Mutant polypeptide and method for producing L-glutamic acid using the same

Abstract

The present application relates to novel mutant polypeptides; polynucleotides encoding the mutant polypeptides; microorganisms comprising the mutant polypeptides, polynucleotides encoding the mutant polypeptides, or vectors comprising the polynucleotides; methods for producing L-glutamic acid, the methods comprising culturing the microorganisms in a medium; and use of the mutant polypeptides or microorganisms for producing L-glutamic acid.

Description

[Technical Field]

[0001] The present application relates to novel mutant polypeptides; polynucleotides encoding the mutant polypeptides; microorganisms comprising the mutant polypeptides, polynucleotides encoding the mutant polypeptides, or vectors comprising the polynucleotides; methods for producing L-glutamic acid, the methods comprising culturing the microorganisms in a medium; and use of the mutant polypeptides or microorganisms for producing L-glutamic acid. [Background technology]

[0002] Glutamic acid is a typical amino acid produced by fermentation. It has a distinctive taste and is one of the important amino acids that is widely used in the food industry, pharmaceuticals, animal feed, and other fields.

[0003] Common methods for producing glutamic acid include fermentation using Coryneform bacteria, including Brevibacterium and Corynebacterium, and mutants thereof (Amino Acid Fermentation, Gakkai Shuppan Center: 195-215, 1986). Other known methods include fermentation using microorganisms such as Escherichia coli, Bacillus, Streptomyces, Penicillium, Klebsiella, Erwinia, and Pantoea (U.S. Patent Publication No. 3,220,929, U.S. Patent Publication No. 6,682,912).

[0004] Furthermore, various research efforts are being made to efficiently produce amino acids, such as the development of microorganisms and fermentation process technologies that can efficiently produce amino acids. Specifically, target-specific approaches have been developed, such as increasing the expression of genes encoding enzymes involved in amino acid biosynthesis in Corynebacterium strains or eliminating genes unnecessary for amino acid biosynthesis (Korean Patent Publication Nos. 10-0924065 and 10-1208480). In addition to these methods, methods for eliminating genes not involved in amino acid production or genes whose specific functions in amino acid production are unknown have also been utilized. However, there remains a growing need for research into methods for efficiently producing L-glutamic acid at high yields. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent Publication No. 3,220,929 [Patent Document 2] U.S. Patent Publication No. 6,682,912 [Patent Document 3] Korean Patent Registration No. 10-0924065 [Patent Document 4] Korean Patent Registration No. 10-1208480 [Patent Document 5] Korean Patent Registration No. 10-0292299 [Patent Document 6] US Patent US 7662943 B2 [Patent Document 7] US Patent US 10584338 B2 [Patent Document 8] US Patent US 10273491 B2 [Patent Document 9] International Patent Publication No. 2008-033001 [Non-patent literature]

[0006] [Non-licensed document 1] Amino Acid Fermentation,Gakkai Shuppan Center:195-215,1986 [Non-licensed document 2] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed document 3] Rice et al., 2000, Trends Genet.16:276-277

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[0007] The present inventors have developed a mutant of a major stimulator superfamily transporter polypeptide, a Corynebacterium glutamicum strain containing the mutant, and a method for producing L-glutamic acid using the strain, and have completed the present application. [Means for solving the problem]

[0008] One aspect of the present application provides a mutant polypeptide in which the amino acid corresponding to position 351 of SEQ ID NO:1 has been substituted with another amino acid.

[0009] In one embodiment, the mutant polypeptide may have the amino acid corresponding to position 351 of SEQ ID NO: 1 substituted with asparagine.

[0010] In one embodiment, the variant polypeptide may comprise a sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO:1.

[0011] In another embodiment, the variant polypeptide may consist of the amino acid sequence of SEQ ID NO:3.

[0012] In yet another embodiment, the variant polypeptide may have major facilitator superfamily transporter activity.

[0013] Another aspect of the present application provides a polynucleotide encoding the mutant polypeptide.

[0014] Another aspect of the present application provides a microorganism comprising said mutant polypeptide, a polynucleotide encoding said mutant polypeptide, or a vector comprising said polynucleotide.

[0015] As a microorganism according to any one of the above-mentioned specific examples, the microorganism may further comprise one or more mutant polypeptides selected from the group consisting of (a) and (b) below; a polynucleotide encoding the mutant polypeptide; or a vector comprising the polynucleotide:

[0016] (a) a mutant polypeptide having TetR family transcriptional regulator activity, in which the amino acid corresponding to position 105 of SEQ ID NO: 5 is substituted with another amino acid; and (b) A mutant polypeptide having isocitrate decomposition enzyme activity, in which the amino acid sequence corresponding to positions 336 to 346 of SEQ ID NO: 9 has been deleted.

[0017] In the microorganism according to any one of the above-mentioned specific examples, the mutant polypeptide having TetR family transcriptional regulator activity of (a) may be a mutant polypeptide having TetR family transcriptional regulator activity in which the amino acid corresponding to position 105 of SEQ ID NO: 5 is substituted with leucine.

[0018] In the microorganism according to any one of the above-mentioned specific examples, the mutant polypeptide having TetR family transcriptional regulator activity of (a) may consist of the amino acid sequence of SEQ ID NO: 7, and the mutant polypeptide having isocitrate decomposition enzyme activity of (b) may consist of the amino acid sequence of SEQ ID NO: 11.

[0019] The microorganism according to any one of the above-mentioned embodiments may have an increased ability to produce L-glutamic acid compared to a microorganism containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same.

[0020] As a microorganism according to any one of the above-mentioned embodiments, the microorganism may be a Corynebacterium microorganism.

[0021] In any one of the above-mentioned embodiments, the Corynebacterium microorganism may be Corynebacterium glutamicum.

[0022] Another aspect of the present application provides a method for producing L-glutamic acid, comprising culturing the microorganism in a medium.

[0023] In one embodiment, the method may further comprise the step of recovering L-glutamic acid from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium.

[0024] Another aspect of the present application provides a composition for producing L-glutamic acid, comprising: the mutant polypeptide; a polynucleotide encoding the mutant polypeptide; a vector comprising the polynucleotide; or a microorganism comprising the mutant polypeptide, a polynucleotide encoding the mutant polypeptide, or a vector comprising the polynucleotide; a culture of the microorganism; or a combination of two or more of these.

[0025] Another aspect of the present application provides use of the mutant polypeptide; a polynucleotide encoding the mutant polypeptide; a vector comprising the polynucleotide; or a microorganism comprising the mutant polypeptide, a polynucleotide encoding the mutant polypeptide, or a vector comprising the polynucleotide; a culture of the microorganism; or a combination of two or more of these, for producing L-glutamic acid. [Effects of the Invention]

[0026] When a microorganism containing the mutant polypeptide of the present application is cultured, it is possible to produce L-glutamic acid at a higher yield than a microorganism containing an existing non-modified polypeptide. DETAILED DESCRIPTION OF THE INVENTION

[0027] This will be explained in more detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the specific descriptions set forth below are not deemed to limit the category of this application. Furthermore, numerous papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the state of the art to which this application pertains and the contents of this application.

[0028] One aspect of the present application provides a mutant polypeptide in which the amino acid corresponding to position 351 of SEQ ID NO:1 has been substituted with another amino acid.

[0029] In this application, the term "major facilitator superfamily transporter (cgmA)" refers to a global transporter, which may be used interchangeably with the term "cgmA," and may refer to, but is not limited to, a major facilitator superfamily transporter encoded by the cgmA gene.

[0030] The gene encoding the major facilitator superfamily transporter may be derived from a microorganism of the genus Corynebacterium, specifically, cgmA derived from Corynebacterium glutamicum, but is not limited thereto.

[0031] Specifically, the major facilitator superfamily transporter protein may include, for example, the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60% or more homology or identity thereto, but is not limited thereto as long as it has major facilitator superfamily transporter activity. Specifically, the amino acid sequence may include SEQ ID NO: 1 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. SEQ ID NO: 1 can be obtained from publicly known databases such as NCBI's GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes). For example, the protein may be derived from the genus Corynebacterium or Corynebacterium glutamicum. More specifically, the protein may be a polypeptide / protein comprising the amino acid sequence set forth in SEQ ID NO: 1, but is not limited thereto. It is also clear that auxiliary proteins having amino acid sequences in which a portion of the sequence has been deleted, modified, substituted or added are also included within the scope of this application, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the protein.

[0032] Furthermore, the major facilitator superfamily transporter protein having the amino acid sequence of SEQ ID NO: 1 may have or contain a nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 2, or may consist of or be encoded by a polynucleotide essentially consisting of the sequence of SEQ ID NO: 2 or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 2, but is not limited thereto.

[0033] As used herein, the term "variant" refers to a polypeptide in which one or more amino acids have been conservatively substituted and / or modified, resulting in a difference from the amino acid sequence of the variant but maintaining its functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the variant. That is, the performance of the variant may be increased, unchanged, or decreased compared to the polypeptide before the modification. Some variants may also include variants in which one or more portions, such as the N-terminal leader sequence or transmembrane domain, have been removed. Other variants may include variants in which portions have been removed from the N- and / or C-termini of the mature protein. The term "mutant" may be used interchangeably with terms such as mutation, variant, mutant polypeptide, mutated protein, mutation, and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited to these terms as long as they are used to mean mutated.

[0034] Variants can also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide can be conjugated to a protein N-terminal signal (or leader) sequence involved in co- or post-translational protein transfer. The polypeptide can also be conjugated to other sequences or linkers that allow the polypeptide to be identified, purified, or synthesized.

[0035] The mutant polypeptide of the present application may be, but is not limited to, a mutant polypeptide in which the amino acid corresponding to position 351 of SEQ ID NO: 1 is substituted with another amino acid.

