Microorganisms with weakened activity of LacI-type DNA-binding transcription regulators and a method for producing L-glutamic acid using the same.

Abstract

The present application relates to a microorganism having weakened activity of a LacI DNA-binding transcriptional regulator protein and a method for producing L-glutamic acid using the same. The Corynebacterium microorganism of the present application having weakened activity of a LacI DNA-binding transcriptional regulator protein has significantly increased L-glutamic acid producing ability, and its use makes it possible to produce L-glutamic acid at a higher yield than existing microorganisms.

Description

Technical Field

[0001] Cross-reference with related applications This application claims the benefit of priority based on Korean Patent Application No. 10-2021-0098072 filed on Jul. 26, 2021, and all the contents disclosed in the document of the Korean Patent Application are incorporated herein by reference.

[0002] This application relates to a microorganism with weakened activity of a LacI family DNA-binding transcriptional regulator and a method for producing L-glutamic acid using the same.

Background Art

[0003] To produce L-amino acids and other useful substances, various studies have been conducted for the development of highly efficient production microorganisms and fermentation process technologies. For example, a target substance-specific approach method such as increasing the expression of genes encoding enzymes involved in the biosynthesis of L-glutamic acid or removing genes unnecessary for biosynthesis is mainly used.

[0004] However, at present, research for increasing the production ability of effective L-glutamic acid is still necessary due to the increasing demand for L-glutamic acid. [[ID=2,4]]

Prior Art Documents

Patent Documents

[0005] <​​​​​​​​​​​​​One objective of this application is to provide a Corynebacterium sp. microorganism in which the activity of LacI family DNA-binding transcriptional regulators is weakened and the L-glutamate production capacity is improved.

[0007] Another object of this application is to provide a method for producing L-glutamic acid, comprising the step of culturing the Corynebacterium microorganisms in a culture medium. [Means for solving the problem]

[0008] This will be explained in detail below. On the other hand, each description and embodiment disclosed in this application is applicable to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not to be considered limited by the specific descriptions below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents as a whole are inserted into this specification by reference to more clearly explain the level of the art to which the present invention belongs and the content of the present invention.

[0009] One aspect of this application provides a Corynebacterium sp. microorganism (or strain, recombinant cell) in which the activity of LacI family DNA-binding transcriptional regulators is attenuated.

[0010] In this application, the LacI family DNA-binding transcription factor protein may be a protein having LacI family transcriptional regulator activity (e.g., a LacI family transcriptional regulator), and may be derived from, for example, Corynebacterium glutamicum ATCC13869 strain, Corynebacterium glutamicum ATCC13032 strain, or Corynebacterium glutamicum ATCC14067 strain, but is not limited thereto. In one example, the LacI family DNA-binding transcription factor protein may be derived from Corynebacterium ATCC13869 strain (its sequence can be obtained from the NCBI GenBank, a known database, for example, GenBank Accession No. WP_060564415.1). In one example, the LacI-type DNA-binding transcription factor protein derived from Corynebacterium glutamicum may have, contain, consist of, or be essentially composed of the amino acid sequence of Sequence ID No. 3. Specifically, the protein may consist of the polypeptide described in the amino acid sequence of Sequence ID No. 3.

[0011] In one example, the LacI-type DNA-binding transcription factor protein may be, but is not limited to, a polypeptide having the activity of a LacI-type DNA-binding transcription factor encoded by a LacI-type DNA-binding transcription factor gene. In one example, the LacI-type DNA-binding transcription factor gene may be derived from the Corynebacterium glutamicum ATCC13869 strain, and specifically may include the nucleic acid sequence of Sequence ID No. 4 (the sequence from position 1,421,016 to position 1,422,125 in the nucleic acid sequence of GenBank Accession No. Sequence ID: CP016335.1, for example, the BBD29_06680 gene).

[0012] In one example, the LacI-type DNA-binding transcription factor protein may contain, or consist of, an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the amino acid sequence described in Sequence ID No. 3. Furthermore, any variant having amino acid sequences with such homology or identity and exhibiting efficacy corresponding to a LacI-type DNA-binding transcription factor protein, including those with deletions, modifications, substitutions, conserved substitutions, or additions to some sequences, is also included in the LacI-type DNA-binding transcription factor protein. For example, this includes cases where the N-terminus, C-terminus, and / or internal amino acid sequence have additions or deletions of sequences that do not alter the function of the variant of this application, spontaneously occurring mutations, silent mutations, or conserved substitutions.

[0013] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little to no effect on the activity of a protein or polypeptide.

[0014] In this application, the phrase "having, including, consisting of, or essentially consisting of a specific nucleic acid sequence (base sequence) or amino acid sequence" of a polynucleotide or polypeptide can mean that the polynucleotide or polypeptide essentially includes the specific nucleic acid sequence (base sequence) or amino acid sequence, and is interpreted to mean that it includes (or does not exclude) a "substantially equivalent sequence" in which mutations (deletions, substitutions, alterations, and / or additions) have been made to the specific nucleic acid sequence (base sequence) or amino acid sequence to the extent that the original function and / or intended function of the polynucleotide or polypeptide is maintained. In one example, the statement that a polynucleotide or polypeptide "has, contains, consists of, or is essentially composed of a specific nucleic acid sequence (base sequence) or amino acid sequence" means that the polynucleotide or polypeptide (i) essentially contains the specific nucleic acid sequence (base sequence) or amino acid sequence, or (ii) consists of, or essentially contains, a nucleic acid sequence or amino acid sequence having 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more homology or identity with the specific nucleic acid sequence (base sequence) or amino acid sequence, and maintains its original function and / or intended function. In one example, the intended function may mean a function that increases (improves) or confers the L-glutamic acid production capacity of microorganisms.

