A bacterial strain for producing high concentrations of L-glutamic acid and a method for producing L-glutamic acid using the same.

Abstract

The present application relates to strains and microorganisms for producing high concentrations of L-glutamic acid and methods of using same.

Description

Technical Field

[0001] The present disclosure relates to a strain for producing high-concentration L-glutamic acid and a method for producing L-glutamic acid using the same.

Background Art

[0002] L-glutamic acid is a typical amino acid produced by fermentation, has a unique taste, and is one of the important amino acids widely used in the food field, as well as in the pharmaceutical field and other animal feed fields. L-glutamic acid can be produced using microorganisms such as the genus Corynebacterium, Escherichia coli, Bacillus, Streptomyces, the genus Penicillum, Klebsiella, Erwinia, or Pantoea (U.S. Patent No. 3,220,929, U.S. Patent No. 6,682,912).

[0003] Currently, various studies are being conducted for the development of microorganisms for highly efficient production of L-glutamic acid and fermentation process technologies. For example, a specific approach to increasing the expression of genes encoding enzymes involved in amino acid biosynthesis in microorganisms or removing genes unnecessary for amino acid biosynthesis is mainly used for improving the production yield of L-glutamic acid.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

[0005] [Non-licensed Document 1] Pearson et al (1988)[Proc. Natl. Acad. Sci. USA 85]:2444 [Non-licensed Document 2] Rice et al., 2000, Trends Genet. 16:276-277 [Non-licensed Document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453 [Non-licensed Document 4] Devereux,J.,et al,Nucleic Acids Research 12:387 (1984) [Non-licensed Document 5] Atschul,[S.] [F.,] [ET AL,J MOLEC BIOL 215]:403 (1990) [Non-licensed Document 6] Guide to Huge Computers,Martin J. Bishop,[ED.,] Academic Press,San Diego, 1994 [Non-licensed Document 7] [CARILLO ETA / . ](1988) SIAM J Applied Math 48:1073 [Non-licensed Document 8] Smith and Waterman,Adv. Appl. Math (1981) 2:482 [Non-licensed Document 9] Schwartz and Dayhoff, eds., Atlas Of Protein Allocation And Structure, National Biomedical Research Foundation, pp. 353-358 (1979)

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Summary of the Invention

Problems to be Solved by the Invention

[0006] One object of the present application is to provide a microorganism, wherein the activity of a protein containing a sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1 is weakened as compared with the inherent activity of the microorganism. Another object of the present application is to provide a microorganism that expresses a recombinant protein containing an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0007] Another object of the present application is to provide a method for producing L-glutamic acid, which includes the step of culturing the microorganism of the present invention in a medium.

[0008] Another object of the present application is to provide a composition for producing L-glutamic acid, which includes the microorganism of the present disclosure; a medium in which the microorganism has been cultured; or a combination thereof.

[0009] Another object of this application is to provide a method for producing a microorganism, comprising the step of weakening the activity of the protein of the present disclosure. In some embodiments, the microorganism is Corynebacterium.

[0010] Another object of this disclosure is to provide a method for increasing L-glutamic acid production by microorganisms, the method comprising the step of weakening the activity of a protein in the microorganism that contains an amino acid sequence having at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1. [Means for solving the problem]

[0011] Each description and embodiment disclosed in this application is applicable to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the categories of this application are not considered to be limited by the specific descriptions set forth below. Moreover, a person with ordinary skill in the art can recognize or confirm many equivalents of the specific aspects of this application described herein by ordinary experimentation alone. Such equivalents are intended to be included in this application.

[0012] One aspect of the present disclosure provides a microorganism in which the activity of a protein comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1 is attenuated. Another aspect of the present disclosure provides a microorganism expressing a recombinant protein comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the microorganism belongs to the genus Corynebacterium.

[0013] Proteins according to some embodiments of this disclosure may have, contain, consist of, or essentially consist of the amino acid sequence described in SEQ ID NO: 1. Specifically, proteins according to some embodiments of this disclosure may consist of the polypeptide described in the amino acid sequence of SEQ ID NO: 1.

[0014] The amino acid sequence of Sequence ID No. 1 can be obtained from the well-known database, the NIH GenBank. In this disclosure, the amino acid sequence of Sequence ID No. 1 may include amino acid sequences that are homologous or identical to the amino acid sequence described in Sequence ID No. 1 by at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 9%, 93%, 94%, 95%, 95.18%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9%. In some embodiments, the microbial protein may be excluded from Sequence ID No. 1. Furthermore, it is obvious that any amino acid sequence having such homology or identity and exhibiting efficacy corresponding to a protein containing the amino acid sequence of Sequence ID No. 1 is also included within the scope of this disclosure, even if it has an amino acid sequence in which some of the sequences are deleted, modified, substituted, conserved substituted, or added.

[0015] For example, the amino acid sequence may have additions or deletions of sequences that do not alter the function of the protein, such as spontaneous mutations, silent mutations, or conservative substitutions, at the N-terminus, C-terminus, and / or within the sequence, as embodied in part of this disclosure.

[0016] The term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions 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.

[0017] In this application, the terms "homology" and "identity" refer to the degree of similarity between two given amino acid sequences or base sequences, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0018] The sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequencing algorithms, and a default gap penalty established by the program used may be applied. Substantially homologous or identical sequences can generally be hybridized with all or part of the sequence under moderate to high stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing codons, or codons considering codon degeneracy in general, in polynucleotides.

[0019] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms such as the "FASTA" program with default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined 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.] [ET AL, J MOLEC BIOL 215]):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.

[0020] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443, as is publicly known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as the total number of symbols in the shorter of two sequences divided by the number of similarly sequenced symbols (i.e., nucleotides or amino acids). 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 NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas of Protein Sequences 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.

[0021] The proteins in some embodiments of this disclosure may be derived from microorganisms. In some embodiments, the microorganisms may be Corynebacterium, Escherichia coli, Bacillus subtilis, Actinomycetes, Penicillium, Klebsiella, Erwinia, or Pantoea. Specifically, the microorganisms may be derived from microorganisms of the genus Corynebacterium, and more specifically, from Corynebacterium glutamicum, Corynebacterium deserti, Corynebacterium crenatum, Corynebacterium efficiens, Corynebacterium suranareeae, etc.

[0022] The protein inactivated by the microorganisms provided in this disclosure is shown in Sequence ID No. 1, but sequences having a similar structure and exhibiting similar activity may be included without limitation.

[0023] For example, the proteins inactivated by the microorganisms of this disclosure include proteins of the genus Corynebacterium having 70% or more identity with SEQ ID NO: 1. Specifically, the proteins may be proteins of Corynebacterium glutamicum having 99% or more identity with SEQ ID NO: 1, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% or more identity. An example of this is the NCBI accession number. WP_060565206.1, QWQ85246.1, WP_065367112.1, WP_038585884.1, WP_040072989.1, WP_0112659 91.1, WP_074492394.1, ARV66101.1, WP_003853812.1, WP_006285921.1, WP_003862956.1, WP_04 Examples of proteins include, but are not limited to, 4027349.1, BAF55605.1, WP_179205783.1, WP_172769145.1, WP_211439424.1, WP_179111498.1, WP_059289862.1, WP_185969420.1, WP_173673681.1, and QDQ21801.1.

[0024] A polynucleotide encoding a protein according to a partial embodiment of this disclosure may include a nucleotide sequence encoding the amino acid sequence described in Sequence ID No. 1. For example, a polynucleotide according to a partial embodiment of this disclosure may be a polynucleotide sequence having a locus tag such as FOL53_14395, B7P23_15040, KaCgl_12360, B5C28_13285, cgR_2591, or BBD29_13140 from the genus Corynebacterium, and may, but is not limited to, BBD29_13140 from Corynebacterium glutamicum ATCC13869. BBD29_13140 may have, contain, consist of, or be essential to the nucleotide sequence described in Sequence ID No. 2.

[0025] In this application, the term "polynucleotide" refers to a polymer of nucleotides in which nucleotide units (monomers) are linked together in a long chain by covalent bonds, and is a DNA or RNA chain of a certain length or longer, and more specifically, a polynucleotide fragment that codes for the aforementioned protein.

[0026] Polynucleotides in some embodiments of this disclosure may undergo various modifications to their coding region, taking into account codon degeneracy or preferred codons in organisms intended to express the proteins described herein, without altering the amino acid sequence of the protein.

