analyzed by PCR using a primer set consisting of M13R(-48) and P18. PCR amplified a fragment of approximately 1563 bp, indicating the recombinant strain: M13R(-48):5'-AGCGGATAACAATTTCACACAGGA-3'.

[0169] The above steps were performed using the overexpression plasmid pXMJ19-BBD29_14900, resulting in the recombinant strain Corynebacterium glutamicum YPG-022. Corynebacterium glutamicum YPG-022 is a recombinant strain in which the BBD29_14900 gene is overexpressed by the plasmid.

[0170] Overexpression plasmid pXMJ19-BBD29_14900 G1114A The recombinant bacterium obtained by carrying out the above steps is Corynebacterium glutamicum YPG-023. Corynebacterium glutamicum YPG-023 contains the plasmid BBD29_14900 G1114A It is a recombinant bacterium that overexpresses genes.

[0171] Example 5. Construction of an engineered strain with the BBD29_14900 gene deleted in the genome I. Construction of knockout plasmids Based on the genome sequence of Corynebacterium glutamicum ATCC 13869 published by NCBI, two pairs of primers were synthesized to amplify both ends of the BBD29_14900 gene coding region and were used to amplify the upstream and downstream homologous arm fragments.

[0172] The primers are as follows (synthesized by Shanghai Yingjun Co., Ltd.): P19 (SEQ ID NO: 23): 5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTCGCTGAAACAGCAGGGGAC-3' P20(SEQ ID NO:24):5'-AGGCGTCGATAAGCAAATTTATCATTTAGCCTTGTTAATC-3' P21(SEQ ID NO:25):5'-GATTAACAAGGCTAAATGATAAATTTGCTTATCGACGCCT-3' P22 (SEQ ID NO: 26): 5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCAACTTGCCATGAGTCGTCTT-3' Using Corynebacterium glutamicum ATCC 13869 as a template, PCR amplification was performed using the primer set P19 / P20 or the primer set P21 / P22 to obtain the upstream homologous arm fragment (approximately 804 bp) and downstream homologous arm fragment (approximately 791 bp), respectively. Simultaneously, PCR amplification was performed using the primer set P19 / P22 to obtain the entire homologous arm fragment (approximately 1555 bp, sequenced as shown in SEQ ID NO: 32) using the upstream and downstream homologous arm fragments as templates. The entire homologous arm fragment was ligated with the shuttle plasmid pkl8mobsacB, which had been digested with XbaI enzyme, using the NEBuider recombination system to obtain a knockout plasmid. The knockout plasmid contained a kanamycin resistance marker.

[0173] II. Production of recombinant bacteria The knockout plasmid was electrotransformed into Corynebacterium glutamicum CGMCC21220, and single colonies grown during cultivation were analyzed by PCR using primer sets P23 and P24 (synthesized by Shanghai Yingjun Co., Ltd.). Strains that amplified bands of 1481 bp and 2660 bp were positive for transformation, while strains that amplified only the 1481 bp band were unsuccessful. Positive strains were screened on 15% sucrose medium and then cultured on plates containing and not containing kanamycin. Strains that grew on the medium without kanamycin but not on the medium containing kanamycin were further identified by PCR using a primer set consisting of P23 and P24. The strain with only one amplification product of 1481 bp was identified as a genetically engineered strain in which the coding region of the BBD29_14900 gene had been knocked out and was named Corynebacterium glutamicum YPG-024. P23(SEQ ID NO:27):5'-TCGCTGAAACAGCAGGGGAC-3' P24(SEQ ID NO:28):5'-AACTTGCCATGAGTCGTCTT-3'

[0174] Example 6: L-glutamic acid fermentation experiment The recombinant bacteria constructed in the above examples and Corynebacterium glutamicum CGMCC21220 were fermented in a BLBI0-5GC-4-H model fermenter (purchased from Shanghai Bailun Biotechnology Co., Ltd.). The fermentation medium formulation is shown in Table 3 (the balance is water), where d1 means 0.1 L. The control process is shown in Table 4. At the initial time of inoculation completion, the bacterial concentration in the system was 15 g / L. During fermentation, the sugar content (residual sugar) of the system was controlled by supplementing 50-55% of glucose solution.

[0175] In each case, three iterations were performed, and the results are shown in Table 5. [Table 3] [Table 4] [Table 5] As shown in Table 5, the point mutation BBD29_14900 in the BBD29_14900 gene coding region in Corynebacterium glutamicum G1114A While overexpression and expression of the gene are advantageous for improving the production of L-glutamic acid, weakening or knocking out the gene is disadvantageous for the accumulation of L-glutamic acid.