[0015] In this application, "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or base sequences, expressed as a percentage. The terms homology and identity are sometimes used interchangeably.

[0016] The sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequencing algorithms, along with a default gap penalty established by the program used. Substantially homologous or identical sequences can generally be hybridized, in whole or in part, under intermediate or highly stringent conditions. It is self-evident that hybridization also includes hybridization with polynucleotides containing codons in general or codons considering codon degeneracy.

[0017] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using a known computer algorithm such as the "FASTA" program with default parameters, for example, as described in Pearson et al (1988) [Proc.Natl.Acad.Sci.USA 85]:2444. Alternatively, the determination may be made using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.][F.,][ETAL, JMOLECBIOL215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San (Including Diego, 1994, and [CARILLO ETA / .](1988)SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Databases.

[0018] The homology, similarity, or identity of polynucleotides or polypeptides is determined by comparing sequence information using a GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443, as is known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, a GAP program can be defined as the number of similarly sequenced symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of two sequences. Default parameters for the GAP program may include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14:6745 (or EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0019] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and may include microorganisms in which a particular mechanism has been weakened or strengthened due to causes such as the insertion of an external gene or the weakening of the activity of an endogenous gene, and which may include genetic modification for the production of a target polypeptide, protein, or product.

[0020] In this application, the term “weakening” of a polypeptide is a concept that encompasses all instances where activity is reduced or absent compared to endogenous activity. Such “weakening” is used interchangeably with terms such as inactivation, deficiency, down-regulation, decline, reduce, and attenuation.

[0021] The aforementioned weakening may include cases where the activity of the polypeptide itself is reduced or eliminated compared to the polypeptide activity originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide (or protein, e.g., LacI-type DNA-binding transcription factor protein), cases where the overall polypeptide activity level and / or concentration (expression level) in the cell is lower than that of the native strain due to inhibition of the gene expression of the polynucleotide encoding it or inhibition of its translation into the polypeptide, cases where the polynucleotide is not expressed at all, and / or cases where the polynucleotide is expressed but the polypeptide is not active. The "intrinsic activity" refers to the activity of a specific polypeptide originally possessed by the parent strain, wild type, or unmodified microorganism before the trait change due to genetic mutation caused by natural or artificial factors. This is used interchangeably with "pre-modification activity." When polypeptide activity is described as "inactivated, deficient, reduced, downregulated, decreased, or attenuated" compared to its intrinsic activity, it means that it has become lower than the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the trait change.

[0022] The attenuation of the activity of such a polypeptide (or protein, for example, LacI family DNA-binding transcriptional regulatory factor protein) is carried out by any method known in the art, but is not limited thereto, and can be achieved by applying various methods well known in the art (for example, Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

[0023] Specifically, the attenuation of a polypeptide (or protein, for example, LacI family DNA-binding transcriptional regulatory factor protein; hereinafter referred to as polypeptide) is 1) Deletion of the whole or part of the gene encoding the polypeptide; 2) Modification of the expression regulatory region (or expression regulatory sequence) so that the expression of the gene encoding the polypeptide is decreased; 3) Modification of the amino acid sequence constituting the polypeptide so that the activity of the polypeptide is removed or attenuated (for example, deletion / substitution / addition of one or more amino acids on the amino acid sequence); 4) Modification of the gene sequence encoding the polypeptide so that the activity of the polypeptide is removed or attenuated (for example, deletion / substitution / addition of one or more nucleobases on the nucleobase sequence of the polypeptide gene so as to encode a polypeptide modified so that the activity of the polypeptide is removed or attenuated); 5) Modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide; 6) Introduction of an antisense oligonucleotide (for example, antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence to the front end of the Shine-Dalgarno sequence of the gene encoding the polypeptide in order to form a secondary structure that cannot be attached by a ribosome. 8) Addition of a promoter that is transcribed in the reverse direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE); or, 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto. For example,

[0024] The deletion of part or all of the gene encoding the polypeptide in 1) above may be the removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacement with a polynucleotide with some nucleotides deleted, or replacement with a marker gene.

[0025] In addition, the modification of the expression regulatory region (or expression regulatory sequence) in 2) above may be the generation of mutations on the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacement with a sequence having weaker activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0026] Furthermore, the modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide in 3) above may be, for example, substitution with a base sequence encoding another start codon having a lower polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0027] Furthermore, the modifications to the amino acid sequence or polynucleotide sequence described in 4) and 5) above may include, but are not limited to, deletion, insertion, non-conservative or conservative substitution or combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to weaken the activity of the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence modified to have weaker activity or an amino acid sequence or polynucleotide sequence modified to have no activity. For example, gene expression can be inhibited or weakened by introducing a mutation into the polynucleotide sequence to form a stop codon, but is not limited to this. The "stop codon" is a codon on mRNA that acts as a signal indicating the end of the protein synthesis process without specifying an amino acid in the codon, and generally, UAA, UAG, and UGA can be used as stop codons.

[0028] For example, the microorganism may be one in which an intrasequence mutation has been introduced into the ORF (open reading frame) of the endogenous gene of SEQ ID NO: 4 to form a termination codon. Alternatively, it may be one in which the codon corresponding to the 310th amino acid (e.g., glutamine, Gln, Q) of a LacI-type DNA-binding transcription factor protein has been replaced with a termination codon. For example, in the nucleic acid sequence of SEQ ID NO: 4, the polynucleotide encoding glutamine at the 310th amino acid may be "CAG," and a mutation may be introduced to replace "CAG" with "TAA," "TAG," or "TGA," respectively, in order to replace it with a termination codon. In this example, the nucleic acid sequence into which the mutation has been introduced to produce the termination codon may be the nucleic acid sequence of SEQ ID NO: 2 (in which the 928th C of the wild-type polynucleotide of SEQ ID NO: 4 is replaced with T; C928T).