[0027] Another aspect of this disclosure is to provide a polynucleotide comprising a nucleic acid sequence encoding the protein, wherein the polynucleotide is not naturally occurring.

[0028] In some embodiments, the polynucleotides of the present disclosure have or contain a nucleotide sequence which has 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98% or more homology or identity with the sequence of SEQ ID NO: 2, or which consist of or are required to consist of a nucleotide sequence which has 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98% or more homology or identity with the sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid sequence includes a start codon that does not occur naturally when encoding an amino acid sequence. In some embodiments, the intrinsic gene encoding the amino acid sequence includes an ATG start codon, which is replaced by GTG. In some embodiments, the intrinsic gene encoding the amino acid sequence includes an ATG start codon, which is replaced by TTG. In some embodiments, the nucleic acid sequence includes a Shine-Dalgarno sequence that does not occur naturally when encoding an amino acid sequence. In some embodiments, the intrinsic gene encoding the amino acid sequence includes a Shine-Dalgarno sequence of CTAGATTGG, which is replaced by CAAGGCCGG. In some embodiments, the intrinsic gene encoding an amino acid includes a Shine-Dalgarno sequence of CTAGATTGG, which is replaced by CGTGACAGG. In some embodiments, the intrinsic gene encoding the amino acid sequence includes a Shine-Dalgarno sequence of CTAGATTGG, which is replaced by CTCCGCTGG. In some implementations, the specific gene encoding the amino acid sequence contains the Shine-Dalgano sequence CTAGATTGG, where CTAGATTGG is replaced by GTCGATTTC.

[0029] Furthermore, polynucleotides as embodiments of certain parts of this disclosure are not limited to any probes that can be prepared from known gene sequences, such as sequences that can hybridize under stringent conditions with complementary sequences to all or part of the polynucleotide sequences of this disclosure. "Stringent conditions" means conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, we can list conditions in which polynucleotides with high homology or identity are hybridized, with 70% or more, 75% or more, 76% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity, and polynucleotides with lower homology or identity are not hybridized. Alternatively, we can list conditions in which the polynucleotides are washed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of normal Southern hybridization: 60°C, 1×SSC, 0.1% SDS, more specifically 60°C, 0.1×SSC, 0.1% SDS, or more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0030] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. The term “complementary” is used to describe the relationships between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides described herein may also include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary throughout the entire sequence.

[0031] Specifically, polynucleotides having homology or identity with the polynucleotides described in this application can be detected using hybridization conditions that include a hybridization step at a Tm value of 55°C, and under the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0032] The appropriate stringency for hybridizing the aforementioned polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (e.g., J. Sambrook et al., ibid.).

[0033] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have undergone genetic modification naturally or artificially, and is a microorganism in which a specific mechanism has been weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of the activity of endogenous genes, and may be a microorganism that has undergone genetic modification for the production of a desired polypeptide, protein, or product.

[0034] In this application, the term "weakening" of polypeptide activity refers to a decrease in activity compared to endogenous activity, and this weakening can be used interchangeably with terms such as down-regulation, decrease, reduce, and attenuation.

[0035] On the other hand, in this disclosure, weakened protein activity may mean a state in which the protein is active but not completely deficient and inactivated, and exhibits lower activity compared to the wild type or parent strain.

[0036] The aforementioned weakening may include cases where the activity of the polypeptide itself decreases compared to the polypeptide activity originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide, or where the overall polypeptide activity and / or concentration (expression level) within the cell is lower than that of the natural strain due to inhibition of gene expression of the polynucleotide encoding it or inhibition of translation into the polypeptide. The "intrinsic activity" refers to the activity of a specific polypeptide that was originally possessed by the parent strain, wild type, or non-mutant microorganism before the trait change due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before the trait change." When polypeptide activity is "weakened, reduced, downregulated, decreased, or attenuated" compared to intrinsic activity, it means that it has decreased compared to the activity of a specific polypeptide that was originally possessed by the parent strain or non-mutant microorganism before the trait change.

[0037] The weakening of the activity of such polypeptides can be achieved by any method known in the art, but is not limited to these methods, and can be achieved by applying a variety of well-known methods in the field (e.g., 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.).

[0038] In some embodiments, the weakening of the polypeptide activity of this application is 1) Deletion of a portion of the genes encoding polypeptides; 2) Modification of the gene expression regulatory region (or gene expression regulatory sequence) so that the expression of the gene encoding the polypeptide is reduced; 3) Modification of the amino acid sequence constituting the polypeptide so as to weaken the polypeptide's activity (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence); 4) Modification of the gene sequence encoding the polypeptide so as to weaken the polypeptide's activity (for example, deletion / substitution / addition of one or more nucleic acid bases in the nucleic acid sequence of the polypeptide gene so as to encode a polypeptide that has been modified to weaken the polypeptide's activity); 5) Modifications of the start codon, Shine-Dalgarno sequence, or nucleotide sequence encoding the 5'UTR region of a polypeptide-encoding gene transcript; 6) Introduction of an antisense oligonucleotide (e.g., 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 before the Shine-Dalgarno sequence in a polypeptide-encoding gene to form a secondary structure that cannot be attached to ribosomes; 8) Addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the polypeptide-coding gene sequence (reverse transcription engineering, RTE); or 9) A combination of two or more selected from items 1) to 8) above is also acceptable, but is not particularly limited thereto.

[0039] In some embodiments of this disclosure, at least a portion of the nucleotide sequence encoding the protein is missing compared to the microorganism's own nucleotide sequence.

[0040] In microorganisms of additional embodiments of the present disclosure, the regulatory expression sequence for the gene encoding the protein is not a sequence unique to the microorganism. In some embodiments, the regulatory expression sequence includes at least one deletion, insertion, or substitution of a unique regulatory expression sequence to reduce the expression of the gene encoding the protein. In some embodiments, the regulatory expression sequence includes at least one sequence selected from the group consisting of a promoter sequence, an operator sequence, a sequence encoding a ribosome binding site, a sequence encoding a transcription termination, and a sequence encoding a translation termination. As used herein, “regulatory expression sequence” means a nucleic acid sequence that directs the transcription of a nucleic acid. The regulatory expression sequence may be a polynucleotide sequence that regulates the expression of a polypeptide encoded by a polynucleotide to which it is functionally (“operably) linked.

[0041] In microorganisms as described in additional embodiments of the present disclosure, the amino acid sequence is not the intrinsic amino acid sequence of the microorganism. In some embodiments, the amino acid sequence includes at least one deletion, insertion, or substitution of the intrinsic amino acid sequence to weaken the activity of the protein.

[0042] In microorganisms as described in additional embodiments of the present disclosure, the nucleotide sequence encoding the protein is not a nucleotide sequence unique to the microorganism. In some embodiments, the nucleotide sequence includes at least one deletion, insertion, or substitution of a unique nucleotide sequence, which weakens the activity of the protein.

[0043] In microorganisms of additional embodiments of the present disclosure, the nucleotide sequence for the start codon, the nucleotide sequence for the stop codon, the Shine-Dalgano sequence, and the 5'UTR region of the protein-coding gene are not intrinsic sequences of the microorganism. In some embodiments, the nucleotide sequence for the start codon of the protein-coding gene is mutated compared to an intrinsic sequence of the microorganism. In some embodiments, the nucleotide sequence for the stop codon of the protein-coding gene is mutated compared to an intrinsic sequence of the microorganism. In some embodiments, the intrinsic gene-coding the protein includes an ATG start codon, where ATG is replaced with GTG. In some embodiments, the intrinsic gene-coding the protein includes an ATG start codon, where ATG is replaced with TTG. In some embodiments, the nucleotide sequence for the Shine-Dalgano of the protein-coding gene is mutated compared to an intrinsic sequence of the microorganism. In some embodiments, the intrinsic gene-coding the protein includes a CTAGATTGG Shine-Dalgano sequence, where CTAGATTGG is replaced with CAAGGCCGG. In some embodiments, the unique gene encoding the protein includes the Shine-Dalgano sequence CTAGATTGG, where CTAGATTGG is replaced by CGTGACAGG. In some embodiments, the unique gene encoding the protein includes the Shine-Dalgano sequence CTAGATTGG, where CTAGATTGG is replaced by CTCCGCTGG. In some embodiments, the unique gene encoding the protein includes the Shine-Dalgano sequence CTAGATTGG, where CTAGATTGG is replaced by GTCGATTTC.