[0036] In one embodiment, the variant polypeptide of the present application may have a sequence identity of 60% or more and less than 100% with the amino acid sequence of SEQ ID NO: 1, specifically, a sequence identity of 80% or more and less than 100%, but is not limited thereto.

[0037] Specifically, the variants of the present application can include amino acid sequences that have at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity to the amino acid sequence set forth in SEQ ID NO: 1, in which the amino acid at position 351 from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid. It is also clear that variants having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also included within the scope of the present application, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the variants of the present application.

[0038] The "other amino acid" is not limited as long as it is an amino acid different from the amino acid before substitution. Meanwhile, when it is expressed in the present application that "a specific amino acid is substituted," it is self-evident that the amino acid is substituted with an amino acid different from the amino acid before substitution, even if it is not specifically stated that the amino acid is substituted with another amino acid.

[0039] Amino acids can generally be classified based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0040] Examples of such classifications include positively charged (basic) amino acids such as arginine, lysine, and histidine; negatively charged (acidic) amino acids such as glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) such as serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, amino acids can be classified into electrically charged amino acids (arginine, lysine, histidine, glutamic acid, and aspartic acid) and uncharged amino acids (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). Phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. Valine, leucine, and isoleucine can be classified as branched amino acids. As another example, the 20 amino acids can be classified by size, and the amino acids can be divided into five groups, starting from the group with the smallest volume: glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, and tyrosine, but are not necessarily limited thereto.

[0041] For example, when it is stated that "the amino acid corresponding to position 351 in SEQ ID NO: 1 is substituted with another amino acid," it means that the amino acid is substituted with, but is not limited to, asparagine, valine, glycine, alanine, glutamate, phenylalanine, arginine, aspartate, cysteine, glutamine, histidine, proline, serine, tyrosine, lysine, tryptophan, methionine, threonine, or leucine, excluding isoleucine.

[0042] Even if the present application describes a "protein having an amino acid sequence set forth in a particular SEQ ID NO," it is clear that proteins having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also used in the present application, so long as they have the same or corresponding activity as a protein consisting of the amino acid sequence of that SEQ ID NO. For example, if they have the same or corresponding activity as the mutant protein, this does not exclude addition of sequences before or after the amino acid sequence that do not change the function of the protein, naturally occurring mutations, silent mutations, or conservative substitutions thereof, and it is clear that even cases in which such additions or mutations of sequences exist fall within the scope of the present application.

[0043] The "Nth position" in the present application can include the Nth position and an amino acid position corresponding to the Nth position. Specifically, it can include an amino acid position corresponding to any amino acid residue in a mature polypeptide disclosed in a specific amino acid sequence. The specific amino acid sequence can be the amino acid sequence of SEQ ID NO: 1.

[0044] As used herein, the term "corresponding to" refers to the amino acid residue at the recited position in the polypeptide, or an amino acid residue that is similar, identical, or homologous to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position may be determining the particular amino acid in a sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0045] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, based on which each amino acid residue in the amino acid sequence can be numbered with reference to the numeric position of the corresponding amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described herein can identify amino acid positions or positions where variations such as substitutions, insertions or deletions occur compared to a query sequence (also referred to as a "reference sequence").

[0046] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) or the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) can be used, but is not limited thereto. Sequence alignment programs, pairwise sequence comparison algorithms, and the like known in the art can also be used appropriately.

[0047] In one embodiment, the mutant polypeptide may have the amino acid corresponding to position 351 of SEQ ID NO: 1 substituted with an amino acid selected from the group consisting of asparagine, valine, glycine, leucine, arginine, alanine, methionine, threonine, glutamine, proline, serine, tryptophan, phenylalanine, histidine, cysteine, tyrosine, lysine, aspartate, and glutamic acid, but is not limited thereto.

[0048] As an example of any of the above-mentioned embodiments, the mutant polypeptide provided in the present application may have the amino acid corresponding to the 351st position from the N-terminus of SEQ ID NO: 1 substituted with an amino acid having a polar or hydrophilic side chain (polar amino acid) selected from serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0049] As an example of any of the above-mentioned embodiments, the variant polypeptide provided in the present application may have the amino acid corresponding to the 351st position from the N-terminus of SEQ ID NO: 1 substituted with an amino acid having an uncharged side chain (also referred to as a neutral amino acid) selected from glycine, alanine, valine, leucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0050] As an example of any of the above-mentioned embodiments, the variant polypeptide provided in the present application may have the amino acid corresponding to the 351st position from the N-terminus of SEQ ID NO: 1 substituted with an amino acid selected from cysteine, proline, threonine, aspartic acid, and asparagine.

[0051] As an example of any of the above-mentioned embodiments, the mutant polypeptide of the present application may be, but is not limited to, a polypeptide in which the amino acid corresponding to position 351 of SEQ ID NO: 1 is substituted with asparagine.

[0052] For example, the variant polypeptide of the present application may include an amino acid sequence in which the asparagine, which is the amino acid corresponding to position 351 in the amino acid sequence set forth in SEQ ID NO: 1, is fixed and which has at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity to SEQ ID NO: 1. It is also clear that variant polypeptides having amino acid sequences in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also within the scope of the present application, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the variant polypeptide of the present application.

[0053] Meanwhile, a person skilled in the art can identify the amino acid corresponding to the 351st position of the amino acid sequence of SEQ ID NO: 1 in the present application in any amino acid sequence through sequence alignment known in the art, and it is obvious that when an "amino acid at a specific position in a particular SEQ ID NO" is mentioned in the present application, it also includes the "amino acid at the corresponding position" in any amino acid sequence, even if not otherwise specified.

[0054] In another embodiment, the variant polypeptide may consist of the amino acid sequence of SEQ ID NO:3.

[0055] Specifically, the variant polypeptides of the present application may have, comprise, consist of, or essentially consist of SEQ ID NO:3 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to said SEQ ID NO:3.

[0056] For example, the amino acid sequence may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally that do not alter the function of the variant of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0057] As used herein, 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 may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartate; aromatic amino acids include phenylalanine, tryptophan, and tyrosine, and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Amino acids can also be classified as those with electrically charged side chains or those with uncharged side chains. Charged amino acids include aspartic acid, glutamic acid, lysine, arginine, and histidine. Uncharged amino acids can be further classified as nonpolar or polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Conservative substitutions typically have little or no effect on the activity of the resulting polypeptide. Conservative substitutions typically have little or no effect on the activity of the resulting protein or polypeptide.

[0058] In another embodiment, the variant polypeptide may have the activity of a major enhancer superfamily enhancer.

[0059] Another aspect of the present application provides a polynucleotide encoding the mutant polypeptide.

[0060] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide units are linked in a long chain by covalent bonds, and a DNA or RNA chain of a certain length or more, and more specifically, a polynucleotide fragment encoding the above-mentioned variant.

[0061] Polynucleotides encoding the variant polypeptides of the present application may include, without limitation, any polynucleotide sequence that encodes the variant polypeptide of the present application. For example, the polynucleotide encoding the variant polypeptide of the present application may be, but is not limited to, a polynucleotide sequence that encodes the amino acid sequence of the variant polypeptide of the present application.

[0062] For example, it may include a nucleic acid sequence that encodes the amino acid sequence set forth in SEQ ID NO: 3. As an example of the present application, the polynucleotide of the present application may have or include SEQ ID NO: 4. Also, the polynucleotide of the present application may consist of or consist essentially of SEQ ID NO: 4.

[0063] The polynucleotides of the present application may have various modifications in the coding region within the scope that does not change the amino acid sequence of the variants of the present application, taking into account codon degeneracy or the codons preferred in the organism in which the variants of the present application are to be expressed. Therefore, it is clear that polynucleotides that can be translated into a polypeptide consisting of the amino acid sequence of the variants of the present application or a polypeptide having homology or identity thereto due to codon degeneracy are also included. For example, the polynucleotide of the present application may be SEQ ID NO: 4 or a degenerated sequence thereof.

[0064] For example, the polynucleotide of the present application may include, but is not limited to, a base sequence having 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity to the sequence of SEQ ID NO: 2, in which the codon encoding isoleucine, which is the amino acid corresponding to position 1052 of SEQ ID NO: 2, is substituted with a codon encoding an amino acid other than isoleucine, such as asparagine. It is also clear that, as long as the polynucleotide sequence has such homology or identity and encodes the amino acid sequence of the mutant polypeptide of the present application, variants having a polynucleotide sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also included within the scope of the present application.

[0065] As another example, the polynucleotide of the present application may have, comprise, or consist essentially of a nucleic acid sequence that is 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to SEQ ID NO: 4, but is not limited thereto. Alternatively, the sequence having the homology or identity may be one in which the codon corresponding to position 351 of SEQ ID NO: 3, which is encoded by SEQ ID NO: 4, is fixed to a codon encoding asparagine.

[0066] Furthermore, the polynucleotides of the present application include, without limitation, probes prepared from known gene sequences, for example, sequences that can hybridize under stringent conditions to a complementary sequence to all or part of the polynucleotide sequences of the present application.

[0067] The term "stringent conditions" refers to conditions that allow 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; F.M.A. Usubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, pp. 9.50-9.51, 11.7-11.8). For example, conditions include those under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, but polynucleotides with lower homology or identity do not hybridize with each other; and conditions include those under which washing is performed once, or specifically two to three times, at a salt concentration and temperature equivalent to those used in conventional Southern hybridization, such as 60°C, 1×SSC, and 0.1% SDS, specifically 60°C, 0.1×SSC, and 0.1% SDS, more specifically 68°C, 0.1×SSC, and 0.1% SDS.

[0068] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to one another. For example, for DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can also include isolated nucleic acid fragments that are complementary to entire sequences, as well as substantially similar nucleic acid sequences.

[0069] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the above-mentioned hybridization conditions, including a hybridization step at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0070] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementation of the polynucleotides, variables well known in the art (e.g., J. Sambrook et al., supra).