[0029] For the introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide (6) above, see, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol.1(1) 1986].

[0030] 7) Adding a sequence complementary to the Shine-Dalgarno sequence to the leading end of the Shine-Dalgarno sequence in a polypeptide-encoding gene to form a secondary structure that cannot be attached to ribosomes may make mRNA translation impossible or reduce its rate.

[0031] The addition of a promoter that is transcribed in the reverse direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE) may be performed to weaken the activity by creating an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide.

[0032] In this application, the term “enhancement” of polypeptide activity means that the polypeptide activity increases compared to its intrinsic activity. Such enhancement is used interchangeably with terms such as activation, upregulation, overexpression, and increase. Here, activation, enhancement, upregulation, overexpression, and increase can all include exhibiting activity that was not originally present, or exhibiting improved activity compared to the intrinsic activity or pre-modification activity. “Intrinsic activity” means the activity of a particular polypeptide that was originally present in the parent strain or unmodified microorganism before the trait change, in cases where the trait changes due to genetic mutation caused by natural or artificial factors. This can be used interchangeably with “pre-modification activity.” “Enhancement,” “upregulation,” “overexpression,” or “increase” of polypeptide activity compared to its intrinsic activity means that the activity and / or concentration (expression level) of a particular polypeptide that was originally present in the parent strain or unmodified microorganism before the trait change.

[0033] The aforementioned enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. Whether or not the polypeptide activity has been enhanced can be confirmed by an increase in the activity level, expression level, or amount of product excreted from the polypeptide.

[0034] The enhancement of the activity of the polypeptide can be achieved by applying a variety of methods well known in the field, and is not limited as long as it can enhance the activity of the target polypeptide compared to the microorganism before modification. Specifically, this may involve, but is not limited to, the use of genetic engineering and / or protein engineering, which are routine methods in molecular biology and are well known to ordinary technicians in the field (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol.2.1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0035] Specifically, the strengthening of the polypeptide in this application is 1) Increased intracellular copy number of polynucleotides encoding polypeptides; 2) Replace the gene expression regulatory region on the chromosome encoding the polypeptide with a highly active sequence; 3) Modification of the nucleotide sequence encoding the start codon or 5'-UTR region of a polypeptide-encoding gene transcript; 4) Modification of the amino acid sequence of the polypeptide so as to enhance its activity; 5) Modification of the polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide's activity (for example, modification of the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified to enhance the polypeptide's activity); 6) Introduction of a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding such activity; 7) Codon optimization of polynucleotides encoding polypeptides; 8) Analyze the tertiary structure of the polypeptide, select exposed sites, and modify or chemically alter them; or 9) Two or more combinations selected from items 1) to 8) above are also acceptable, but are not particularly limited to these.

[0036] More specifically, The increase in the intracellular copy number of the polynucleotide encoding the polypeptide described in 1) above may be achieved by introducing into the host cell a vector that can replicate and function independently of the host, on which the polynucleotide encoding the polypeptide is operably linked. Alternatively, it may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into the chromosomes within the host cell. The introduction into the chromosomes is performed by introducing into the host cell a vector that can insert the polynucleotide into the chromosomes within the host cell, but is not limited to this.

[0037] 2) Replacing a gene expression regulatory region (or expression regulatory sequence) on a chromosome encoding a polypeptide with a more active sequence may, for example, involve deletion, insertion, non-conservative or conservative substitution or a combination thereof to induce mutagenesis in the sequence, or replacement with a sequence having stronger activity, in order to further enhance the activity of the expression regulatory region. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and decoding. For example, the original promoter may be replaced with a stronger promoter.

[0038] Known examples of strong promoters include, but are not limited to, the CJ1-CJ7 promoters (US Registered Patent US7662943B2), the lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Registered Patent US10584338B2), O2 promoter (US Registered Patent US10273491B2), tkt promoter, and yccA promoter.

[0039] The nucleotide sequence modification encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide (as described in 3) above) may, for example, involve substitution with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0040] The modifications of the amino acid sequence or polynucleotide sequence described in 4) and 5) above may be, but are not limited to, deletion, insertion, non-conservative or conservative substitution or combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, or replacement with an improved amino acid sequence or polynucleotide sequence to have stronger activity or an improved amino acid sequence or polynucleotide sequence to increase activity. Specifically, such replacement is carried out by inserting a polynucleotide into the chromosome by homologous recombination, but is not limited to this. The vector used in this case may additionally include a selection marker to confirm the presence or absence of a chromosomal insertion.

[0041] The introduction of a foreign polynucleotide exhibiting polypeptide activity (6) above may be the introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same or similar activity as the polypeptide into the host cell. The foreign polynucleotide is not restricted in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be appropriately selected by a person skilled in the art using a known transformation method, and the introduction of the polynucleotide into the host cell can generate the polypeptide and increase its activity.

[0042] The codon optimization of the polynucleotide encoding the polypeptide described in 7) above may be codon optimization of the endogenous polynucleotide so that transcription or translation increases in the host cell, or optimization of the codon of the exogenous polynucleotide so that optimized transcription or translation occurs in the host cell.