[0044] In a microorganism according to an additional embodiment of the present disclosure, the microorganism includes an antisense oligonucleotide that is complementaryly bound to a transcript of the gene encoding the protein. In some embodiments, the antisense oligonucleotide includes antisense RNA. In microorganisms of additional embodiments of the present disclosure, a sequence complementary to the Shine-Dalgano sequence of the gene encoding the protein is inserted before the Shine-Dalgano sequence to form a secondary structure. In some embodiments, the secondary structure does not bind to a ribosome and reduce or delay mRNA translation.

[0045] In microorganisms of additional embodiments of the present disclosure, a promoter for reverse transcription (RTE) is inserted at the 3' end of the open reading frame (ORF) of the gene encoding the protein. In some embodiments, the activity of the protein is weakened by the production of an antisense nucleotide molecule complementary to at least a portion of the gene encoding the protein.

[0046] for example, The deletion of a portion of the gene encoding the polypeptide described in 1) above may be replaced with a polynucleotide in which some nucleotides are missing in the polynucleotide encoding the endogenous target polypeptide within the chromosome, or with a marker gene.

[0047] Furthermore, the modification of the expression regulatory region (or expression regulatory sequence) described in 2) above may result in a mutation on the expression regulatory region (or expression regulatory sequence) through 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 that regulates the termination of transcription and decoding.

[0048] The modifications of the amino acid sequence or polynucleotide sequence described in 3) and 4) above may be, but are not limited to, the introduction of mutations in the sequence, 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, by deletion, insertion, non-conservative or conservative substitution or a combination thereof, so as to weaken the activity of the polypeptide. For example, gene expression can be inhibited or weakened by introducing mutations in the polynucleotide sequence to form a termination codon, but are not limited to this.

[0049] The modification of the start codon or the nucleotide sequence encoding the 5'UTR region of the polypeptide-encoding gene transcript described in 5) above may, but is not limited to, substitution with, for example, a nucleotide sequence encoding another start codon with a lower polypeptide expression rate compared to the endogenous start codon.

[0050] 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].

[0051] 7) In order to form a secondary structure that ribosomes cannot attach to, the addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in the polypeptide-encoding gene may make mRNA translation impossible or reduce its rate.

[0052] Furthermore, the addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (Reverse transcription engineering, RTE) may weaken the activity by creating complementary antisense nucleotides in the transcript of the gene encoding the polypeptide.

[0053] In this application, the term “enhancement” of polypeptide activity means that the activity of a polypeptide increases compared to its intrinsic activity. Such enhancement may be 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-mutation activity. “Intrinsic activity” means the activity of a specific polypeptide that was originally present in the parental strain or non-mutant microorganism before the trait change, in cases where the trait has been altered by a genetic mutation due to natural or artificial factors. This may be used interchangeably with “pre-mutation 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 specific polypeptide that was originally present in the parental strain or non-mutant microorganism before the trait change is improved.

[0054] 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's activity has been enhanced can be confirmed by an increase in the polypeptide's activity level, expression level, or the amount of product excreted from the polypeptide.

[0055] The enhancement of the polypeptide activity can be achieved by applying various 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 deformation. 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 this 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.).

[0056] In some embodiments, the enhancement of polypeptide activity in this application is 1) Increase in the intracellular copy number of polynucleotides encoding polypeptides; 2) Replace gene expression regulatory regions on chromosomes encoding polypeptides with potent active sequences; 3) Modifications 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 polypeptide activity; 5) Modification of the polynucleotide sequence encoding the polypeptide so as to enhance polypeptide activity (for example, modification of the polynucleotide sequence of the polypeptide gene so as to encode the modified polypeptide so as to enhance polypeptide activity); 6) Introduction of a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding it; 7) Codon optimization of polynucleotides encoding polypeptides; 8) Analyze the tertiary structure of the polypeptide, select exposed sites to deform or chemically modify; or 9) A combination of two or more selected from items 1) to 8) above is also acceptable, but is not particularly limited thereto.

[0057] In additional concrete examples, 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 can be 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 these methods.

[0058] The replacement of a gene expression regulatory region (or regulatory sequence) on a chromosome encoding a polypeptide with a more potent sequence may, for example, involve generating a sequence mutation by deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacement with a sequence having stronger activity, in order to further enhance the activity of the regulatory region. The regulatory region may include, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences that regulate transcription and decoding termination. For example, the original promoter may be replaced with a potent promoter, but is not limited to these.

[0059] Examples of well-known powerful promoters include, but are not limited to, the CJ1-CJ7 promoters (US Patent No. 7662943 B2), 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 Patent No. 10584338 B2), O2 promoter (US Patent No. 10273491 B2), tkt promoter, and yccA promoter.

[0060] The sequence modification encoding the start codon or 5'UTR region of the polypeptide-encoding gene transcript described in 3) above may, but is not limited to, substitution with a sequence encoding another start codon that has a higher polypeptide expression rate compared to the endogenous start codon.

[0061] 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, thereby causing a sequence mutation, or replacement with an improved amino acid sequence or polynucleotide sequence that has stronger activity or an improved amino acid sequence or polynucleotide sequence that has increased activity, in order to enhance the activity of the polypeptide. Specifically, such replacement can be 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 further include a selection marker for confirming the presence or absence of a chromosomal insertion.

[0062] 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. A person skilled in the art can appropriately select a known transformation method as the method used for the introduction. When the introduced polynucleotide is expressed in the host cell, the polypeptide is produced and its activity increases.

[0063] 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.

[0064] 8) Analyzing the tertiary structure of a polypeptide and selecting exposed sites to deform or chemically modify may, for example, involve comparing the sequence information of the polypeptide to be analyzed with a database containing sequence information of known proteins to determine candidate template proteins according to the degree of sequence similarity, confirming the structure based on that, and selecting exposed sites to deform or chemically modify.

[0065] 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 of the polypeptide expressed in the wild-type or pre-mutation microorganism, or an increase in the amount of product produced from said polypeptide.

[0066] Modification of some or all polynucleotides in microorganisms as embodied in part of this disclosure may be induced by (a) homologous recombination using a chromosome insertion vector within the microorganism or genome editing using an engineered nuclease (e.g., CRISPR-Cas9) and / or (b) light and / or chemical treatment such as ultraviolet light and radiation. Methods for modifying some or all of the genes may include methods using DNA recombination techniques. For example, deletion of some or all of the genes 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 include, but is not limited to, a dominant selection marker.

[0067] Another aspect of this disclosure is to provide a vector comprising the polynucleotide.

[0068] Vectors embodying certain aspects of this disclosure may include a DNA product comprising a polynucleotide sequence encoding the target polypeptide, operably linked to a suitable regulatory region (or regulatory sequence) so as to enable expression of the target polypeptide in a suitable host. The regulatory region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences regulating the termination of transcription and decoding. After being transformed into a suitable host cell, the vector can replicate or function independently of the host genome and can be integrated into the genome itself.

[0069] The vectors used in this disclosure are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0070] As an example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a chromosome insertion vector within a cell. The insertion of the polynucleotide into the chromosome may be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker may further be included to confirm the presence or absence of the chromosome insertion. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the target nucleic acid molecule insertion, and markers conferring selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface polypeptides can be used. Transformed cells can be selected because, in an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes.

[0071] In this application, the term "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them, regardless of whether they are inserted into or outside the chromosomes of the host cell, as long as they can be expressed in the host cell. The polynucleotide also includes DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the host cell in any form that can be introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene structure containing all the elements necessary for its expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to the sequences necessary for expression in the host cell, but is not limited thereto.

[0072] Furthermore, the term "operably linked" in the foregoing means that a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target polypeptide of this application is functionally linked to the polynucleotide sequence.

[0073] The microorganisms in some embodiments of this disclosure may be, but are not limited to, microorganisms in which the activity of the protein or the polynucleotide encoding it described herein is attenuated; or microorganisms genetically modified through a vector to attenuate the activity of the protein or the polynucleotide encoding it (e.g., recombinant microorganisms). The vector is as described above.

[0074] The microorganisms in some embodiments of this disclosure may be microorganisms that have the ability to produce L-glutamic acid.

[0075] The microorganisms in some embodiments of this disclosure may be, but are not limited to, microorganisms that naturally possess L-glutamate production ability or microorganisms in which the activity of the protein of this application or the polynucleotide encoding it is attenuated to confer L-glutamate production ability to a parent strain that does not possess L-glutamate production ability.