[0176] The present invention has been described in detail above. Those skilled in the art can practice the present invention in a broader range using equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without undue experimentation. While the present invention provides specific examples, it should be understood that the present invention can be further improved. In short, in accordance with the principles of the present invention, this application is intended to cover any modifications, uses, or improvements of the present invention, including modifications using conventional techniques known in the art that deviate from the scope disclosed in this application. Applications of some basic features may be made within the scope of the following appended claims. [Industrial Applicability]

[0177] The present invention has discovered that weakening or knocking out the BBD29_14900 gene affects the ability to produce L-glutamic acid through the product encoded by the gene. It has also disclosed that introducing point mutations into the coding sequence or increasing the copy number or overexpressing the gene results in a recombinant strain, which is advantageous in producing high concentrations of glutamic acid compared to unmodified strains. [Accession number]

[0178] Species name: Corynebacterium glutamicum, Strain number: YPGLU001, Accession number: CGMCC 21220

Claims

1. A bacterium for producing L-glutamic acid, the bacterium contains a modified protein comprising an amino acid sequence in which the aspartic acid at position 372 of SEQ ID NO: 3 is substituted with asparagine in the amino acid sequence of SEQ ID NO: 3 or a homologous sequence having 95% or more sequence identity thereto, and the modified protein is overexpressed; the L-glutamic acid production ability is improved compared to the case where a protein having the amino acid sequence of SEQ ID NO: 3 is overexpressed instead of the modified protein under the same conditions, The bacterium for producing L-glutamic acid is Corynebacterium glutamicum.

2. The bacterium described in claim 1, characterized in that the gene encoding the modified protein comprises a nucleotide sequence in which the guanine (G) at the 1114th base of the nucleotide sequence shown in SEQ ID NO: 1 is replaced with adenine (A).

3. A nucleic acid characterized by encoding a protein comprising an amino acid sequence in which the 372nd aspartic acid in the amino acid sequence shown in SEQ ID NO: 3 is substituted with asparagine.

4. The nucleic acid of claim 3, wherein the protein comprises the amino acid sequence shown in SEQ ID NO:

4.

5. The nucleic acid of claim 3, comprising a nucleotide sequence having a mutation at the 1114th base of the nucleotide sequence shown in SEQ ID NO: 1, wherein the mutation is a mutation from guanine (G) to adenine (A) at the 1114th base of the nucleotide sequence shown in SEQ ID NO:

1.

6. A protein characterized by comprising the amino acid sequence shown in SEQ ID NO:

4.

7. A recombinant vector characterized by comprising the nucleic acid described in claim 3.

8. A recombinant microorganism comprising the nucleic acid of claim 3 and being Corynebacterium glutamicum.

9. A method for producing L-glutamic acid, comprising culturing the bacterium according to any one of claims 1 to 2 and recovering L-glutamic acid from the culture.

10. A protein designated BBD29_14900 D372N protein, comprising the following amino acid sequence: (a1) the amino acid sequence shown in SEQ ID NO: 3; and (a2) an amino acid sequence having 95% or more sequence identity with the amino acid sequence of (a1); a protein having an amino acid sequence in which the aspartic acid corresponding to the 372nd amino acid residue in the amino acid sequence of SEQ ID NO: 3 is substituted with asparagine; and A protein that, when overexpressed in Corynebacterium glutamicum YPGLU001 (Accession No.: CGMCC 21220), improves L-glutamic acid production ability compared to overexpressing a protein having the amino acid sequence of SEQ ID NO: 3 instead of the protein under the same conditions.

11. A nucleic acid encoding the protein described in claim 10.

12. An expression cassette, a recombinant vector, or a recombinant bacterium comprising the nucleic acid of claim 11, The expression cassette, recombinant vector or recombinant bacterium, wherein the recombinant bacterium is Corynebacterium glutamicum.

13. A protein described in claim 10, a nucleic acid described in claim 11, or an expression cassette, recombinant vector, or recombinant bacterium described in claim 12 for use in producing glutamic acid.

14. A recombinant bacterium that overexpresses a protein having the amino acid sequence set forth in SEQ ID NO:4, wherein the overexpression of said protein is caused by an increased copy number of a gene in said bacterium that encodes said protein; A recombinant bacterium, wherein the bacterium is Corynebacterium glutamicum.

15. A recombinant bacterium described in claim 14 for use in producing glutamic acid.

16. A method for improving the production of glutamic acid in a bacterium, the method comprising the step of replacing a gene having the nucleotide sequence shown in SEQ ID NO: 1 in the bacterial genome of the bacterium Corynebacterium glutamicum with a gene having the nucleotide sequence shown in SEQ ID NO:

2.

17. A method for improving glutamic acid production in a bacterium that is Corynebacterium glutamicum, comprising the step of overexpressing a gene having the nucleotide sequence shown in SEQ ID NO:2 in the bacterium by increasing the copy number of the gene.

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