[0043] 8) Analyzing the tertiary structure of a polypeptide and selecting exposed sites for modification or chemical modification may, for example, involve comparing the sequence information of the polypeptide to be analyzed with a database containing sequence information of proteins, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on this, and selecting exposed sites to be modified or chemically modified.

[0044] Such enhancement of polypeptide activity may, but is not limited to, an increase in the activity or concentration expression level of the corresponding polypeptide relative to the activity or concentration expressed in the wild-type or pre-modified microbial strain, or an increase in the amount of product produced from the polypeptide.

[0045] Modification of part or all of a polynucleotide in the microorganism of this application (e.g., modification to encode the protein variant described above) may be induced by (a) homologous recombination using a chromosome insertion vector within the microorganism or genome editing using gene scissors (engineered nuclease, e.g., CRISPR-Cas9), and / or (b) light and / or chemical treatment such as ultraviolet light and radiation. Methods for modifying part or all of the gene may include methods using DNA recombination techniques. For example, deletion of part or all of the gene may be achieved by injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination. The injected nucleotide sequence or vector may, but is not limited to, contain a dominant selection marker.

[0046] In one example, the weakening of the polypeptide (or protein, e.g., LacI-type DNA-binding transcription factor protein; hereinafter referred to as polypeptide) may be caused by the recombination method. The recombination method may include homologous recombination. The homologous recombination method can induce homologous recombination between the sequence of the polypeptide and an endogenous gene in the microorganism by transforming the microorganism with a vector containing a portion of the sequence of the gene encoding the polypeptide and culturing it in the presence of a selection marker product.

[0047] The microorganisms of this application may be, but are not limited to, microorganisms in which the LacI-type DNA-binding transcription factor protein or the polynucleotide encoding it is inactivated or weakened; or microorganisms genetically modified by a vector to inactivate or weaken the LacI-type DNA-binding transcription factor protein or the polynucleotide encoding it (e.g., recombinant microorganisms).

[0048] The microorganisms (or strains, recombinant cells) of this application may be microorganisms that have the ability to produce L-glutamate or have improved L-glutamate production ability (or production volume).

[0049] The microorganisms of this application may be, but are not limited to, microorganisms that naturally possess L-glutamate production ability, or microorganisms in which the activity of LacI-type DNA-binding transcription factor proteins is weakened and / or L-glutamate production ability is conferred or improved from a parent strain that lacks L-glutamate production ability.

[0050] The statement that the microorganism (or strain, recombinant cell) has improved L-glutamate production ability (or production volume) or possesses L-glutamate production ability means that the microorganism (or strain, recombinant cell) has improved L-glutamate production ability compared to the unmodified microorganism, pre-recombination cells, parent strain, and / or wild-type strain, or that it has been given L-glutamate production ability, unlike the unmodified microorganism, pre-recombination cells, parent strain, and / or wild-type strain that lack L-glutamate production ability.

[0051] For example, a microorganism in which the activity of a LacI-type DNA-binding transcription factor protein is weakened may have improved (increased) L-glutamate production capacity compared to an unmodified microorganism of the same species. In this application, "unmodified microorganism" may mean a wild-type strain or a naturally occurring strain itself, or a strain before its characteristics are altered by genetic mutations due to natural or artificial factors, rather than excluding strains containing spontaneously occurring mutations in microorganisms. For example, the unmodified microorganism may mean, for example, a strain in which the activity of a LacI-type DNA-binding transcription factor protein is not weakened or is not weakened (or a strain in which a mutation that induces weakening of the activity of a LacI-type DNA-binding transcription factor protein is not introduced or is not introduced). The term "unmodified microorganism" is interchangeable with "pre-modification strain," "pre-modification microorganism," "non-mutant strain," "unmodified strain," "non-mutant microorganism," or "reference microorganism." The weakening of the activity of the LacI-type DNA-binding transcription factor protein is as described above. In one example, the unmodified microorganism used as the target strain for comparing the presence or absence of the increase in L-glutamic acid production capacity may be, but is not limited to, the Corynebacterium glutamicum ATCC13032 strain, Corynebacterium glutamicum ATCC13869 strain, Corynebacterium glutamicum ATCC14067 strain, a strain in which the odhA gene is deleted in the wild type of Corynebacterium glutamicum (for example, the ATCC13869△odhA strain), or the Corynebacterium glutamicum BL2 strain (KFCC11074, Korean Registered Patent No. 10-0292299), which is known as an L-glutamic acid-producing NTG mutant strain.

[0052] The microorganism (or strain, recombinant cell) may contain mutations that further increase L-glutamate production, and the location of the mutation and / or the gene and / or protein to which it is mutated are not limited as long as they increase L-glutamate production. The recombinant cell may be any cell capable of transformation.