[0076] As an example, the recombinant microorganisms of this disclosure may include all microorganisms that have been transformed through a vector such that the activity of the protein described herein or the polynucleotide encoding it is attenuated, and which have attenuated activity of the protein described herein or the polynucleotide encoding it, and which are capable of producing L-glutamic acid.

[0077] For the purposes of the embodiments of this disclosure, the recombinant microorganisms of the embodiments of this disclosure may be, but are not limited to, natural wild-type microorganisms or microorganisms that produce L-glutamic acid containing the protein or polynucleotide encoding the protein described herein, in which the activity of said protein or polynucleotide encoding the protein is weakened, and the L-glutamic acid production capacity is increased compared to the natural wild-type microorganisms or microorganisms that produce L-glutamic acid containing the protein or polynucleotide encoding the protein described herein. As an example, the non-mutated microorganisms whose protein activity is not weakened and which are used as target strains for comparing the presence or absence of increased L-glutamic acid production capacity, as described in this application, may be, but are not limited to, the Corynebacterium glutamicum ATCC13869 strain, which is known as an L-glutamic acid-producing strain and lacks the odhA gene, 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.

[0078] As an example, the recombinant strain with increased production capacity has an L-glutamic acid production capacity of approximately 0.3% or more compared to the parent strain or non-mutated microorganism before mutation. Specifically, this includes approximately 0.5% or more, approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% 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 10.7% or more, approximately 11% or more, approximately 12% or more, approximately 12.3% or more, approximately 13% or more, approximately 14% or more, and approximately The increase may be 14.4% or more, approximately 15% or more, approximately 15.1% or more, approximately 16% or more, or approximately 16.9% or more (there are no special restrictions on the upper limit; for example, it may be approximately 200% or less, approximately 150% or less, approximately 100% or less, approximately 50% or less, approximately 40% or less, approximately 30% or less, or approximately 20% or less), but it is not limited to these as long as it has a positive increase compared to the productivity of the parent strain or non-mutated microorganism before mutation. In other examples, the microorganisms with increased production capacity showed L-glutamic acid production capacity of approximately 1.005 times or more, approximately 1.01 times or more, approximately 1.02 times or more, approximately 1.03 times or more, approximately 1.04 times or more, approximately 1.05 times or more, approximately 1.06 times or more, approximately 1.07 times or more, approximately 1.08 times or more, approximately 1.09 times or more, approximately 1.10 times or more, approximately 1.107 times or more, and approximately 1.11 times or more compared to the parent strain or non-mutated microorganism. The increase may be more than double, approximately 1.12 times or more, approximately 1.123 times or more, approximately 1.13 times or more, approximately 1.14 times or more, approximately 1.144 times or more, approximately 1.15 times or more, approximately 1.151 times or more, approximately 1.16 times or more, or approximately 1.169 times or more (there is no special limit 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), but is not limited to these.

[0079] In this application, the term "non-myxoid microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but rather means the wild-type or natural-type strain itself, or a strain before phenotypic changes due to genetic mutations caused by natural or anthropogenic factors. For example, the non-myxoid microorganism may mean a strain in which the activity of the protein described herein has not been weakened, or before it has been weakened. The term "non-myxoid microorganism" may be used interchangeably with "pre-deformation strain," "pre-deformation microorganism," "non-mutant strain," "non-myxoid strain," "non-mutant microorganism," or "reference microorganism."

[0080] As another example of the microorganisms of this disclosure, the microorganisms of this disclosure include Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, and Corynebacterium striatum. It may also be Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, and more specifically, it may be, but is not limited to, Corynebacterium glutamicum.

[0081] As another example, the recombinant microorganisms of this disclosure may be microorganisms in which the activity of some proteins in the L-glutamate biosynthesis pathway is further enhanced, or the activity of some proteins in the L-glutamate degradation pathway is further inactivated, thereby enhancing L-glutamate production capacity.

[0082] Specifically, the microorganisms in some embodiments of this disclosure may be microorganisms in which the OdhA protein is further inactivated or the odhA gene is further deficient. More specifically, the microorganisms in some embodiments of this disclosure may be Corynebacterium glutamicum in which the OdhA protein has been inactivated with Corynebacterium glutamicum ATCC13869, or microorganisms in which the odhA gene is deficient with said Corynebacterium glutamicum ATCC13869. The “OdhA protein” can have its sequence obtained from the NCBI GenBank, a known database, and may, for example, include the amino acid sequence of NCBI sequence ID WP_060564343.1, and the odhA gene may include, but is not limited to, the nucleotide sequence of NCBI GenBank BBD29_06050, and any protein exhibiting the same activity may be included without limitation.

[0083] However, the inactivation of the OdhA protein or the deletion of the OdhA gene is just one example and is not limited thereto. The microorganisms described herein 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.

[0084] Another aspect of the present disclosure provides a method for producing L-glutamic acid, comprising the step of culturing the microorganism of the present disclosure in a culture medium.

[0085] A method for producing L-glutamic acid according to some embodiments of this disclosure may include the step of culturing in a culture medium a microorganism in which the activity of the protein described in this application or the polynucleotide encoding it has been weakened; or a Corynebacterium microorganism that has been genetically modified through a vector so as to weaken the activity of the protein of this application or the polynucleotide encoding it.

[0086] In this disclosure, the term "culture" means growing the microorganisms described herein under appropriately controlled environmental conditions. The culture process described in some embodiments of this disclosure can be carried out according to suitable culture media and culture conditions known in the art. Such a culture process can be readily adapted and used by those skilled in the art depending on the selected microorganism. Specifically, the culture may be batch, continuous, and / or fed-batch.

[0087] In this application, the term "culture medium" means a substance mixed mainly with nutrients necessary for culturing the microorganisms described herein, supplying nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganisms described herein are not particularly limited and any culture medium used for culturing ordinary microorganisms can be used. However, the microorganisms described herein can be cultured under aerobic conditions in an ordinary culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, while adjusting the temperature, pH, etc.

[0088] Specifically, culture media for microorganisms of the genus Corynebacterium can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0089] In this disclosure, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, 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.

[0090] The nitrogen sources may 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, peptone, NZ-amine, beef extract, yeast extract, malt extract, corn maceration, casein hydrolysate, 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.

[0091] 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, and may also include amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the culture medium in batches or continuously, but are not limited thereto.

[0092] In some embodiments of this disclosure, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture medium in an appropriate manner during microbial culture to adjust the pH of the medium. Furthermore, during culture, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble formation. In addition, oxygen or oxygen-containing gases can be injected into the culture medium to maintain an aerobic state, or nitrogen, hydrogen, or carbon dioxide gases can be injected without gas injection, or to maintain anaerobic and microaerobic states, although this is not limited to these methods.

[0093] In some embodiments of this disclosure, other culture temperatures can be maintained at 20-45°C, specifically 25-40°C, and culture can be performed for approximately 10-160 hours, but are not limited to these.

[0094] L-glutamic acid produced by culturing according to some embodiments of this disclosure is either secreted into the culture medium or remains within the cells.

[0095] A method for producing L-glutamic acid according to some embodiments of this disclosure may further include, for example, a step of preparing the microorganism described herein, a step of preparing a culture medium for culturing the microorganism, or a combination thereof (in any order), for example, before the culturing step.

[0096] A method for producing L-glutamic acid according to some embodiments of this disclosure may further include a step of recovering L-glutamic acid from the culture medium (the medium in which the culture was performed) or from the cultured microorganism. The recovery step may further include a step after the culture step.

[0097] The aforementioned recovery may involve collecting the desired L-glutamic acid using appropriate methods known in the art, such as the microbial culture methods described in this application, for example, batch, continuous, or fed-batch culture methods. 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 thereof can be used to recover the desired L-glutamic acid from the culture medium or microorganism using appropriate methods known in the art.

[0098] A method for producing L-glutamic acid according to some embodiments of the present disclosure may further 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-glutamic acid according to the present disclosure includes both a recovery step and a purification step, the recovery step and the purification step can be carried out sequentially or discontinuously, regardless of order, or simultaneously or integrated into a single step.

[0099] In the methods described in some embodiments of this disclosure, the proteins, polynucleotides, vectors, and microorganisms are as described in the other embodiments described above.