[0071] As used herein, the term "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0072] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences can generally hybridize to all or part of the sequence under moderately or highly stringent conditions. Hybridization obviously includes hybridization to polynucleotides containing common codons in polynucleotides or codons that take codon degeneracy into account.

[0073] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms such as the "FASTA" program using 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 implemented 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), as implemented in the 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 Diego, 1994, and [CARILLO ETA / .] (1988) SIAM J Applied Math 48:1073. For example, BLAST or ClustalW from the National Database Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0074] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that of Needleman et al. (1970), J Mol Biol. 48:443, as known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines a match as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, the terms "homology" or "identity," as used herein, indicate the relatedness between sequences.

[0075] Another aspect of the present application provides a vector comprising the polynucleotide of the present application. The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a microorganism.

[0076] In this application, the term "vector" may also include a DNA construct containing a base sequence of a polynucleotide encoding a polypeptide of interest operably linked to a suitable expression control region (or expression control sequence) so as to enable the expression of the polypeptide of interest in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable microorganism, the vector can replicate or function independently of the host genome, or it can be integrated into the genome itself.

[0077] The vectors used in this application are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage 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.

[0078] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome of a cell using a vector for chromosomal integration. The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of insertion into the chromosome may also be included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of insertion of the target nucleic acid molecule. A marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide, may be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0079] The term "transformation" as used herein refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a microorganism or microorganisms, thereby enabling the expression of the polypeptide encoded by the polynucleotide in the microorganism. A transformed polynucleotide may include any polynucleotide, whether it is inserted into the chromosome of the microorganism or located extrachromosomally, as long as it is expressible in the microorganism. The polynucleotide may also include DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form that can be introduced and expressed in the microorganism. For example, the polynucleotide may be introduced into the microorganism in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own 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 microorganism in its own form and operably linked to sequences necessary for its expression in the microorganism, but is not limited thereto.

[0080] In addition, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant of the present application.

[0081] Another aspect of the present application provides a microorganism comprising the mutant polypeptide, a polynucleotide encoding the mutant polypeptide, or a vector comprising the polynucleotide.

[0082] In one embodiment, the microorganism of the present application may be a microorganism capable of producing L-glutamic acid.

[0083] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially, and may be a microorganism in which a specific mechanism has been weakened or enhanced by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and may also be a microorganism that has been genetically modified for the production of a desired polypeptide, protein, or product. In this application, the terms "microorganism," "strain," and "microorganism" may be used interchangeably and without limitation.

[0084] In this application, the term "L-glutamic acid (L-glutamate)" refers to a type of amino acid, classified as a non-essential amino acid. It is known as the most common excitatory neurotransmitter in the central nervous system, and monosodium glutamate (MSG) has been developed and is widely used as a seasoning due to its rich flavor.

[0085] In this application, the term "microorganism capable of producing L-glutamic acid" refers to a prokaryotic or eukaryotic microbial strain capable of producing L-glutamic acid in vivo, and includes both microorganisms in which the ability to produce L-glutamic acid has been imparted to a parent strain that does not have the ability to produce L-glutamic acid, and microorganisms that have the ability to produce L-glutamic acid endogenously. The ability to produce L-glutamic acid can be imparted or enhanced by species improvement.

[0086] In one embodiment, the microorganism of the present application may be, but is not limited to, a microorganism that naturally has the ability to produce the mutant polypeptide of the present application or L-glutamic acid; or a microorganism into which the mutant of the present application or a polynucleotide encoding it (or a vector containing the polynucleotide) has been introduced into a parent strain that does not have the ability to produce the mutant polypeptide of the present application or L-glutamic acid, and / or which has been conferred the ability to produce L-glutamic acid.

[0087] In one embodiment, the microorganisms of the present application include, but are not limited to, microorganisms in which the chromosomal gene encoding the mutant polypeptide of the present application has been mutated to contain the mutant polypeptide sequence of the present application and / or microorganisms in which a vector containing a polynucleotide encoding the mutant polypeptide of the present application has been introduced to contain the mutant polypeptide of the present application.

[0088] In the present application, the term "non-modified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type or naturally occurring strain itself, or a strain before its traits are changed due to genetic mutations caused by natural or artificial factors. For example, the non-modified microorganism refers to a strain before a mutant polypeptide described herein has been introduced or has been introduced. The term "non-modified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "non-mutated strain," "non-modified strain," "non-mutated microorganism," or "reference microorganism."

[0089] The microorganism capable of producing L-glutamic acid of the present application may be, but is not limited to, a microorganism comprising one or more of the mutant of the present application, the polynucleotide of the present application, and the vector comprising the polynucleotide of the present application; a microorganism that has been modified to express the mutant of the present application or the polynucleotide of the present application; a microorganism (e.g., a recombinant strain) that expresses the mutant of the present application or the polynucleotide of the present application; or a microorganism (e.g., a recombinant strain) that has the activity of the mutant of the present application.

[0090] For example, the strain of the present application is a cell or microorganism that has been transformed with a vector containing a polynucleotide encoding the polynucleotide of the present application or a polynucleotide encoding the variant of the present application and expresses the variant of the present application. The strain of the present application can include all microorganisms capable of producing L-glutamic acid, including the variant of the present application. For example, the microorganism of the present application may be a recombinant strain having increased L-glutamic acid production ability, where a polynucleotide encoding the variant of the present application is introduced into a naturally occurring wild-type microorganism or a microorganism capable of producing L-glutamic acid, thereby expressing the mutant polypeptide. The recombinant strain having increased L-glutamic acid production ability may be, but is not limited to, a naturally occurring wild-type microorganism or a microorganism not modified with a major facilitator superfamily transporter (e.g., a microorganism expressing a wild-type major facilitator superfamily transporter or a microorganism not expressing the variant of the present application). For example, the microorganism of the present application having increased L-glutamic acid production ability may be, but is not limited to, a microorganism having increased L-glutamic acid production ability compared to a microorganism containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same. For example, the untransformed microorganism, which is the subject strain for comparing the increase in L-glutamic acid production ability, may be, but is not limited to, the wild-type Corynebacterium glutamicum strain ATCC13869 or ATCC13032, or the glutamic acid-producing strain KFCC11074 (KR 10-0292299 B1).

[0091] The microorganisms of the present application may include all microorganisms that can express the mutant polypeptides of the present application by various known methods other than the introduction of the nucleic acid or vector.

[0092] For example, the microorganism having increased L-glutamic acid production ability has an L-glutamic acid production ability that is about 1% or more, specifically about 1% or more, about 2% or more, about 3% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 46% or more, about 47% or more, about 48% or more, about 49% or more, about 50% or more, compared to the L-glutamic acid production ability of the parent strain or untransformed microorganism before mutation. % or more, about 51% or more, or about 52% or more (the upper limit is not particularly limited, and may be, for example, about 200% or less, about 150% or less, about 100% or less, about 90% or less, about 80% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, or about 53% or less), but is not limited thereto as long as there is an increase in the + value compared to the productivity of the parent strain or untransformed microorganism before mutation. In another example, the recombinant strain having increased L-glutamic acid producing ability may have an increased L-glutamic acid producing ability of about 1.01-fold or more, about 1.02-fold or more, about 1.03-fold or more, about 1.05-fold or more, about 1.1-fold or more, about 1.15-fold or more, about 1.20-fold or more, about 1.25-fold or more, about 1.30-fold or more, about 1.35-fold or more, about 1.40-fold or more, about 1.45-fold or more, about 1.46-fold or more, about 1.47-fold or more, about 1.48-fold or more, about 1.49-fold or more, about 1.50-fold or more, about 1.51-fold or more, or about 1.52-fold or more (the upper limit is not particularly limited, and may be, for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, about 2-fold or less, or about 1.53-fold or less) compared to the parent strain or untransformed microorganism before mutation, but is not limited thereto. The term "about" refers to a range that includes, but is not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​in a range that is equal to or similar to the numerical value following the term "about."

[0093] As a microorganism according to any one of the above-mentioned specific examples, the microorganism of the present application may be a microorganism belonging to the genus Corynebacteria, Escherichia, Erwinia, Serratia, Providencia, Pseudomonas, Leptospira, Salmonella, Brevibacteria, Hypomonas, Chromobacterium, or Norcardia, or a microorganism belonging to fungi or yeast, specifically, a microorganism of the genus Corynebacterium, but is not limited thereto.

[0094] As an example of the present application, the microorganism of the present application may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganism of the present application may be a Corynebacterium microorganism, more specifically, Corynebacterium glutamicum, but is not limited thereto.

[0095] The microorganism capable of producing L-glutamic acid of the present application may further comprise, but is not limited to, one or more mutant polypeptides selected from the group consisting of the following (a) and (b): a polynucleotide encoding the mutant polypeptide; or a vector comprising the polynucleotide: (a) a mutant polypeptide having TetR family transcriptional regulator activity, in which the amino acid corresponding to position 105 of SEQ ID NO: 5 is substituted with another amino acid; and (b) A mutant polypeptide having isocitrate decomposition enzyme activity, in which the amino acid sequence corresponding to positions 336 to 346 of SEQ ID NO: 9 has been deleted.

[0096] As an example of the present application, the microorganism capable of producing L-glutamic acid of the present application may further comprise a mutant polypeptide having TetR family transcriptional regulator activity, in which the amino acid corresponding to position 105 of SEQ ID NO: 5 has been substituted with another amino acid; a polynucleotide encoding the mutant polypeptide; or a vector comprising the polynucleotide, but is not limited thereto.

[0097] As used herein, the term "mutant polypeptide having TetR family transcriptional regulator activity" means a mutant polypeptide having TetR family transcriptional regulator activity which comprises one or more amino acid substitutions in the amino acid sequence of a polypeptide having TetR family transcriptional regulator activity; or a mutant of a polypeptide having TetR family transcriptional regulator activity which comprises one or more amino acid substitutions in the parent sequence, which is the amino acid sequence of a polypeptide having TetR family transcriptional regulator activity.