[0053] As an example, the microorganism (or strain, recombinant cell) with improved (increased) production capacity (or production volume) is approximately 1% or more, approximately 2.5% or more, approximately 5% or more, approximately 6% or more, approximately 7% or more, approximately 8% or more, approximately 9% or more, approximately 10% or more, approximately 10.5% or more, approximately 11% or more, approximately 11.5% or more, approximately 12% or more, and approximately 1% or more compared to the pre-mutation parent strain or unmodified microorganism. 2.5% or more, approximately 13% or more, approximately 13.5% or more, approximately 14% or more, approximately 14.5% or more, approximately 15% or more, approximately 15.5% or more, approximately 16% or more, approximately 16.5% or more, approximately 17% or more, approximately 17.4% or more, approximately 17.5% or more, approximately 18% or more, approximately 18.5% or more, approximately 19% or more, approximately 19.5% or more, approximately 20% or more, approximately 20.5% or more, approximately 21% or more, approximately 21.1% or more, approximately 21.5% % or more, approximately 21.5% or more, approximately 22% or more, approximately 22.5% or more, approximately 23% or more, approximately 23.5% or more, approximately 24% or more, approximately 24.5% or more, approximately 25% or more, approximately 25.5% or more, approximately 26% or more, approximately 26.5% or more, approximately 27% or more, approximately 27.5% or more, approximately 28% or more, approximately 28.5% or more, approximately 29% or more, approximately 29.5% or more, approximately 30% or more, approximately 31% or more, approximately 32% or more, approximately 33 The increase may be % or more, approximately 34% or more, or approximately 35% or more (the upper limit is not specifically restricted and may be, for example, approximately 200% or less, approximately 150% or less, approximately 100% or less, approximately 50% or less, approximately 45% or less, approximately 40% or less, or approximately 35% or less), or in one example, an increase of approximately 17.2% or more, approximately 21.4% or more, or approximately 21.6% or more. In other examples, the microorganism (or strain, recombinant cell) with increased production capacity (or production volume) may have increased L-glutamic acid production capacity (or production volume) by approximately 1.1 times or more, approximately 1.12 times or more, approximately 1.13 times or more, 1.15 times or more, 1.16 times or more, 1.17 times or more, 1.18 times or more, 1.19 times or more, approximately 1.2 times or more, approximately 1.21 times or more, approximately 1.22 times or more, 1.25 times or more, or approximately 1.3 times or more (there are no special restrictions on the upper limit, for example, it may be approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, or approximately 2 times or less), or in one example, it may have increased by approximately 1.172 times or more, approximately 1.214 times or more, or approximately 1.216 times or more.More specifically, the recombinant strain with increased production capacity (or production volume) may have an L-glutamic acid production capacity of approximately 17.2%, 21.4%, or 21.6% (or approximately 1.17 times, 1.21 times, or 1.22 times) compared to the pre-mutation parent strain or the unmodified microorganism, but is not limited to this. The term "about" includes, but is not limited to, all numerical values ​​within a range equivalent to or similar to the numerical value that follows the term, such as ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc.

[0054] In one example, the Corynebacterium sp. microorganisms include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, and Corynebacterium striatum. It may also be Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, and / or Corynebacterium flavescens.

[0055] As another example, the recombinant microorganisms of this application may be microorganisms in which the activity of some proteins in the L-glutamate biosynthesis pathway is additionally enhanced, or the activity of some proteins in the L-glutamate degradation pathway is additionally inactivated, thereby enhancing L-glutamate production capacity.

[0056] Specifically, the microorganisms of this application may be microorganisms in which the OdhA protein is additionally inactivated or in which the odhA gene is additionally deleted. More specifically, the microorganisms of this application may be Corynebacterium glutamicum in which the OdhA protein is inactivated with Corynebacterium glutamicum ATCC13869, or microorganisms in which the odhA gene is deleted with Corynebacterium glutamicum ATCC13869. The OdhA protein may include the amino acid sequence of NCBISequenceIDWP_060564343.1 (for example, the amino acid sequence of SEQ ID NO: 23). The OdhA protein may be a protein having the activity of a multifunctional oxoglutarate decarboxylase / oxoglutarate dehydrogenase thiamine pyrophosphate-binding subunit / dihydrolipoyllysine-residue succinyl transferase subunit derived from the Corynebacterium glutamicum strain. The OdhA gene may be derived from the Corynebacterium glutamicum ATCC13869 strain, and more specifically, may contain the nucleic acid sequence of Sequence ID 24 (the sequence from positions 1,276,170 to 1,279,787 in the nucleic acid sequence of GenBank Accession No. Sequence ID: CP016335.1, for example, the BBD29_06050 gene).

[0057] However, the inactivation of the OdhA protein or deletion of the OdhA gene is just one example, and is not limited thereto. The microorganisms of this application may also be microorganisms in which the activity of proteins in various known L-glutamate biosynthesis pathways is enhanced, or the activity of proteins in degradation pathways is inactivated or weakened.

[0058] Another aspect of this application provides a method for producing L-amino acids, comprising the step of culturing the microorganism of this application in a culture medium.

[0059] The method for producing L-amino acids described in this application may include the step of culturing the microorganism described in this application in a culture medium. The microorganism described in this application is as described above.

[0060] In addition, the L-amino acid in this application may be L-glutamic acid.

[0061] In this application, “cultivation” means growing the Corynebacterium glutamicum strain of this application under appropriately controlled environmental conditions. The cultivation process of this application is carried out using suitable culture media and cultivation conditions known in the art. Such a cultivation process can be easily adapted and used by those skilled in the art depending on the selected strain. Specifically, the cultivation may be batch, continuous, and / or fed-batch.

[0062] In this application, "culture medium" refers to a mixture mainly composed of nutrients necessary for culturing the Corynebacterium glutamicum strain of this application, supplying nutrients and growth factors, including water, which is essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the Corynebacterium glutamicum strain of this application can be any culture medium used for culturing ordinary microorganisms without any special restrictions. However, the Corynebacterium glutamicum strain of this application can be cultured in an ordinary culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, under aerobic conditions, while adjusting the temperature, pH, etc.

[0063] Specifically, the culture medium for Corynebacterium strains can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0064] In this application, the carbon sources include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, sugarcane meal, and corn maceration can also be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and other appropriate amounts of carbon sources can be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0065] The nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptones, NZ-amines, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their decomposition products, defatted soy cake or its decomposition products. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0066] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or their corresponding sodium-containing salts. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate, as well as amino acids, vitamins, and / or suitable precursors. These components or precursors are added to the culture medium in batches or continuously, but are not limited to these methods.