[0100] Another aspect of the present disclosure provides a composition for L-glutamic acid production comprising a Corynebacterium microorganism in which the activity of the protein of the present disclosure is weakened; a culture medium in which the same is cultured; or a combination thereof.

[0101] The compositions according to some embodiments of this disclosure may further include any suitable excipients commonly used in compositions for L-glutamic acid production, such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0102] Another aspect of the present disclosure provides a method for producing a Corynebacterium microorganism, comprising the step of weakening the activity of the protein of the present disclosure.

[0103] Another aspect of the present disclosure provides the use of Corynebacterium microorganisms with weakened protein activity for L-glutamic acid production.

[0104] The aforementioned proteins, weakening agents, and microorganisms of the genus Corynebacterium are as described in the other sections above. [Effects of the Invention]

[0105] The L-glutamic acid-producing Corynebacterium microorganisms with weakened protein activity described herein can produce L-glutamic acid in high yield and can be usefully utilized for the industrial production of L-glutamic acid. [Modes for carrying out the invention]

[0106] Examples The present application will be described in more detail below through experimental examples. However, the following embodiments are merely preferred embodiments for illustrative purposes of 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 performed by a person of ordinary skill who is skilled in the art of this application or a similar art.

[0107] Example 1. Production of a recombinant vector for modifying the start codon of the BBD29_13140 gene. When the start codon of the BBD29_13140 gene (SEQ ID NO: 2) on the chromosome of Corynebacterium strains was changed from ATG to GTG and TTG, respectively, changes in L-glutamic acid production capacity were observed.

[0108] Specifically, to construct a start codon modification vector, gene fragments were obtained by PCR using Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template, with primer pairs for SEQ ID NOs. 3 and 4, and primer pairs for SEQ ID NOs. 5 and 6. The polymerase used was Solg TM Using Pfu-X DNA polymerase, PCR amplification was performed by denaturing at 95°C for 5 minutes, denaturing at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes.

[0109] The amplified gene fragment and the chromosome transformation vector pDCM2 (Republic of Korea Publication No. 10-2020-0136813), which was cleaved 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) to obtain a recombinant plasmid, which was named the pDCM2-BBD29_13140(a1g) vector. 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.

[0110] Using Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template, gene fragments were obtained by PCR using primer pairs of SEQ ID NOs. 3 and 7, and primer pairs of SEQ ID NOs. 8 and 6. The polymerase used was Solg TM Using Pfu-X DNA polymerase, PCR amplification was performed by denaturing at 95°C for 5 minutes, denaturing at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerizing at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes.

[0111] The amplified gene fragments and the chromosome transformation vector pDCM2, which had been cleaved with SmaI restriction enzyme, were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named the pDCM2-BBD29_13140(a1t) vector. 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.

[0112] The pDCM2-BBD29_13140(a1g) vector and pDCM2-BBD29_13140(a1t) vector, prepared in this manner, were introduced into the bacterial strain in the following example.

[0113] Example 2. Production of a Corynebacterium glutamicum strain with altered start codon of the BBD29_13140 gene. Example 2-1. Production of a Corynebacterium glutamicum strain with L-glutamic acid production ability derived from wild-type Corynebacterium glutamicum. To create a strain of Corynebacterium glutamicum ATCC13869 capable of producing L-glutamic acid, we constructed a Corynebacterium glutamicum ATCC13869ΔodhA strain lacking the odhA gene, based on previous literature (Appl Environ Microbiol. 2007 Feb;73(4):1308-19. Epub 2006 Dec 8.). The sequence of the OdhA protein was obtained from the NCBI GenBank, a well-known database (GenBank accession number: WP_060564343.1).

[0114] Specifically, due to the odhA deficiency, Corynebacterium glutamicum ATCC13869 chromosomal DNA was used as a template, and the upstream and downstream regions of the odhA gene were obtained by PCR using primer pairs of SEQ ID NOs. 9 and 10, and primer pairs of SEQ ID NOs. 11 and 12, respectively. The polymerase used was Solg TMUsing Pfu-X DNA 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.

[0115] Recombinant plasmids were obtained by cloning the amplified odhA upstream and downstream regions, along with the chromosome transformation plasmid vector pDCM2 cleaved with SmaI restriction enzyme, using the Gibson assembly method, and named pDCM2-ΔodhA. 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.

[0116] The constructed pDCM2-ΔodhA vector was used to transform Corynebacterium glutamicum ATCC13869 strain by electroporation. Following a secondary crossover process, a strain lacking the odhA gene on the chromosome was obtained. The presence or absence of the odhA gene deletion was confirmed by PCR using primer pairs of SEQ ID NOs. 13 and 14, and by genome sequencing. The resulting strain was named ATCC13869ΔodhA.

[0117] Example 2-2. Production of a strain of Corynebacterium glutamicum with weakened BBD29_13140 gene that possesses L-glutamic acid production ability. The Corynebacterium glutamicum ATCC13869ΔodhA strain, prepared in Example 2-1, was subjected to the introduction of the pDCM2-BBD29_13140(a1g) vector and the pDCM2-BBD29_13140(a1t) vector, prepared in Example 1, to confirm the effect on L-glutamic acid production capacity.

[0118] After transforming Corynebacterium glutamicum ATCC13869ΔodhA strains with the aforementioned vectors by electroporation, strains were obtained in which the start codon of the BBD29_13140 gene on the chromosome was changed from ATG to GTG and TTG, respectively, through a secondary crossover process. The genetic manipulation was confirmed by PCR using primer pairs of SEQ ID NOs. 15 and 16, and by genome sequencing. The resulting strains were named ATCC13869ΔodhA-BBD29_13140(a1g) and ATCC13869ΔodhA-BBD29_13140(a1t), respectively.

[0119] To confirm the L-glutamic acid production ability of the ATCC13869ΔodhA-BBD29_13140(a1g), ATCC13869ΔodhA-BBD29_13140(a1t) strains and the ATCC13869ΔodhA strain prepared as described above, they were cultured using the same method as described below, with the latter being used as a control group.

[0120] The bacterial strain was inoculated onto a plate medium consisting of the following seed medium and incubated at 30°C for 20 hours. Subsequently, the bacterial strain was inoculated onto a 250 ml corner baffle flask containing 25 ml of the following production medium using one platinum loop, and incubated at 30°C for 40 hours with shaking at 200 rpm.

[0121] <Seedling medium> Glucose 1%, beef extract 0.5%, polypeptone 1%, sodium chloride 0.25%, yeast extract 0.5%, agar 2%, urea 0.2%, pH 7.2

[0122] <Production culture medium> Raw sugar 6%, calcium carbonate 5%, ammonium sulfate 2.25%, monopotassium phosphate 0.1%, magnesium sulfate 0.04%, ferrous sulfate 10 mg / L, thiamine hydrochloride 0.2 mg / L, biotin 50 μg / L

[0123] After the culture was completed, the L-glutamic acid production capacity was measured using high-performance liquid chromatography (HPLC), and the measurement results are shown in Table 1 below.

[0124] [Table 1]

[0125] As shown in Table 1 above, we confirmed that the L-glutamic acid concentration increased by approximately 7% and 15.1% in the strains ATCC13869ΔodhA-BBD29_13140(a1g) and ATCC13869ΔodhA-BBD29_13140(a1t), in which the start codon of the BBD29_13140 gene was weakened from ATG to GTG or TTG, compared to the wild-type ATCC13869ΔodhA strain.

[0126] Example 3. Preparation of a recombinant vector with a modified ribosome binding site (RBS) of BBD29_13140. To weaken the BBD29_13140 gene on the chromosome of Corynebacterium strains, the Shine-Dalgano sequence (SD) was predicted from the 35 base pairs at the top of the gene and the 35 base pairs from the N-terminus of the open reading frame (ORF).

[0127] Based on the aforementioned nucleotide sequences, we predicted a total of four candidate ribosome binding sites—RBS1, RBS2, RBS3, and RBS4—using the RBS Calculator (GitHub). As a result, these four candidate ribosome binding sites are sequences predicted to exhibit 20%, 60%, 80%, and 90% reduced expression compared to existing ribosome binding sites, respectively.

[0128] The predicted ribosome binding sites are shown in Table 2 below.

[0129] [Table 2]

[0130] To determine whether altering the ribosome binding site of BBD29_13140 on the chromosome of Corynebacterium strains to weaken the gene improves L-glutamate production, recombinant vectors were created for each of the four previously predicted ribosome binding site alterations.