[0098] In the present application, the term "TetR-family transcriptional regulator (mmpLR)" refers to a global regulator having activities involved in efflux, cell division, and stress response, and may be used interchangeably with the term "mmpLR." It may refer to, but is not limited to, a TetR-family transcriptional regulator encoded by the mmpLR gene.

[0099] The gene encoding the TetR family transcriptional regulator may be derived from a microorganism of the genus Corynebacterium, specifically, mmpLR derived from Corynebacterium glutamicum, but is not limited thereto.

[0100] Specifically, the TetR family transcriptional regulator protein may include, for example, the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having 60% or more homology or identity thereto, but is not limited thereto as long as it has TetR family transcriptional regulator protein activity. Specifically, the amino acid sequence may include SEQ ID NO: 5 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. SEQ ID NO: 5 can be obtained from publicly known databases such as NCBI's GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes). For example, the protein may be derived from the genus Corynebacterium or Corynebacterium glutamicum. More specifically, the protein may be a polypeptide / protein comprising the amino acid sequence set forth in SEQ ID NO: 5, but is not limited thereto. It is also clear that auxiliary proteins having amino acid sequences in which a portion of the sequence has been deleted, modified, substituted or added are also included within the scope of this application, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the protein.

[0101] Furthermore, the TetR family transcriptional regulatory factor protein having the amino acid sequence of SEQ ID NO: 5 may have or contain the sequence of SEQ ID NO: 6 or a nucleotide sequence that has 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 6, or may consist of or be encoded by a polynucleotide that essentially consists of the sequence of SEQ ID NO: 6 or a nucleotide sequence that has 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 6, but is not limited thereto.

[0102] The mutant polypeptide having TetR family transcriptional regulator activity of the present application may be, but is not limited to, a mutant polypeptide having TetR family transcriptional regulator activity in which the amino acid corresponding to position 105 of SEQ ID NO: 5 has been substituted with another amino acid.

[0103] In one embodiment, the mutant polypeptide of the present application having TetR family transcriptional regulator activity may have a sequence identity of 60% or more and less than 100%, specifically, 80% or more and less than 100%, to the amino acid sequence of SEQ ID NO: 5, but is not limited thereto.

[0104] Specifically, the mutant polypeptides of the present application having TetR family transcriptional regulatory factor activity can include those having an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity to the amino acid sequence set forth in SEQ ID NO: 5, in which the amino acid at position 105 from the N-terminus of SEQ ID NO: 5 is substituted with another amino acid. It is also clear that mutants having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also included within the scope of the present application, so long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the mutants of the present application.

[0105] In one specific example, the mutant polypeptide of the present application having TetR family transcriptional regulator activity may be, but is not limited to, a polypeptide in which the amino acid corresponding to position 105 of SEQ ID NO: 5 is substituted with leucine.

[0106] For example, a variant polypeptide having TetR family transcriptional regulatory factor activity of the present application may include an amino acid sequence in which the leucine amino acid corresponding to position 105 in the amino acid sequence set forth in SEQ ID NO: 5 is fixed and which has at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity to SEQ ID NO: 5. It is clear that variant polypeptides having an amino acid sequence with such homology or identity, in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, are also included within the scope of the present application, so long as the amino acid sequence has the corresponding efficacy to the variant polypeptide of the present application.

[0107] In another embodiment, the mutant polypeptide having TetR family transcriptional regulator activity may consist of the amino acid sequence of SEQ ID NO:7.

[0108] Specifically, the mutant polypeptide having TetR family transcriptional regulator activity of the present application may have, comprise, consist of, or be essentially composed of SEQ ID NO: 7 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to SEQ ID NO: 7.

[0109] For example, the amino acid sequence may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally that do not alter the function of the variant of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0110] A polynucleotide encoding a mutant polypeptide having TetR family transcriptional regulator activity of the present application may include, but is not limited to, a nucleotide sequence having 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity to the sequence of SEQ ID NO: 6, in which the codon encoding proline, the amino acid corresponding to position 314 of SEQ ID NO: 6, is substituted with a codon encoding an amino acid other than proline, such as leucine. It is clear that the scope of the present application also includes polynucleotide sequences having such homology or identity and encoding the amino acid sequence of a mutant polypeptide having TetR family transcriptional regulator activity of the present application, in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added.

[0111] In another example, a polynucleotide encoding a mutant polypeptide having TetR family transcriptional regulator activity of the present application may have, comprise, or consist essentially of a nucleic acid sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to SEQ ID NO: 8, but is not limited thereto. Alternatively, the sequence having homology or identity may be one in which the codon corresponding to position 105 of SEQ ID NO: 7, encoded by SEQ ID NO: 8, is fixed to a codon encoding leucine.

[0112] As an example of the present application, the microorganism capable of producing L-glutamic acid of the present application may further comprise: a mutant polypeptide having isocitrate degrading enzyme activity, in which the amino acid sequence corresponding to positions 336 to 346 of SEQ ID NO: 9 has been deleted; a polynucleotide encoding the mutant polypeptide; or a vector containing the polynucleotide; but is not limited thereto.

[0113] As used herein, the term "variant polypeptide with isocitrate degrading activity" refers to a variant polypeptide with isocitrate degrading activity that contains one or more amino acid substitutions in the amino acid sequence of a polypeptide with isocitrate degrading activity; or a variant of a polypeptide with isocitrate degrading activity that contains one or more amino acid substitutions in the parent sequence, which is the amino acid sequence of a polypeptide with isocitrate degrading activity.

[0114] In the present application, the term "isocitrate lyase (aceA)" refers to an enzyme that produces succinate from isocitrate as a substrate, and may be used interchangeably with the term "aceA." The term may refer to an isocitrate lyase encoded by the aceA gene, but is not particularly limited thereto.

[0115] The gene encoding the isocitrate decomposing enzyme may be derived from a microorganism of the genus Corynebacterium, specifically, aceA derived from Corynebacterium glutamicum, but is not limited thereto.

[0116] Specifically, the isocitrate degrading enzyme protein may include, for example, the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having 60% or more homology or identity thereto, but is not limited thereto as long as it has isocitrate degrading enzyme protein activity. Specifically, the amino acid sequence may include SEQ ID NO: 9 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. The sequence of SEQ ID NO: 9 can be obtained from publicly known databases such as NCBI's GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes). For example, the isocitrate degrading enzyme protein may be derived from the genus Corynebacterium or Corynebacterium glutamicum. More specifically, the isocitrate degrading enzyme protein may be a polypeptide / protein comprising the amino acid sequence set forth in SEQ ID NO: 9, but is not limited thereto. It is also clear that auxiliary proteins having amino acid sequences in which a portion of the sequence has been deleted, modified, substituted or added are also included within the scope of this application, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the protein.

[0117] Furthermore, the isocitrate degrading enzyme protein having the amino acid sequence of SEQ ID NO: 9 may have or contain a nucleotide sequence of SEQ ID NO: 10 or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 10, or may consist of or be encoded by a polynucleotide essentially consisting of the sequence of SEQ ID NO: 10 or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 10, but is not limited thereto.

[0118] The mutant polypeptide having isocitrate decomposition enzyme activity of the present application may be, but is not limited to, a mutant polypeptide having isocitrate decomposition enzyme activity in which the amino acid sequence corresponding to positions 336 to 346 of SEQ ID NO: 9 has been deleted.

[0119] In one embodiment, the variant polypeptide having isocitrate degrading enzyme activity of the present application may have a sequence identity of 60% or more and less than 97% with the amino acid sequence of SEQ ID NO: 9, specifically, a sequence identity of 80% or more and less than 97%, but is not limited thereto.

[0120] Specifically, the mutant polypeptides of the present application having isocitrate degrading enzyme activity can include amino acid sequences that are at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 9, but lacking the amino acid sequence corresponding to positions 336 to 346 from the N-terminus of SEQ ID NO: 9. It is clear that the scope of the present application also includes mutants having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the mutants of the present application.

[0121] In another embodiment, the mutant polypeptide having isocitrate degrading enzyme activity may consist of the amino acid sequence of SEQ ID NO:11.

[0122] Specifically, the mutant polypeptide having isocitrate degrading enzyme activity of the present application may have, comprise, consist of, or essentially consist of SEQ ID NO:11 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to said SEQ ID NO:11.

[0123] For example, the amino acid sequence may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally that do not alter the function of the variant of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0124] A polynucleotide encoding a mutant polypeptide having isocitrate degrading enzyme activity of the present application may include, but is not limited to, a nucleotide sequence having 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity to the sequence of SEQ ID NO: 10, but lacking the nucleotide sequence corresponding to positions 1006 to 1038 of SEQ ID NO: 10. It is clear that the scope of the present application also includes variants having a polynucleotide sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, as long as the polynucleotide sequence has such homology or identity and encodes the amino acid sequence of a mutant polypeptide having isocitrate degrading enzyme activity of the present application.

[0125] In other examples, a polynucleotide encoding a variant polypeptide with isocitrate degrading enzyme activity of the present application may have, comprise, consist of, or consist essentially of a nucleic acid sequence that is 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to SEQ ID NO:12, but is not limited to this.

[0126] As a microorganism according to any one of the above-mentioned specific examples, the microorganism capable of producing L-glutamic acid of the present application may be a microorganism having increased L-glutamic acid production ability, including, but not limited to, a mutant polypeptide in which the amino acid corresponding to position 351 of SEQ ID NO: 1 is substituted with another amino acid; a mutant polypeptide having TetR family transcriptional regulator activity in which the amino acid corresponding to position 105 of SEQ ID NO: 5 is substituted with another amino acid; and a mutant polypeptide having isocitrate degrading enzyme activity in which the amino acid sequence corresponding to positions 336 to 346 of SEQ ID NO: 9 is deleted.