[0067] Furthermore, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture medium in an appropriate manner during the culture of the Corynebacterium strain of this application. In addition, the generation of bubbles can be suppressed during culture by using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, oxygen or oxygen-containing gas can be injected into the culture medium to maintain an aerobic state, or anaerobic and non-aerobic states can be maintained without gas injection, or by injection of nitrogen, hydrogen, or carbon dioxide gas, but is not limited to these.

[0068] In the culture described in this application, the culture temperature can be maintained at 20-45°C, specifically 25-40°C, and the culture can be performed for approximately 10-160 hours, but is not limited to this.

[0069] The L-amino acids (e.g., L-glutamic acid) produced by the culture method described in this application can be secreted into the culture medium or remain within the cells.

[0070] The method for producing L-amino acids of this application may additionally include, for example, the step of preparing the microorganism (strain) of this application, the step of preparing a culture medium for culturing the microorganism, or a combination thereof (in any order), before the culturing step.

[0071] The method for producing L-amino acids according to this application may additionally include a step of recovering L-amino acids from the culture medium (the culture medium in which the culture was performed) or microorganisms (Corynebacterium strains). The recovery step is additionally included after the culture step.

[0072] The aforementioned recovery may involve collecting the target L-amino acids using a suitable method known in the art, such as a batch, continuous, or fed-batch culture method for culturing microorganisms as described in this application. For example, various chromatography methods such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination of these methods can be used to recover the target L-amino acids from the culture medium or microorganisms using a suitable method known in the art.

[0073] Furthermore, the method for producing L-amino acids of this application may additionally include a purification step. The purification can be carried out using a suitable method known in the art. For example, if the method for producing L-amino acids of this application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out sequentially or discontinuously, in any order, simultaneously, or integrated into a single step.

[0074] Another aspect of this application provides a composition for producing L-amino acids (e.g., L-glutamic acid) comprising the microorganism of this application; a culture medium in which the microorganism is cultured; or two or more combinations thereof.

[0075] The compositions of this application may further contain any suitable excipients commonly used in compositions for amino acid production, such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0076] In the composition of this application, the microorganism (bacterial strain), culture medium, and L-amino acids are as described in the other embodiments described above.

[0077] Another aspect of this application provides uses for the production of L-amino acids (e.g., L-glutamic acid) of the microorganism of this application; culture media in which the microorganism is cultured; or two or more combinations thereof.

[0078] Another aspect of this application provides uses for the microorganisms of this application; culture media in which they are cultured; or combinations of two or more thereof for use in the production of compositions for the production of L-amino acids (e.g., L-glutamic acid). [Effects of the Invention]

[0079] Microorganisms of the genus Corynebacterium in which the activity of the LacI family DNA-binding transcriptional regulator described in this application is weakened exhibit a significant increase in L-glutamic acid production capacity. Using these microorganisms, it is possible to produce L-glutamic acid in a higher yield compared to existing microorganisms. [Modes for carrying out the invention]

[0080] The present application will be described in more detail below with reference to examples. However, the following examples are merely preferred embodiments for illustrating the present application and are not intended to limit the scope of the rights of this application. On the other hand, technical matters not described herein can be easily understood and implemented by an ordinary person skilled in the art of this application or a similar art.

[0081] Example 1: Vector construction for the expression of LacI-type DNA-binding transcription factor protein mutants in microorganisms

[0082] In this example, to confirm the effect of a mutant (Q310*; sequence of sequence number 1, 309) in which the corresponding codon of glutamine (Gln, Q) at position 310 of the amino acid sequence of the LacI-type DNA-binding transcription factor protein (amino acid sequence of sequence number 3) is replaced with a stop codon (*), a vector for creating an expressing strain was prepared as described below.

[0083] Using the gDNA (genomic DNA) of wild-type Corynebacterium glutamicum ATCC13869 as a template, PCR was performed using primer pairs for the sequences described in SEQ ID NOs. 5 and 6, and primer pairs for the sequences described in SEQ ID NOs. 7 and 8, respectively. Using a mixture of the two resulting fragments as a template, overlapping PCR was performed again using primer pairs for the sequences of SEQ ID NOs. 5 and 8 to obtain another fragment. The polymerase used was Solg TM Using Pfu-X DNA polymerase, PCR was performed by denaturing at 95°C for 5 minutes, followed by denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute 30 seconds, repeated 30 times, and then polymerization reaction at 72°C for 5 minutes.

[0084] The amplified gene fragment and the chromosome transformation vector pDCM2 (Republic of Korea Publication No. 10-2020-0136813), which had been cut with SmaI restriction enzyme, were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix). Cloning was performed by mixing the Gibson assembly reagent with each gene fragment in the calculated moles and then storing at 50°C for 1 hour. The bacterial strain was streaked onto LB solid medium containing kanamycin (25 mg / l). After selecting colonies with the target gene inserted, the vector was obtained using a commonly known plasmid (vector) extraction method. The vector was named pDCM2-BBD29_06680(Q310*). The sequences of the primers used in this example are shown in Table 1 below.

[0085] [Table 1]

[0086] Example 2: Preparation of L-glutamic acid-producing strains from wild-type Corynebacterium glutamicum and introduction of LacI-type DNA-binding transcription factor protein mutants.

[0087] Example 2-1: Preparation of a Corynebacterium glutamicum strain with L-glutamic acid production ability derived from wild-type Corynebacterium glutamicum.