[0131] Specifically, using wild-type Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template, gene fragments were obtained by PCR using primer pairs for SEQ ID NOs. 17 and 18, SEQ ID NOs. 19 and 20, SEQ ID NOs. 17 and 21, SEQ ID NOs. 22 and 20, SEQ ID NOs. 17 and 23, SEQ ID NOs. 24 and 20, SEQ ID NOs. 17 and 25, and SEQ ID NOs. 26 and 20. The polymerase used was Solg TM Using Pfu-X DNA polymerase, PCR amplification was performed by denaturing at 95°C for 5 minutes, denaturing at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerizing at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes.

[0132] The amplified gene fragments and the chromosome transformation vector pDCM2, which had been cleaved with SmaI restriction enzyme, were cloned using the Gibson assembly method to obtain recombinant plasmids, which were named pDCM2-RBS1, pDCM2-RBS2, pDCM2-RBS3, and pDCM2-RBS4 vectors, respectively. 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.

[0133] Example 4. Synthesis of recombinant strains for modifying the ribosome binding site (RBS) of BBD29_13140 The ATCC13869ΔodhA strain produced in Example 2-1 was subjected to the introduction of the pDCM2-RBS1, pDCM2-RBS2, pDCM2-RBS3, and pDCM2-RBS4 vectors produced in Example 3, and their effects on L-glutamic acid production capacity were investigated.

[0134] Specifically, the four vectors described above were used to transform Corynebacterium glutamicum ATCC13869ΔodhA strains by electroporation. Following a secondary crossover process, strains were obtained in which the ribosome binding sequences of the BBD29_13140 gene on the chromosome were modified to RBS1, RBS2, RBS3, and RBS4, respectively.

[0135] The genetic manipulation was confirmed through PCR and genome sequencing using primer pairs of SEQ ID NO: 27 and SEQ ID NO: 28, which can amplify the homologous recombination upstream and downstream regions, respectively. These were named ATCC13869ΔodhA-RBS1, ATCC13869ΔodhA-RBS2, ATCC13869ΔodhA-RBS3, and ATCC13869ΔodhA-RBS4, respectively.

[0136] To confirm the L-glutamic acid production ability of the ATCC13869ΔodhA-RBS1, ATCC13869ΔodhA-RBS2, ATCC13869ΔodhA-RBS3, ATCC13869ΔodhA-RBS4 and the ATCC13869ΔodhA strain prepared as described above, they were cultured in the same manner as in Example 2-2 above.

[0137] After the culture was completed, the L-glutamic acid production capacity was measured using high-performance liquid chromatography (HPLC), and the measurement results are shown in Table 3 below.

[0138] [Table 3]

[0139] As shown in Table 3 above, we confirmed that the concentration of L-glutamic acid increased in strains in which the ribosome binding sequence of the BBD29_13140 gene was weakened by RBS compared to the wild-type ATCC13869ΔodhA strain.

[0140] Example 5. Production of a BBD29_13140 gene deletion vector. In addition to the weakening effect of BBD29_13140 observed in the above example, a deletion of the BBD29_13140 gene in Corynebacterium strains was used to investigate how L-glutamic acid production capacity changes when the BBD29_13140 gene is deleted, and to confirm how this affects L-glutamic acid production. A deletion vector was then prepared.

[0141] To produce a recombinant vector deleting the aforementioned gene, gene fragments were obtained by PCR using Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template, with primer pairs of SEQ ID NOs. 3 and 29, and primer pairs of SEQ ID NOs. 30 and 31. The polymerase used was Solg TM Using Pfu-X DNA polymerase, PCR amplification was performed by denaturing at 95°C for 5 minutes, denaturing at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerizing at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes.

[0142] The amplified gene fragments and the chromosome transformation vector pDCM2, which had been cleaved with SmaI restriction enzyme, were cloned using the Gibson assembly method to obtain a recombinant plasmid named pDCM2-ΔBBD29_13140. 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.

[0143] The pDCM2-ΔBBD29_13140 vector prepared in this manner was introduced into the bacterial strain in the following example.

[0144] Example 6. Production of a Corynebacterium glutamicum strain lacking the BBD29_13140 gene. The ATCC13869ΔodhA strain prepared in Example 2-1 was subjected to the introduction of the pDCM2-ΔBBD29_13140 vector prepared in Example 5, and its effect on L-glutamic acid production capacity was confirmed.

[0145] Specifically, the pDCM2-ΔBBD29_13140 vector was used to transform Corynebacterium glutamicum ATCC13869ΔodhA strain by electroporation. Following a secondary crossover process, a strain lacking the BBD29_13140 gene on the chromosome was obtained. This genetic manipulation was confirmed by PCR using primer pairs of SEQ ID NOs. 32 and 33, and by genome sequencing. This strain was named ATCC13869ΔodhAΔBBD29_13140.

[0146] The ATCC13869ΔodhAΔBBD29_13140 strain prepared as described above was cultured in the same manner as in Example 2-2 to confirm its L-glutamic acid production ability, with the ATCC13869ΔodhA strain as the control group.

[0147] After the culture was completed, the L-glutamic acid production capacity was measured using high-performance liquid chromatography (HPLC), and the measurement results are shown in Table 4 below.

[0148] [Table 4]

[0149] As shown in Table 4 above, we confirmed that ATCC13869ΔodhAΔBBD29_13140, which lacks the BBD29_13140 gene, exhibited inhibited growth compared to the wild-type ATCC13869ΔodhA strain, and conversely, the L-glutamic acid concentration decreased to approximately 49.5%.

[0150] Example 7. Production of a Corynebacterium glutamicum strain with L-glutamic acid production ability derived from a Corynebacterium glutamicum strain with a weakened BBD29_13140 gene and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutation. In addition to strains derived from wild-type Corynebacterium, to confirm whether the aforementioned gene exhibits a similar effect in strains derived from NTG mutant Corynebacterium with increased L-glutamate production capacity, the pDCM2-BBD29_13140(a1t) vector prepared in Example 1 was introduced into the KFCC11074 strain (Korean Registered Patent No. 10-0292299), known as an L-glutamate-producing NTG mutant strain, and its effect on L-glutamate production capacity was confirmed.

[0151] After transforming the KFCC11074 strain with the aforementioned vector by electroporation, a strain was obtained in which the start codon of the BBD29_13140 gene on the chromosome was changed from ATG to TTG via a secondary crossover process. This genetic manipulation was confirmed by PCR using primer pairs of SEQ ID NOs. 15 and 16, and by genome sequencing, and this strain was named KFCC11074-BBD29_13140(a1t).

[0152] The fermentation titer experiments were conducted on the fabricated KFCC11074-BBD29_13140(a1t) and Corynebacterium glutamicum KFCC11074 strains using the method specified below.

[0153] The bacterial strain was inoculated onto a plate medium consisting of the seed medium described below and cultured at 30°C for 20 hours. Subsequently, the bacterial strain was inoculated onto a 250 ml corner baffle flask containing 25 ml of the production medium described below using one platinum loop, and cultured at 30°C for 40 hours with shaking at 200 rpm.

[0154] <Seedling medium> Glucose 1%, beef extract 0.5%, polypeptone 1%, sodium chloride 0.25%, yeast extract 0.5%, agar 2%, urea 0.2%, pH 7.2

[0155] <Production culture medium> Raw sugar 6%, calcium carbonate 5%, ammonium sulfate 2.25%, monopotassium phosphate 0.1%, magnesium sulfate 0.04%, ferrous sulfate 10 mg / L, thiamine hydrochloride 0.2 mg / L, biotin 500 μg / L

[0156] After the culture was completed, the L-glutamic acid production capacity was measured using high-performance liquid chromatography (HPLC), and the measurement results are shown in Table 5 below.

[0157] [Table 5]

[0158] As shown in Table 5 above, we confirmed that the L-glutamic acid concentration increased by approximately 16.9% in the KFCC11074-BBD29_13140(a1t) strain, in which the start codon of the BBD29_13140 gene was weakened from ATG to TTG, compared to the KFCC11074 strain.