[0127] As a microorganism according to any one of the above-mentioned specific examples, the microorganism capable of producing L-glutamic acid of the present application may be a microorganism having increased L-glutamic acid production ability, including, but not limited to, a mutant polypeptide in which the amino acid corresponding to position 351 of SEQ ID NO: 1 is substituted with asparagine; a mutant polypeptide having TetR family transcriptional regulator activity in which the amino acid corresponding to position 105 of SEQ ID NO: 5 is substituted with leucine; and a mutant polypeptide having isocitrate degrading enzyme activity in which the amino acid sequence corresponding to positions 336 to 346 of SEQ ID NO: 9 is deleted.

[0128] Meanwhile, the microorganisms capable of producing L-glutamic acid in the present application include all of the following: natural wild-type microorganisms themselves; microorganisms that have improved L-glutamic acid production ability by strengthening or weakening the activity of a polypeptide related to the L-glutamic acid production mechanism; and microorganisms that have improved L-glutamic acid production ability by introducing or strengthening the activity of an external polypeptide.

[0129] While it has been known that microorganisms of the genus Corynebacterium can produce L-glutamic acid, their productivity is significantly low, and the genes and mechanisms underlying the production mechanism have not yet been fully elucidated. Therefore, the Corynebacterium microorganisms of the present application that are capable of producing L-glutamic acid may include natural wild-type microorganisms themselves, Corynebacterium microorganisms that have improved L-glutamic acid production ability by strengthening or weakening the activity of a polypeptide involved in the L-glutamic acid production mechanism, or Corynebacterium microorganisms that have improved L-glutamic acid production ability by introducing or strengthening the activity of an exogenous polypeptide.

[0130] As used herein, the term "enhancement" of a polypeptide activity means that the activity of the polypeptide is enhanced compared to its endogenous activity. The term "enhancement" may be used interchangeably with terms such as activation, up-regulation, overexpression, and enhancement.

[0131] The enhancement may include any of the following: exhibiting an activity that the molecule did not originally have, or exhibiting an activity that is improved compared to the intrinsic activity or the activity before modification.

[0132] For example, the "substance that exhibits an activity not originally possessed" may be, but is not limited to, "introduction of a protein." The introduction of a protein means that a gene not originally possessed by a microorganism is expressed in the microorganism to exhibit the activity of a specific protein, or that the activity of the protein is enhanced or improved compared to the endogenous activity or activity of the protein before modification. For example, a polynucleotide encoding a specific protein may be introduced into a chromosome of the microorganism, or a vector containing a polynucleotide encoding a specific protein may be introduced into the microorganism, and the activity may be exhibited.

[0133] The term "intrinsic activity" refers to the activity of a specific polypeptide that a parent strain or an unaltered microorganism originally possessed before the trait is changed due to genetic mutation caused by natural or artificial factors. This term may be used interchangeably with "activity before transformation."

[0134] The activity of a polypeptide being enhanced compared to its endogenous activity means that the activity and / or concentration (expression level) of a specific polypeptide is improved compared to that originally possessed by the parent strain or non-transformed microorganism before transformation.

[0135] By way of example, the enhancement may be, but is not limited to, an enhancement of the activity or concentration of the corresponding protein relative to the activity or concentration of the wild-type protein or the initial microbial strain, generally by about 1%, about 10%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%, up to about 1000% or about 2000% or more.

[0136] The activity of the polypeptide can be enhanced by introducing an exogenous polypeptide or by enhancing the activity of an endogenous polypeptide. Whether or not the activity of the polypeptide is enhanced can be confirmed by enhancing the activity, expression level, or amount of a product secreted from the polypeptide.

[0137] The activity of the polypeptide can be enhanced by various methods well known in the art, and is not limited thereto, as long as the activity of the polypeptide of interest can be enhanced compared to that of the microorganism before transformation. Specifically, the method may utilize genetic engineering and / or protein engineering, which are routine methods in molecular biology and well known to those skilled in the art, but is not limited thereto (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.).

[0138] Specifically, the activity of the polypeptide of the present application is enhanced by: 1) enhancing the intracellular copy number of a polynucleotide encoding a polypeptide; 2) Modification of the expression regulatory region of the gene on the chromosome encoding the polypeptide (e.g., mutation within the expression regulatory region, replacement with a sequence having stronger activity, or insertion of a sequence having stronger activity); 3) a modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the polypeptide activity is enhanced; 5) modifying a polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide's activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide's activity); 6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same; 7) codon optimization of the polynucleotide encoding the polypeptide; 8) Analyzing the tertiary structure of polypeptides and selectively deforming or chemically modifying exposed sites; 9) Regulation of the cellular localization of proteins (polypeptides); or 10) It may be a combination of two or more selected from the above 1) to 9), but is not particularly limited thereto.

[0139] for example, The 1) intracellular copy number of a polynucleotide encoding a polypeptide can be increased by introducing into a host cell a vector operably linked to the polynucleotide encoding the polypeptide, which can replicate and function independently of the host. Alternatively, one or more copies of the polynucleotide encoding the polypeptide can be introduced into a chromosome in the host cell. The introduction into a chromosome can be achieved by, but is not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell. The vector is as described above.

[0140] The replacement of the expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide with a sequence with stronger activity may involve, for example, mutation of the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or replacement with a sequence with stronger activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. For example, the original promoter may be replaced with a strong promoter, but this is not limited thereto.

[0141] Examples of known strong promoters include, but are not limited to, the cj1 to cj7 promoters (U.S. Patent No. 7,662,943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (U.S. Patent No. 10,584,338 B2), the O2 promoter (U.S. Patent No. 10,273,491 B2), the tkt promoter, and the yccA promoter.

[0142] The nucleotide sequence modification of the start codon or 5'-UTR region of the gene encoding the polypeptide may be, for example, but is not limited to, substituting an alternative start codon that results in a higher polypeptide expression rate than the endogenous start codon.

[0143] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above may be, but is not limited to, a sequence mutation such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, or an amino acid sequence or polynucleotide sequence modified to have stronger activity or to enhance the activity. Specifically, the replacement can be achieved by, but is not limited to, inserting the polynucleotide into a chromosome by homologous recombination. The vector used in this case may further contain a selection marker for detecting the presence or absence of insertion into the chromosome. The selection marker is as described above.

[0144] The introduction of an exogenous polynucleotide that exhibits the activity of a polypeptide (6) may be the introduction of an exogenous polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide into a host cell. The exogenous polynucleotide is not limited in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be any known transformation method appropriately selected by those skilled in the art. The introduced polynucleotide may be expressed in a host cell to produce a polypeptide, and its activity may be enhanced.

[0145] The codon optimization of the polynucleotide encoding the polypeptide (7) may be that of an endogenous polynucleotide that has been codon-optimized to enhance transcription or translation in a host cell, or that of an exogenous polynucleotide that has been codon-optimized to optimize transcription and translation in a host cell.

[0146] 8) Analyzing the tertiary structure of a polypeptide and selecting and modifying or chemically modifying exposed sites may involve, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and modifying exposed sites to be modified or chemically modified. The 9) intracellular localization of a protein (polypeptide) may be targeted to a specific organelle or specific intracellular space within a cell, for example, to the periplasm or cytoplasm through the addition or removal of a leader sequence that functions in targeting the protein (polypeptide), but is not limited thereto.

[0147] Such enhanced polypeptide activity may be, but is not limited to, an enhanced activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in a wild-type or untransformed microbial strain, or an increased amount of a product produced from the polypeptide.

[0148] In the present application, the term "weakening" of a polypeptide activity refers to a concept that encompasses all of the following: weakening of the activity compared to the endogenous activity, or absence of activity. The term "weakening" may be used interchangeably with terms such as deficiency, inactivation, deletion, disruption, down-regulation, decrease, attenuation, repression, and reduction.

[0149] For example, the attenuation means a state in which a protein exhibits activity but is not completely inactivated by deletion, and the activity of the protein is weakened compared to that of a non-transformed microorganism, a wild-type strain, or a parent strain, but is not limited thereto.

[0150] For example, the attenuation may be, but is not limited to, inactivation, which means that the protein is not expressed at all, or is expressed but has no or attenuated activity, compared to the parent strain or an unmodified strain.

[0151] The weakening may include cases where the activity of the polypeptide itself is weakened or eliminated compared to the activity of the polypeptide originally possessed by the microorganism due to a mutation in the polynucleotide encoding the polypeptide, cases where the overall polypeptide activity in the cell is lower than that of a wild-type strain due to inhibition of expression of the gene encoding it or inhibition of translation into the polypeptide, cases where the gene is not expressed at all, and cases where the gene is expressed but the polypeptide has no activity.

[0152] "Polypeptide activity is weakened compared to its intrinsic activity" means that the activity of a specific polypeptide is lower than that originally possessed by the parent strain or non-transformed microorganism before transformation. Whether or not the activity of the polypeptide is weakened can be confirmed by examining the activity, expression level, or amount of a product secreted from the polypeptide.

[0153] By way of example, the attenuation may be, but is not limited to, that the activity of the protein is less than about 100%, about 90% or less, about 80% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 5% or less, or 0% of the activity of the protein in the parent strain or untransformed microorganism before transformation.

[0154] For example, the inactivation means that the protein is not expressed at all, or is expressed but has no or weakened activity compared to an untransformed microorganism.

[0155] The activity of such a polypeptide can be attenuated by any method known in the art, but is not limited thereto, and can be achieved by applying various methods well known in the art (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.).