[0088] To create a strain of Corynebacterium glutamicum ATCC13869 capable of producing L-glutamic acid, we created a Corynebacterium glutamicum ATCC13869△odhA strain lacking the odhA gene (GenBank Accession No. WP_060564343.1, SEQ ID NO: 24) based on prior literature (Appl Environ Microbiol. 2007 Feb;73(4):1308-19. Epub 2006 Dec 8.).

[0089] Specifically, using Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template due to the odhA deletion, the upstream and downstream regions of the odhA gene were obtained by PCR using primer pairs of SEQ ID NOs. 17 and 18, and primer pairs of SEQ ID NOs. 19 and 20, respectively. The polymerase used was Solg. TM Using Pfu-XDNA polymerase, the PCR amplification conditions were as follows: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes.

[0090] A recombinant vector, named pDCM2-△odhA, was obtained by cloning the amplified odhA upstream and downstream regions, along with the chromosome transformation vector pDCM2 cleaved with SmaI restriction enzyme, using the Gibson assembly method. Cloning was performed by mixing the Gibson assembly reagent with each gene fragment in the calculated moles and then storing the mixture at 50°C for 1 hour.

[0091] The prepared pDCM2-△odhA vector was used to transform Corynebacterium glutamicum ATCC13869 strain by electroporation. Following a secondary cross-reaction, a strain with a deletion of the odhA gene on the chromosome was obtained. The presence or absence of the odhA gene deletion was confirmed by PCR and genome sequencing using sequence numbers 21 and 22. The resulting strain was named ATCC13869△odhA. The primer sequences used in this example are shown in Table 2 below.

[0092] [Table 2]

[0093] Example 2-2: Preparation of bacterial strains introducing LacI-type DNA-binding transcription factor protein mutants

[0094] The vector pDCM2-BBD29_06680(Q310*) prepared in Example 1 was transformed into ATCC13869△odhA prepared in Example 2-1 by electroporation. After secondary cross-reactivity, a strain with the BBD29_06680(Q310*) mutation introduced on the chromosome was obtained. The strain with the BBD29_06680(Q310*) mutation was confirmed by PCR and genome sequencing using SEQ ID NOs. 9 and 10, and the resulting strain was named CA02-1626. The CA02-1626 strain was named Corynebacterium glutamicumCA02-1626 and deposited with the Korean Culture of Microorganisms (KCCM) under the Budapest Convention on January 18, 2021, with deposit number KCCM12930P.

[0095] The primer sequences used in this example are shown in Table 3 below.

[0096] [Table 3]

[0097] Examples 2-3: Comparison of L-glutamate production capacity of bacterial strains expressing LacI-type DNA-binding transcription factor protein mutants.

[0098] The L-glutamic acid production ability of the bacterial strains prepared in section 2-2 above was to be confirmed using the ATCC13869△odhA strain as a control group. The control group and the CA02-1626 strain were cultured using the following method.

[0099] Each bacterial strain was inoculated into a 250 ml corner baffle flask containing 25 ml of seed medium and cultured with shaking at 30°C for 20 hours at 200 rpm. Subsequently, 1 ml of seed culture solution was inoculated into a 250 ml corner baffle flask containing 25 ml of production medium and cultured with shaking at 30°C for 40 hours at 200 rpm. After the culture period, the amount of L-glutamic acid produced was measured using high-performance liquid chromatography (HPLC), and the results are shown in Table 4 below.

[0100] <Seedling medium> Glucose 1%, meat juice 0.5%, polypeptone 1%, sodium chloride 0.25%, yeast extract 0.5%, urea 0.2%, pH 7.2

[0101] <Production culture medium> Raw sugar 6%, calcium carbonate 5%, ammonium sulfate 2.25%, potassium monophosphate 0.1%, magnesium sulfate 0.04%, ferrous sulfate 10 mg / L, thiamine hydrochloride 0.2 mg / L, biotin 50 μg / L

[0102] [Table 4]

[0103] As shown in Table 4 above, we confirmed that the L-glutamic acid concentration increased by approximately 21.6% in the CA02-1626 strain, which was introduced with the BBD29_06680(Q310*) mutation, compared to the control group's ATCC13869△odhA strain.

[0104] Example 3: Preparation of LacI-type DNA-binding transcription factor protein-deficient bacterial strains and measurement of L-glutamate production capacity

[0105] Example 3-1: Preparation of a LacI-type DNA-binding transcription factor gene deletion vector

[0106] In the above example, it was confirmed that L-glutamic acid production improved when the corresponding codon of glutamine (Gln, Q), the 310th amino acid of the LacI-type DNA-binding transcription factor protein, was replaced with a stop codon. Therefore, in this example, we aimed to confirm the effect of deletion of the LacI-type DNA-binding transcription factor (BBD29_06680) gene on L-glutamic acid production.

[0107] Specifically, due to the BBD29_06680 deletion, the upstream and downstream regions of the BBD29_06680 gene were obtained by PCR using gDNA (genomic DNA) of Corynebacterium glutamicum ATCC13869 as a template, and primer pairs for sequences described in SEQ ID NOs. 11 and 12, and SEQ ID NOs. 13 and 14, respectively. The polymerase used was Solg. TM Using Pfu-X DNA polymerase, PCR amplification was performed under the following conditions: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes. The amplified DNA fragments were cloned using the Gibson assembly method with the chromosome transformation vector pDCM2, which had been cut with SmaI restriction enzyme, to obtain a recombinant vector named pDCM2-△BBD29_06680. Cloning was performed by mixing the Gibson assembly reagent with each gene fragment in the calculated moles and then storing at 50°C for 1 hour.