[0159] From the above description, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

[0160] Specific concrete examples Example 1-1. A Corynebacterium microorganism in which the activity of a protein containing the amino acid sequence of Sequence ID No. 1 is weakened compared to its endogenous activity. Example 1-2. In Example 1-1, the Corynebacterium microorganism is a microorganism capable of producing L-glutamic acid. Implemented Example 1-3. In Implemented Example 1-1, the protein is encoded by a polynucleotide described in the base sequence of Sequence ID No. 2, and is a microorganism. Implemented Example 1-4. In Implemented Example 1-1, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum. Implemented Examples 1-5. In Implemented Example 1-1, the microorganism is characterized by increased L-glutamic acid production capacity compared to a parent strain or wild type in which the activity of the protein containing the amino acid sequence of Sequence ID No. 1 is not weakened compared to its endogenous activity. Examples 1-6. A method for producing L-glutamic acid, comprising the step of culturing a Corynebacterium microorganism in which the activity of a protein containing the amino acid sequence of SEQ ID NO: 1 is weakened compared to its endogenous activity, in a culture medium. Examples 1-7. A method for producing the protein in Examples 1-6, wherein the protein is encoded by a polynucleotide described by the base sequence of Sequence ID No. 2. Implemented Example 1-8. A production method in which, in Implemented Example 1-6, the weakening of the protein's activity is the weakening of the polynucleotide described by the base sequence of Sequence ID No. 2. Examples 1-9. A production method in which, in Examples 1-6, the Corynebacterium microorganism is Corynebacterium glutamicum. Implemented Example 1-10. In Implemented Example 1-6, the production method further comprises the step of recovering L-glutamic acid from the cultured microorganism or culture medium. Example 2-1. A microorganism in which the activity of a protein containing a sequence having at least 85% sequence identity with the amino acid sequence of Sequence ID No. 1 is weakened compared to the intrinsic activity of the microorganism. Example 2-2. A microorganism expressing a recombinant protein containing an amino acid sequence that has at least 85% sequence identity with the amino acid sequence of SEQ ID NO: 1. Example 2-3. A microorganism in which, in Example 2-1 or 2-2, the protein contains an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1. Example 2-4. A microorganism in which, in any one of Examples 2-1 to 2-3, the protein contains the amino acid sequence of SEQ ID NO: 1. Example 2-5. A microorganism in which, in any one of Examples 2-1 to 2-3, the protein does not contain the amino acid sequence of Sequence ID No. 1. Implemented Example 2-6. In any one of Implemented Examples 2-1 to 2-3, the protein is NCBI accession number WP_060565206.1, QWQ85246.1, WP_065367112.1, WP_038585884.1, WP_040072989.1, WP_011265991.1, WP_074492394.1, ARV66101.1, WP_003853812.1, WP_006285921.1, WP Microorganisms having sequences selected from the group consisting of _003862956.1, WP_044027349.1, BAF55605.1, WP_179205783.1, WP_172769145.1, WP_211439424.1, WP_179111498.1, WP_059289862.1, WP_185969420.1, WP_173673681.1, and QDQ21801.1. Example 2-7. A microorganism in which, in any one of Examples 2-1 to 2-6, the microorganism has the ability to produce L-glutamic acid. Implemented Example 2-8. A microorganism in which, in any one of Implemented Examples 2-1 to 2-7, the protein is encoded by a nucleotide molecule containing a sequence that has at least 85% sequence identity with the nucleotide sequence of Sequence ID No. 2 or its degenerate sequence. Implemented Example 2-9. A microorganism in which, in any one of Implemented Examples 2-1 to 2-8, the protein is encoded by a nucleotide molecule containing a sequence that has at least 95% sequence identity with the nucleotide sequence of Sequence ID No. 2 or its degenerate sequence. Implemented Example 2-10. A microorganism in which, in any one of Implemented Examples 2-1 to 2-9, the protein is encoded by a nucleotide molecule containing a sequence that has at least 85% sequence identity with the nucleotide sequence of SEQ ID NO: 2. Example 2-11. A microorganism in which, in any one of Examples 2-1 to 2-10, the protein is encoded by a nucleotide molecule containing a sequence that has at least 95% sequence identity with the nucleotide sequence of Sequence ID No. 2. Example 2-12. A microorganism in which, in any one of Examples 2-1 to 2-11, the protein is encoded by a nucleotide molecule containing the nucleotide sequence of Sequence ID No. 2. Example 2-13. A microorganism in which, in any one of Examples 2-1 to 2-12, the protein is encoded by a nucleotide molecule that does not contain the nucleotide sequence of Sequence ID No. 2. Example 2-14. A microorganism in which, in any one of Examples 2-1 to 2-13, the microorganism is Corynebacterium. Example 2-15. In any one of Examples 2-1 to 2-14, the microorganism is Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singular, Corynebacterium halotolerans, Corynebacterium striatum Microorganisms selected from the group consisting of Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, Corynebacterium flavescens, Corynebacterium crenatum, and Corynebacterium suranareeae. Example 2-16. A microorganism in which, in any one of Examples 2-1 to 2-15, the microorganism is Corynebacterium glutamicum. Example 2-17. A microorganism in which, in any one of Examples 2-1 to 2-16, the L-glutamic acid production capacity is increased compared to the parent strain or wild type in which the activity of the protein containing the amino acid sequence of SEQ ID NO: 1 is not weakened, compared to its intrinsic activity. Example 2-18. A microorganism in which, in any one of Examples 2-1 to 2-17, at least a portion of the nucleotide sequence encoding the protein is missing when compared to the microorganism's own nucleotide sequence. Example 2-19. A microorganism in which the nucleotide sequence encoding the protein described in Example 2-18 is missing. Example 2-20. A microorganism in which, in any one of Examples 2-1 to 2-19, the expression regulatory sequence for the gene encoding the protein is not a sequence unique to the microorganism. Implemented Example 2-21. A microorganism in which, in Implemented Example 2-20, the expression regulatory sequence comprises deletion, insertion, or substitution of at least one specific expression regulatory sequence to reduce the expression of the gene encoding the protein. Example 2-22. A microorganism in which, in Example 2-20 or 2-21, the expression regulatory sequence includes at least one sequence selected from the group consisting of a promoter sequence, an operator sequence, a sequence encoding a ribosome binding site, a sequence encoding transcription termination, and a sequence encoding translation termination. Example 2-23. A microorganism in which, in any one of Examples 2-1 to 2-22, the amino acid sequence is not the intrinsic amino acid sequence of the microorganism. Example 2-24. A microorganism in which, in Example 2-23, the amino acid sequence includes at least one deletion, insertion, or substitution of a specific amino acid sequence, thereby weakening the activity of the protein. Example 2-25. A microorganism in which, in any one of Examples 2-1 to 2-24, the nucleotide sequence encoding the protein is not the microorganism's own nucleotide sequence. Example 2-26. A microorganism in which, in Example 2-25, the nucleotide sequence includes at least one deletion, insertion, or substitution of a specific nucleotide sequence, thereby weakening the activity of the protein. Implemented in 2-27. A microorganism in which, in any one of Implemented Examples 2-1 to 2-26, at least one sequence selected from the group consisting of a nucleotide sequence for a start codon, a nucleotide sequence for a stop codon, a Shine-Dalgano sequence, and the 5'UTR region of the gene encoding the protein is not an intrinsic sequence of the microorganism. Implemented Example 2-28. A microorganism in which, in Implemented Example 2-27, the nucleotide sequence for the start codon of the gene encoding the protein has been mutated compared to the microorganism's own sequence. Implemented Example 2-29. A microorganism in which, in Implemented Example 2-27 or 2-28, the nucleotide sequence for the stop codon of the gene encoding the protein is mutated compared to the microorganism's intrinsic sequence. Implemented Example 2-30. A microorganism in which, in any one of Implemented Examples 2-27 to 2-29, the unique gene encoding the protein contains the start codon of ATG, and ATG is replaced by GTG. Implemented Example 2-31. A microorganism in which, in any one of Implemented Examples 2-27 to 2-30, the unique gene encoding the protein contains the start codon of ATG, and ATG is replaced by TTG. Implemented Example 2-32. A microorganism in which, in any one of Implemented Examples 2-27 to 2-30, the nucleotide sequence for the Shine-Dalgano sequence of the gene encoding the protein is mutated compared to the microorganism's intrinsic sequence. Implemented Example 2-33. A microorganism in which, in any one of Implemented Examples 2-27 to 2-32, the unique gene encoding the protein contains the Shine-Dalgano sequence CTAGATTGG, where CTAGATTGG is replaced by CAAGGCCGG. Implemented Example 2-34. A microorganism in which, in any one of Implemented Examples 2-27 to 2-32, the unique gene encoding the protein contains the Shine-Dalgano sequence CTAGATTGG, where CTAGATTGG is replaced by CGTGACAGG. Implemented Example 2-35. A microorganism in which, in any one of Implemented Examples 2-27 to 2-32, the unique gene encoding the protein contains the Shine-Dalgano sequence CTAGATTGG, where CTAGATTGG is replaced by CTTCGCTGG. Implemented Example 2-36. A microorganism in which, in any one of Implemented Examples 2-27 to 2-32, the unique gene encoding the protein contains the Shine-Dalgano sequence CTAGATTGG, where CTAGATTGG is replaced by GTCGATTTC. Implemented Example 2-37. A microorganism in any one of Implemented Examples 2-1 to 2-36, wherein the microorganism contains an antisense oligonucleotide that is complementarily bound to the transcript of the gene encoding the protein. Example 2-38. A microorganism in which, in Example 2-37, the antisense oligonucleotide contains antisense RNA. Implemented Example 2-39. A microorganism in which, in any one of Implemented Examples 2-1 to 2-38, a sequence complementary to the Shine-Dalgano sequence of the gene encoding the protein is inserted before the Shine-Dalgano sequence to form a secondary structure. Example 2-40. A microorganism in which, in Example 2-39, the secondary structure binds to a ribosome and does not reduce or delay mRNA translation. Example 2-41. A microorganism in which, in any one of Examples 2-1 to 2-40, the promoter for reverse transcription (RTE) is inserted at the 3' end of the open reading frame (ORF) of the gene encoding the protein. Example 2-42. A microorganism in which, as in Example 2-41, the activity of a protein is weakened by the production of an antisense nucleotide molecule complementary to at least a portion of the gene encoding the protein. Example 2-43. A microorganism in which the odhA gene is deficient in any one of Examples 2-1 to 2-42. Example 2-44. In any one of Examples 2-1 to 2-43, the microorganism is ATCC13869 lacking the odhA gene. Example 2-45. A microorganism in which, in Example 2-43 or 2-44, the odhA gene contains a nucleotide sequence encoding NCBI sequence ID WP_060564343.1. Example 2-46. A microorganism in which, in Example 2-43 or 2-44, the odhA gene contains NCBI GenBank BBD29_06050. Example 2-47. A microorganism in which, in any one of Examples 2-1 to 2-46, the microorganism contains an N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutant strain. Implemented Example 2-48. Use of a microorganism according to any one of Implemented Examples 2-1 to 2-47 for the production of L-glutamic acid. Example 2-49. A method for producing L-glutamic acid, comprising the step of culturing a microorganism according to any one of Examples 2-1 to 2-45. Example 2-50. In Example 2-49, the protein is encoded by the nucleotide sequence of SEQ ID NO: 2, and the activity of the nucleotide sequence of SEQ ID NO: 2 is weakened compared to the intrinsic activity. Example 2-51. In Example 2-49 or 2-50, the production method further comprises the step of recovering L-glutamic acid from the cultured microorganism or culture medium. Example 2-52. A method relating to any one of Examples 2-49 to 2-51, wherein the production method further comprises the step of recovering L-glutamic acid from the cultured microorganism. Implemented Example 2-53. In any one of Implemented Examples 2-49 to 2-52, the production method further comprises the step of recovering L-glutamic acid from the culture medium in which the microorganism was cultured. Example 2-54. A method relating to any one of Examples 2-49 to 2-53, wherein the cultivation is carried out using at least one carbon source selected from the group consisting of carbohydrates, sugar alcohols, organic acids, amino acids, starch hydrolysates, sugar solutions (molasses), blackstrap molasses, rice winter, cassava, bagasse, and corn maceration. Example 2-55. A method relating to any one of Examples 2-49 to 2-54, wherein the culture is carried out using at least one nitrogen source selected from the group consisting of ammonia, ammonium sulfate, ammonium hydrochloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, glutamic acid, methionine, glutamine, peptone, NZ-amine, beef extract, yeast extract, malt extract, corn maceration, casein hydrolysate, fish or their degraded products, and defatted soybean meal or its degraded products. Example 2-56. A method in which, in any one of Examples 2-49 to 2-55, the culture is carried out using at least one selected from the group consisting of monopotassium phosphate, dipotassium phosphate, their corresponding sodium-containing salts, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Example 2-57. A method for increasing L-glutamic acid production by a microorganism, comprising the step of weakening the activity of a protein containing an amino acid sequence that has at least 85% sequence identity with the amino acid sequence of Sequence ID No. 1 in the microorganism. Example 2-58. A method in which, in Example 2-57, the microorganism is one of the microorganisms described in any one of Examples 2-1 to 2-45. Example 2-59. A polynucleotide comprising a nucleic acid sequence encoding an amino acid sequence having at least 85% sequence identity with the amino acid sequence of Sequence ID No. 1, wherein the polynucleotide is not spontaneously generated. Implemented Example 2-60. In Implemented Example 2-59, the amino acid sequence is a polynucleotide having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1. Example 2-61. A polynucleotide in which, in Example 2-59 or 2-60, the amino acid molecule contains the amino acid sequence of SEQ ID NO: 1. Example 2-62. A polynucleotide in which, in Example 2-59 or 2-60, the amino acid molecule does not contain the amino acid sequence of SEQ ID NO: 1. Implemented Example 2-63. In any one of Implemented Examples 2-59 to 2-62, the nucleic acid sequence is a polynucleotide containing a start codon that is not spontaneously generated when encoding an amino acid sequence. Example 2-64. A polynucleotide in which, in any one of Examples 2-59 to 2-63, the polynucleotide contains the start codon GTG. Example 2-65. A polynucleotide in which, in any one of Examples 2-59 to 2-63, the polynucleotide includes a start codon, TTG. Example 2-66. A polynucleotide in which, in any one of Examples 2-59 to 2-65, the nucleic acid sequence includes a Shine-Dalgano sequence that is not spontaneously generated when encoding an amino acid sequence. Example 2-67. A polynucleotide in which, in any one of Examples 2-59 to 2-66, the polynucleotide contains a mutant Shine-Dalgano sequence, CAAGGCCGG. Example 2-68. A polynucleotide in which, in any one of Examples 2-59 to 2-66, the polynucleotide contains a mutant Shine-Dalgano sequence, CGTGACAGG. Example 2-69. A polynucleotide in which, in any one of Examples 2-59 to 2-66, the polynucleotide contains a mutant Shine-Dalgano sequence, CTTCGCTGG. Example 2-70. In any one of Examples 2-59 to 2-66, the polynucleotide is a polynucleotide that is a mutant Shine-Dalgano sequence, GTCGATTTC. Example 2-71. A vector containing a polynucleotide according to any one of Examples 2-59 to 2-70. Example 2-72. Use, process, or system of a compositional product characterized by one or more elements disclosed in this application.