[0156] Specifically, the attenuation of the activity of the polypeptide of the present application is 1) Deletion of all or part of the gene encoding the polypeptide; 2) modification of the expression control region (or expression control sequence) so that expression of the gene encoding the polypeptide is attenuated; 3) modification of the amino acid sequence constituting the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence) so as to eliminate or attenuate the activity of the polypeptide; 4) modifying a polynucleotide sequence encoding a polypeptide so that the activity of the polypeptide is eliminated or attenuated (e.g., deleting / substituting / adding one or more nucleic acid bases in the nucleic acid base sequence of the polypeptide gene to encode a modified polypeptide so that the activity of the polypeptide is eliminated or attenuated); 5) a modification of the initiation codon or 5'-UTR region nucleotide sequence of a gene encoding a polypeptide; 6) introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementary 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 of a gene encoding a polypeptide in order to form a secondary structure that prevents ribosome attachment; 8) Addition of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the polynucleotide sequence encoding the polypeptide (reverse transcription engineering, RTE); 9) Regulation of the cellular localization of proteins (polypeptides); or 10) It may be a combination of two or more selected from the above 1) to 9), but is not particularly limited thereto.

[0157] for example, The deletion of a part or the whole of the gene encoding the polypeptide (1) may be removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacement with a polynucleotide having a partial deletion of the nucleotide sequence, or replacement with a marker gene.

[0158] Methods for deleting a portion or the entire polynucleotide include, but are not limited to, deleting the polynucleotide through homologous recombination using a vector for chromosomal insertion in a microorganism, or inducing mutations using light such as ultraviolet light or chemicals, and then selecting a strain in which the target gene is deleted from the resulting mutants. Methods for deleting a portion or the entire gene include DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene can be injected into the microorganism to cause homologous recombination, thereby deleting a portion or the entire gene. The injected nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

[0159] Furthermore, the modification of the expression regulatory sequence (2) may involve the generation of a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacement with a sequence having a weaker activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0160] Furthermore, the modification of the amino acid sequence or polynucleotide sequence in 3) and 4) above may be, but is not limited to, the generation of a mutation in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to attenuate the activity of the polypeptide, or the replacement with an amino acid sequence or polynucleotide sequence that has been improved to have weaker activity or an amino acid sequence or polynucleotide sequence that has been improved to have no activity. For example, but not limited to, the introduction of a mutation in the polynucleotide sequence to form a stop codon may inhibit or attenuate gene expression.

[0161] Furthermore, the nucleotide sequence modification of the start codon or 5'-UTR region of the gene encoding the polypeptide may be, for example, but is not limited to, substituting another start codon that has a lower polypeptide expression rate than the endogenous start codon.

[0162] 6) The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide can be carried out by referring to, 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].

[0163] 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure that prevents ribosome attachment may disable or slow down mRNA translation.

[0164] 8) Addition of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of a polynucleotide sequence encoding a polypeptide (reverse transcription engineering, RTE) may be performed to attenuate activity by creating an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide.

[0165] 9) Regulation of the intracellular location of a protein (polypeptide) may be targeting the protein (polypeptide) to a specific organelle or specific intracellular space within a cell, for example, but not limited to, targeting the protein (polypeptide) to the periplasm or cytoplasm through the addition or removal of a leader sequence that functions in targeting the protein (polypeptide).

[0166] Such attenuation of polypeptide activity may mean, but is not limited to, attenuation of the activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in a wild-type or untransformed microbial strain, or an increase in the amount of product produced from the polypeptide.

[0167] Another aspect of the present application provides a method for producing L-glutamic acid, comprising culturing the microorganism of the present application in a medium.

[0168] Specifically, the method for producing L-glutamic acid of the present application may include, but is not limited to, a step of culturing a microorganism containing a mutant of the present application, a polynucleotide of the present application, or a vector of the present application in a medium.

[0169] In the present application, the term "culturing" refers to growing the microorganism of the present application under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using appropriate media and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be, but is not limited to, a batch, continuous, and / or fed-batch culture.

[0170] As used herein, the term "culture medium" refers to a mixture of nutrients, primarily those required for culturing the microorganisms of the present application, and provides nutrients, including water, and growth factors essential for survival and growth. Specifically, the culture medium and other culture conditions used to culture the microorganisms of the present application can be any medium commonly used for culturing microorganisms, without any particular limitations. The microorganisms of the present application can be cultured under aerobic conditions in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while adjusting the temperature, pH, and other parameters. For example, culture media for Corynebacterium strains can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0171] In the present application, examples of the carbon source include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; amino acids such as glutamic acid, methionine, and lysine; and glycerol and propanediol. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can be used. A variety of other suitable carbon sources can also be used without limitation. These carbon sources can be used alone or in combination, and are not limited thereto.

[0172] Examples of the nitrogen source 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, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its hydrolyzed products, defatted soybean cake or its hydrolyzed products, etc. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0173] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or the corresponding sodium-containing salts. The inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and other compounds, including amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the culture medium in a batch or continuous manner. However, the present invention is not limited thereto.

[0174] During the cultivation of the microorganism of the present application, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner. Furthermore, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, to maintain an aerobic state in the medium, oxygen or an oxygen-containing gas can be injected into the medium, and to maintain an anaerobic or microaerobic state, no gas can be injected or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limiting.

[0175] In the culture of the present invention, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and the culture can be carried out for about 10 to 160 hours, but is not limited thereto.

[0176] The L-glutamic acid produced by the culture of the present application is either secreted into the medium or remains intracellularly.

[0177] In one embodiment, the method for producing L-glutamic acid of the present application may further include a step of preparing the microorganism of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order), for example, before the culturing step.

[0178] In one embodiment, the method for producing L-glutamic acid of the present application may further include a step of recovering L-glutamic acid from the culture medium (the medium in which the culture was performed) or the microorganism of the present application. The recovery step may be performed after the culturing step.

[0179] The recovery may involve collecting L-glutamic acid using a suitable method known in the art based on the microbial culture method of the present application, such as a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination of these methods can be used to recover L-glutamic acid from the medium or the microorganism using a suitable method known in the art.

[0180] The method for producing L-glutamic acid of the present application may further include a purification step. The purification can be performed using a suitable method known in the art. In one example, when the method for producing L-glutamic acid of the present application includes both a recovery step and a purification step, the recovery step and the purification step can be performed continuously or discontinuously, in any order, simultaneously, or integrated into one step, but are not limited thereto.

[0181] In the methods of the present application, the mutant polypeptide, polynucleotide, L-glutamic acid, etc. are as described above in other aspects.

[0182] Another aspect of the present application provides a composition for producing L-glutamic acid, comprising: a mutant polypeptide of the present application; a polynucleotide encoding the mutant polypeptide; a vector comprising the polynucleotide; or a microorganism comprising the mutant polypeptide of the present application, a polynucleotide encoding the mutant polypeptide, or a vector comprising the polynucleotide; a culture of the microorganism; or a combination of two or more of these.

[0183] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing L-glutamic acid, such as, but not limited to, a preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer, or isotonic agent.

[0184] In one embodiment, each component present in the compositions of the present application can be included in a microbiologically effective amount, or in an amount that can be suitably present in a production composition.

[0185] In the composition of the present application, the variant polypeptide, polynucleotide, L-glutamic acid, etc. are as described above in other aspects.

[0186] Another aspect of the present application provides the use of the mutant polypeptide of the present application in the production of L-glutamic acid.

[0187] The variant polypeptides of the present application, L-glutamic acid, etc., are as described above in other aspects.

[0188] Another aspect of the present application provides use of the microorganism of the present application for producing L-glutamic acid.

[0189] The variant polypeptides of the present application, L-glutamic acid, etc., are as described above in other aspects.

[0190] The present application will be described in more detail below through experimental examples. However, the following examples are merely preferred embodiments for illustrating the present application, and are not intended to limit the scope of the present application. Meanwhile, technical matters not described in this specification can be fully understood and easily performed by those of ordinary skill in the technical field of the present application or a similar technical field.

[0191] Example 1. Screening of mutants with increased glutamic acid production through artificial mutagenesis Example 1-1. Induction of artificial mutations through UV irradiation To select mutant strains with improved glutamic acid production, the target product of fermentation, wild-type Corynebacterium glutamicum (ATCC 13869) was first spread onto a nutrient medium containing agar and cultured at 30°C for 16 hours. Several hundred colonies thus obtained were then irradiated with UV light at room temperature to induce random mutations in the genome of the strain.

[0192] Example 1-2. Fermentation titer test of mutagenized strains and selection of strains Fermentation titer was tested using the mutant strains randomly mutated in Example 1-1.

[0193] Each colony was subcultured in nutrient medium and then cultured in fermentation medium for 5 hours. Then, 25% Tween 40 was added to each medium at a concentration of 0.4%, and each colony was cultured for an additional 32 hours.

[0194] <Nutrient medium> Glucose 1%, meat juice 0.5%, polypeptone 1%, sodium chloride 0.25%, yeast extract 0.5%, agar 2%, urea 0.2%, pH 7.2

[0195] <Fermentation medium> Glucose 6%, calcium carbonate 5%, ammonium sulfate 2.25%, monopotassium phosphate 0.1%, magnesium sulfate 0.04%, ferrous sulfate 10 mg / L, biotin 0.3 mg / L, thiamine hydrochloride 0.2 mg / L

[0196] Each colony was cultured under the above conditions, and mutant strains that produced L-glutamic acid equivalent to or greater than that of wild-type Corynebacterium glutamicum (ATCC 13869) were selected. The L-glutamic acid concentrations of the selected mutant strains were then measured using HPLC. The measured L-glutamic acid concentrations are shown in Table 1 below.

[0197] [Table 1]

[0198] Referring to Table 1, "ATCC13869-C4" and "ATCC13869-C7" were selected as mutant strains that exhibited increased glutamic acid production compared to the wild-type strain.

[0199] Example 2. Confirmation of mutations through gene sequencing To confirm the genetic mutations of the mutant strains, the genes of the ATCC13869-C4 and ATCC13869-C7 strains selected in Example 1-2 were compared with those of the wild-type strain.