[0108] The primer sequences used in this example are shown in Table 5 below.

[0109] [Table 5]

[0110] Example 3-2: Preparation of bacterial strains lacking LacI-type DNA-binding transcription factor proteins

[0111] The vector pDCM2-△BBD29_06680 prepared in Example 3-1 was used to transform ATCC13869△odhA prepared in Example 2-1 by electroporation. After a secondary crossover, a strain with a deletion of the BBD29_06680 gene on the chromosome was obtained, which was confirmed by PCR and genome sequencing using primer pairs of SEQ ID NOs. 15 and 16. The selected strain was named CA02-1627. The sequences of the primers used in this example are listed in Table 6 below.

[0112] [Table 6]

[0113] Example 3-3: Measurement of L-glutamate production capacity of LacI-type DNA-binding transcription factor protein-deficient bacterial strains

[0114] Using the ATCC13869△odhA strain prepared in Example 2-1 as a control group, the L-glutamic acid production ability of the CA02-1627 strain was evaluated using the fermentation titer evaluation method of Example 2-3. After the completion of cultivation, the amount of L-glutamic acid produced was measured using high-performance liquid chromatography (HPLC), and the measurement results are shown in Table 7 below.

[0115] [Table 7]

[0116] As shown in Table 7 above, we confirmed that the concentration of L-glutamic acid increased by approximately 21.4% in CA02-1627, which lacked the BBD29_06680 gene, compared to the control group ATCC13869△odhA strain.

[0117] Example 4: Preparation of bacterial strains into which LacI-type DNA-binding transcription factor protein mutants derived from NTG mutants have been introduced, and measurement of L-glutamic acid production capacity.

[0118] To confirm whether the BBD29_06680(Q310*) mutant exhibits the same effect in strains of Corynebacterium genus with increased L-glutamic acid production capacity (NTG (N-Methyl-N'-nitro-N-nitrosoguanidine) mutants), the mutant was introduced into Corynebacterium glutamicum BL2 strain (KFCC11074, Korean Patent No. 10-0292299), which is known as an L-glutamic acid-producing NTG mutant strain.

[0119] The pDCM2-BBD29_06680(Q310*) vector prepared in Example 1 was used to transform the KFCC11074 strain by electroporation. After secondary cross-reactivity, strains with the BBD29_06680(Q310*) mutant introduced onto the chromosome were selected. This was confirmed by PCR and genome sequencing using SEQ ID NOs. 9 and 10, and the resulting strain was named CA02-1630.

[0120] Fermentation titer experiments were conducted using the prepared CA02-1630 and Corynebacterium glutamicum KFCC11074 strains according to the following specified method.

[0121] Each bacterial strain was inoculated into a 250 ml corner baffle flask containing 25 ml of seed medium and cultured with shaking at 30°C for 20 hours at 200 rpm. Subsequently, 1 ml of seed culture solution was inoculated into a 250 ml corner baffle flask containing 25 ml of production medium and cultured with shaking at 30°C for 40 hours at 200 rpm. After the culture period, the amount of L-glutamic acid produced was measured using HPLC, and the results are shown in Table 8 below.

[0122] <Seedling medium> Glucose 1%, meat juice 0.5%, polypeptone 1%, sodium chloride 0.25%, yeast extract 0.5%, urea 0.2%, pH 7.2

[0123] <Production culture medium> Raw sugar 6%, calcium carbonate 5%, ammonium sulfate 2.25%, potassium monophosphate 0.1%, magnesium sulfate 0.04%, ferrous sulfate 10 mg / L, thiamine hydrochloride 0.2 mg / L, biotin 500 μg / L

[0124] [Table 8]

[0125] As shown in Table 8 above, the CA02-1630 strain was found to have an approximately 17.2% increase in L-glutamic acid concentration compared to the control group KFCC11074 strain.

[0126] From the above description, a person skilled in the art will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, the embodiments described above should be understood to be illustrative and not limiting in all respects. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, which are described below in more detail, and their equivalent concepts.

[0127] [Accession Number] Depository name: Korea Microbial Conservation Center Accession number: KCCM12930P Date of acceptance: 20210118 JPEG0007863608000009.jpg240169

Claims

1. A method for producing L-glutamic acid, comprising the step of culturing a Corynebacterium microorganism in which the activity of LacI-type DNA-binding transcription factor proteins is weakened in a culture medium, Here, the Corynebacterium microorganisms exhibit increased L-glutamate production compared to parent strains or wild-type strains in which the activity of LacI-type DNA-binding transcription factor proteins is not weakened. The LacI-type DNA-binding transcription factor protein contains the amino acid sequence of SEQ ID NO: 3, The weakened activity of the LacI-type DNA-binding transcription factor protein is (1) Deletion of a gene encoding a LacI-type DNA-binding transcription factor protein, 、 (2) The codon corresponding to the 310th amino acid in the amino acid sequence of SEQ ID NO: 3 is mutated and terminates A method of producing L-glutamic acid by forming stop codons.

2. The method for producing L-glutamic acid according to claim 1, further comprising the step of recovering L-glutamic acid from the culture medium or microorganisms obtained by the culture.

3. The method for producing L-glutamic acid according to claim 1, wherein the LacI-type DNA-binding transcription factor protein is derived from Corynebacterium glutamicum.

4. The method for producing L-glutamic acid according to claim 1, wherein the LacI-type DNA-binding transcription factor protein is encoded by the polynucleotide described in the base sequence of Sequence ID No.

4.

5. The method for producing L-glutamic acid according to claim 1, wherein the Corynebacterium microorganism is Corynebacterium glutamicum.

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