Claims

1. A modified microorganism in which the expression level of a protein containing the amino acid sequence of SEQ ID NO: 1 is reduced compared to the intrinsic expression level of the protein in the unmodified microorganism, The modified microorganism is a microorganism that has increased L-glutamic acid production compared to the unmodified microorganism.

2. The modified microorganism expresses a recombinant protein containing the amino acid sequence of SEQ ID NO: 1, according to claim 1.

3. The microorganism according to claim 1, wherein the protein is encoded by a nucleotide molecule containing the nucleotide sequence of SEQ ID NO:

2.

4. The microorganism according to claim 1, wherein the regulatory expression sequence for the gene encoding the protein is not the unmodified, intrinsic sequence of the microorganism.

5. The microorganism according to claim 1, wherein the modified microorganism is Corynebacterium.

6. The modified microorganism according to claim 1, wherein the modified microorganism has the ability to produce L-glutamic acid.

7. A method for producing L-glutamic acid, comprising the step of culturing the microorganism described in claim 1.

8. A method for increasing L-glutamic acid production by a microorganism, comprising the step of reducing the expression level of a protein containing the amino acid sequence of SEQ ID NO: 1 in the microorganism described in claim 1.

9. A polynucleotide that contains a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1, but is not spontaneously occurring. The nucleic acid sequence includes a start codon GTG or a start codon TTG, or The polynucleotide is a polynucleotide comprising a mutated Shine-Dalgano sequence CAAGGCCGG, CGTGACAGG, CTTCGCCTGG, or GTCGATTTC.

10. Use of the microorganism described in claim 1 for L-glutamic acid production.

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