[0200] As a result, it was confirmed that the ATCC13869-C4 and ATCC13869-C9 strains contain the same mutation (the 1052nd nucleotide of the polynucleotide sequence shown in SEQ ID NO: 2 is replaced with A) at a specific position in the gene cgmA (SEQ ID NO: 2), which encodes a major facilitator superfamily transporter.

[0201] Therefore, in the following Examples 3 and 4, it was determined whether the mutations affect the amount of glutamic acid produced by Corynebacterium microorganisms.

[0202] Example 3. Construction of mutant strains and confirmation of glutamic acid production - 1 Example 3-1. Construction of a strain with mutations introduced - 1 We constructed a mutant strain incorporating the mutation identified in Example 2. Specifically, we introduced the mutation (a substitution of A at nucleotide 1052 of the polynucleotide sequence shown in SEQ ID NO: 2) into wild-type Corynebacterium glutamicum (ATCC13869 and ATCC13032) and constructed a gene replacement vector to replace the isoleucine at nucleotide 351 of the major facilitator superfamily transporter shown in SEQ ID NO: 1 with asparagine. The gene fragment for constructing the vector was obtained by PCR using ATCC13869 genomic DNA as a template. Primers containing the polynucleotides shown in SEQ ID NOs: 13, 14, 15, and 16 were constructed based on information on the Corynebacterium glutamicum (ATCC13869) gene and its surrounding nucleotide sequence registered in the National Institutes of Health GenBank.

[0203] PCR was performed by denaturing at 95°C for 5 minutes, followed by 30 cycles of denaturing at 95°C for 20 seconds, annealing at 55°C for 20 seconds, and polymerizing at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. More specifically, a 500-bp polynucleotide was obtained by amplified using primers with SEQ ID NOs: 13 and 14, and another 500-bp polynucleotide was obtained by amplified using primers with SEQ ID NOs: 15 and 16. The two resulting gene fragments were ligated using infusion enzyme into pDZ vector (Korean Patent Registration No. 10-0924065 and International Patent Publication No. 2008-033001) digested with restriction enzymes BamHI and SalI to construct a gene replacement vector, designated "pDZ-cgmA(I351N)." The primer sequences used to construct this vector are listed in Table 2 below.

[0204] [Table 2]

[0205] The gene replacement vector was then transformed into a wild-type strain by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector had been integrated into the chromosome through recombination of the homologous sequences were selected on a medium containing 25 mg / L of kanamycin. Gene sequence analysis of the Corynebacterium glutamicum transformants after the secondary recombination confirmed that the targeted mutation had been introduced into the strains. The mutated strains were designated "ATCC13869::cgmA(I351N)" and "ATCC13032::cgmA(I351N)."

[0206] Example 3-2. Confirmation of glutamic acid production amount - 1 The mutant strains ATCC13869::cgmA(I351N) and ATCC13032::cgmA(I351N) prepared in Example 3-1 and their wild-type Corynebacterium glutamicum strains (ATCC13869 and ATCC13032) were cultured in the same manner as in Example 1-2.

[0207] After the cultivation was completed, the concentration of L-glutamic acid in each medium was measured. The measured L-glutamic acid concentrations are shown in Table 3 below.

[0208] [Table 3]

[0209] As shown in Table 3, the concentration of L-glutamic acid produced by the mutant Corynebacterium glutamicum ATCC13869::cgmA(I351N) strain was approximately 1.5 g / L (approximately 21%) higher than that produced by wild-type Corynebacterium glutamicum ATCC13869.

[0210] Furthermore, the concentration of L-glutamic acid produced by the mutant Corynebacterium glutamicum ATCC13032::cgmA(I351N) strain was approximately 0.8 g / L (approximately 22%) higher than that produced by wild-type Corynebacterium glutamicum ATCC13032.

[0211] That is, it was confirmed that the mutant of the present application increases the L-glutamic acid-producing ability of a microorganism.

[0212] Example 4. Construction of mutant strains and confirmation of glutamic acid production - 2 Example 4-1. Construction of a mutant strain - 2 A TerR family transcriptional regulator (mmpLR) mutant was further introduced into the mutant strains ATCC13869::cgmA(I351N) and ATCC13032::cgmA(I351N) constructed in Example 3-1.

[0213] Specifically, the mmpLR mutation (replacement of nucleotide 314 of the polynucleotide sequence shown in SEQ ID NO: 6 with T) was introduced into wild-type Corynebacterium glutamicum (ATCC13869 and ATCC13032), and a gene replacement vector was constructed to replace proline at position 105 of the TerR family transcriptional regulator shown in SEQ ID NO: 5 with leucine. The gene fragment for constructing the vector was obtained by PCR using ATCC13869 genomic DNA as a template. Primers containing the polynucleotides shown in SEQ ID NOs: 17, 18, 19, and 20 were constructed based on information on the Corynebacterium glutamicum (ATCC13869) gene and surrounding nucleotide sequence registered in the National Institutes of Health GenBank.

[0214] PCR was performed by denaturing at 95°C for 5 minutes, followed by 30 cycles of denaturing at 95°C for 20 seconds, annealing at 55°C for 20 seconds, and polymerizing at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. More specifically, a 500 bp polynucleotide was obtained by amplified using primers of SEQ ID NOs: 17 and 18, and another 500 bp polynucleotide was obtained by amplified using primers of SEQ ID NOs: 19 and 20. The two resulting gene fragments were ligated using infusion enzyme into pDZ vector (Korean Patent Registration No. 10-0924065 and International Patent Publication No. 2008-033001) digested with restriction enzymes BamHI and SalI to construct a gene replacement vector, designated "pDZ-mmpLR(P105L)." The primer sequences used to construct this vector are listed in Table 4 below.

[0215] [Table 4]

[0216] The gene replacement vector was then transformed into the ATCC13869::cgmA(I351N) and ATCC13032::cgmA(I351N) strains by homologous recombination. Strains in which the vector had been integrated into the chromosome through recombination of the homologous sequences were selected on a medium containing 25 mg / L kanamycin. Gene sequence analysis of the Corynebacterium glutamicum transformants after the secondary recombination confirmed that the targeted mutation had been introduced into the strains. The mutated strains were designated "ATCC13869::cgmA(I351N)_mmpLR(P105L)" and "ATCC13032::cgmA(I351N)_mmpLR(P105L)."

[0217] Example 4-2. Confirmation of glutamic acid production amount - 2 The mutant strains ATCC13869::cgmA(I351N)_mmpLR(P105L) and ATCC13032::cgmA(I351N)_mmpLR(P105L) prepared in Example 4-1 and their respective wild-type Corynebacterium glutamicum strains (ATCC13869 and ATCC13032) were cultured in the same manner as in Example 1-2.

[0218] After the cultivation was completed, the concentration of L-glutamic acid in each medium was measured. The measured L-glutamic acid concentrations are shown in Table 5 below.

[0219] [Table 5]

[0220] As shown in Table 5, the concentration of L-glutamic acid produced by the mutant Corynebacterium glutamicum ATCC13869::cgmA(I351N)_mmpLR(P105L) strain was found to be approximately 2.4 g / L (approximately 34%) higher than that produced by wild-type Corynebacterium glutamicum ATCC13869.

[0221] Furthermore, the concentration of L-glutamic acid produced by the mutant Corynebacterium glutamicum ATCC13032::cgmA(I351N)_mmpLR(P105L) strain was approximately 1.1 g / L (approximately 31%) higher than that produced by wild-type Corynebacterium glutamicum ATCC13032.

[0222] That is, it was confirmed that when a mutant polypeptide having TetR family transcriptional regulatory factor activity is further introduced into a microorganism containing the mutant polypeptide of the present application, the L-glutamic acid productivity is further increased.

[0223] Example 5. Construction of mutant strains and confirmation of glutamic acid production - 3 Example 5-1. Construction of a strain with mutations introduced - 3 An isocitrate decomposition enzyme (aceA) mutant was further introduced into the mutant strains ATCC13869::cgmA(I351N)_mmpLR(P105L) and ATCC13032::cgmA(I351N)_mmpLR(P105L) constructed in Example 4-1.

[0224] Specifically, a gene replacement vector was constructed to introduce the aceA mutation (a deletion of the nucleotide sequence corresponding to positions 1006 to 1038 of the polynucleotide sequence shown in SEQ ID NO: 10) into wild-type Corynebacterium glutamicum (ATCC13869 and ATCC13032) and delete the lysine corresponding to position 336 to the glutamine corresponding to position 346 of the isocitrate hydrolase enzyme shown in SEQ ID NO: 9 contained in the strain. The gene fragment for constructing the vector was obtained by PCR using ATCC13869 genomic DNA as a template. Primers containing the polynucleotides shown in SEQ ID NOs: 21, 22, 23, and 24 were constructed based on information on the Corynebacterium glutamicum (ATCC13869) gene and surrounding nucleotide sequence registered in the National Institutes of Health GenBank.

[0225] PCR was performed by denaturing at 95°C for 5 minutes, followed by 30 cycles of denaturing at 95°C for 20 seconds, annealing at 55°C for 20 seconds, and polymerizing at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. More specifically, a 500 bp polynucleotide was obtained by amplified using primers with SEQ ID NOs: 21 and 22, and another 500 bp polynucleotide was obtained by amplified using primers with SEQ ID NOs: 23 and 24. The two resulting gene fragments were ligated using infusion enzyme into pDZ vector (Korean Patent Registration No. 10-0924065 and International Patent Publication No. 2008-033001) digested with restriction enzymes BamHI and SalI to construct a gene replacement vector, designated "pDZ-aceA_deletion." The primer sequences used to construct this vector are listed in Table 6 below.

[0226] [Table 6]

[0227] The gene replacement vector was then transformed into the ATCC13869::cgmA(I351N)_mmpLR(P105L) and ATCC13032::cgmA(I351N)_mmpLR(P105L) strains by homologous recombination. Strains in which the vector had been integrated into the chromosome by homologous recombination were selected on a medium containing 25 mg / L kanamycin. Subsequently, gene sequence analysis was performed on the Corynebacterium glutamicum transformants after the secondary recombination was completed, and it was confirmed that the target mutation had been introduced into the strains. The strains into which the mutation had been introduced were named "ATCC13869::cgmA(I351N)_mmpLR(P105L)_aceA_deletion" and "ATCC13032::cgmA(I351N)_mmpLR(P105L)_aceA_deletion."

[0228] Example 5-2. Confirmation of glutamic acid production amount - 3 The mutant strains ATCC13869::cgmA(I351N)_mmpLR(P105L)_aceA_deletion and ATCC13032::cgmA(I351N)_mmpLR(P105L)_aceA_deletion prepared in Example 5-1 and their wild-type Corynebacterium glutamicum strains (ATCC13869 and ATCC13032) were cultured in the same manner as in Example 1-2.

[0229] After the cultivation was completed, the concentration of L-glutamic acid in each medium was measured. The measured L-glutamic acid concentrations are shown in Table 7 below.

[0230] [Table 7]

[0231] As shown in Table 7, the concentration of L-glutamic acid produced by the mutant Corynebacterium glutamicum ATCC13869::cgmA(I351N)_mmpLR(P105L)_aceA_deletion strain was found to be approximately 3.3 g / L (47%) higher than that produced by wild-type Corynebacterium glutamicum ATCC13869.

[0232] Furthermore, the concentration of L-glutamic acid produced by the mutant Corynebacterium glutamicum ATCC13032::cgmA(I351N)_mmpLR(P105L)_aceA_deletion strain was approximately 1.6 g / L (approximately 44%) higher than that produced by wild-type Corynebacterium glutamicum ATCC13032.

[0233] That is, it was confirmed that when a mutant polypeptide having TetR family transcriptional regulator activity and a mutant polypeptide having isocitrate decomposition enzyme activity are further introduced into a microorganism containing the mutant polypeptide of the present application, the L-glutamic acid production ability is further increased.

[0234] Example 6. Confirmation of glutamic acid production by the mutant KFCC11074 strain Example 6-1. Construction of a strain with mutations introduced - 1 To confirm whether the mutation would have a similar effect on strains with increased glutamic acid production, other than the wild-type strain, the mutation was introduced into the KFCC11074 strain (Korean Patent Registered Publication No. 10-0292299), which is known to be a glutamic acid-producing strain.

[0235] Specifically, the pDZ-cgmA(I351N) vector prepared in Example 3-1 was transformed into the KFCC11074 strain by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector had been integrated into the chromosome through recombination of the homologous sequences were selected in a medium containing 25 mg / L kanamycin. Subsequently, gene sequence analysis of the Corynebacterium glutamicum transformant strain after the secondary recombination was performed confirmed that the targeted mutation had been introduced into the strain, and the mutated strain was named "KFCC11074_cgmA(I351N)."

[0236] Example 6-2. Construction of a strain with mutations introduced - 2 A TerR family transcriptional regulator (mmpLR) mutant was further introduced into the mutant strain KFCC11074_cgmA(I351N) constructed in Example 6-1.

[0237] Specifically, the pDZ-mmpLR(P105L) vector prepared in Example 4-1 was transformed into the KFCC11074_cgmA(I351N) strain by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector had been integrated into the chromosome through recombination of the homologous sequences were selected in a medium containing 25 mg / L kanamycin. Subsequently, gene sequence analysis of the Corynebacterium glutamicum transformant strain after the secondary recombination was performed confirmed that the targeted mutation had been introduced into the strain, and the mutated strain was named "KFCC11074_cgmA(I351N)_mmpLR(P105L)."

[0238] Example 6-3. Construction of a strain with introduced mutations - 3 An isocitrate decomposition enzyme (aceA) mutant was further introduced into the mutant strain KFCC11074_cgmA(I351N)_mmpLR(P105L) constructed in Example 6-2.

[0239] Specifically, the pDZ-aceA_deletion vector prepared in Example 5-1 was transformed into the KFCC11074_cgmA(I351N)_mmpLR(P105L) strain by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector had been integrated into the chromosome through recombination of the homologous sequences were selected in a medium containing 25 mg / L kanamycin. Subsequently, gene sequence analysis of the Corynebacterium glutamicum transformant after the secondary recombination was performed confirmed that the targeted mutation had been introduced into the strain. The mutated strain was designated "KFCC11074_cgmA(I351N)_mmpLR(P105L)_aceA_deletion."

[0240] Example 7. Confirmation of glutamic acid production by the mutant KFCC11074 strain Example 7-1. Confirmation of glutamic acid production amount - 1 The mutant strain KFCC11074_cgmA(I351N) prepared in Example 6-1 and the wild-type KFCC11074 strain were cultured in the same manner as in Example 1-2.

[0241] After the cultivation was completed, the concentration of L-glutamic acid in each medium was measured. The measured L-glutamic acid concentrations are shown in Table 8 below.

[0242] [Table 8]

[0243] As shown in Table 8, the concentration of L-glutamic acid produced by the mutant Corynebacterium glutamicum KFCC11074_cgmA(I351N) strain was found to be approximately 1.3 g / L (approximately 23%) higher than the concentration of L-glutamic acid produced by the unmutated Corynebacterium glutamicum KFCC11074.

[0244] That is, it was confirmed that the mutations of the present application increase the L-glutamic acid productivity of microorganisms even in strains with increased glutamic acid productivity.

[0245] Example 7-2. Confirmation of glutamic acid production amount - 2 The mutant strains KFCC11074_cgmA(I351N) and KFCC11074_cgmA(I351N)_mmpLR(P105L) prepared in Examples 6-1 and 6-2, respectively, and the wild-type KFCC11074 strain were cultured in the same manner as in Example 1-2.

[0246] After the cultivation was completed, the concentration of L-glutamic acid in each medium was measured. The measured L-glutamic acid concentrations are shown in Table 9 below.

[0247] [Table 9]

[0248] As shown in Table 9, the concentration of L-glutamic acid produced by the mutant Corynebacterium glutamicum KFCC11074_cgmA(I351N)_mmpLR(P105L) strain was found to be approximately 2.2 g / L (approximately 39%) higher than the concentration of L-glutamic acid produced by the unmutated Corynebacterium glutamicum KFCC11074.

[0249] That is, when a mutant polypeptide having TetR family transcriptional regulator activity is further introduced into a microorganism containing the mutant polypeptide of the present application, it was confirmed that the L-glutamic acid production ability of the microorganism is further increased even in a strain with increased glutamic acid production ability.

[0250] Example 7-3. Confirmation of glutamic acid production amount - 3 The mutant strains KFCC11074_cgmA(I351N), KFCC11074_cgmA(I351N)_mmpLR(P105L), and KFCC11074_cgmA(I351N)_mmpLR(P105L)_aceA_deletion prepared in Examples 6-1, 6-2, and 6-3, respectively, and the wild-type KFCC11074 strain were cultured in the same manner as in Example 1-2.

[0251] After the cultivation was completed, the concentration of L-glutamic acid in each medium was measured. The measured L-glutamic acid concentrations are shown in Table 10 below.

[0252] [Table 10]

[0253] As shown in Table 10, the concentration of L-glutamic acid produced by the mutant Corynebacterium glutamicum KFCC11074_cgmA(I351N)_mmpLR(P105L)_aceA_deletion strain was found to be approximately 3.1 g / L (approximately 52%) higher than the concentration of L-glutamic acid produced by the unmutated Corynebacterium glutamicum KFCC11074.

[0254] That is, when a mutant polypeptide having TetR family transcriptional regulator activity and a mutant polypeptide having isocitrate degrading enzyme activity are further introduced into a microorganism containing the mutant polypeptide of the present application, it was confirmed that the L-glutamic acid production ability of the microorganism is further increased even in a strain with increased glutamic acid production ability.

[0255] From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including within the meaning and scope of the claims below, and any modifications or variations derived from the equivalent concepts thereof, rather than the above detailed description.

Claims

1. A mutant polypeptide in which the amino acid corresponding to position 351 of SEQ ID NO: 1 has been replaced with another amino acid.

2. 2. The mutant polypeptide of claim 1, wherein the amino acid corresponding to position 351 of SEQ ID NO: 1 is substituted with asparagine.

3. The mutant polypeptide of claim 1, wherein the mutant polypeptide comprises a sequence having 80% or more sequence identity with the amino acid sequence of SEQ ID NO:

1.

4. A polynucleotide encoding the mutant polypeptide of any one of claims 1 to 3.

5. A microorganism comprising the mutant polypeptide according to any one of claims 1 to 3, a polynucleotide encoding said mutant polypeptide, or a vector comprising said polynucleotide.

6. The microorganism according to claim 5, further comprising any one or more mutant polypeptides selected from the group consisting of (a) and (b): a polynucleotide encoding the mutant polypeptide; or a vector comprising the polynucleotide: (a) a mutant polypeptide having TetR family transcriptional regulator activity, in which the amino acid corresponding to position 105 of SEQ ID NO: 5 is substituted with another amino acid; and (b) A mutant polypeptide having isocitrate decomposition enzyme activity, in which the amino acid sequence corresponding to positions 336 to 346 of SEQ ID NO:9 has been deleted.

7. The microorganism described in claim 6, wherein the mutant polypeptide (a) having TetR family transcriptional regulator activity is a mutant polypeptide having TetR family transcriptional regulator activity in which the amino acid corresponding to position 105 of SEQ ID NO: 5 is substituted with leucine.

8. The mutant polypeptide having TetR family transcriptional regulatory factor activity of (a) above consists of the amino acid sequence of SEQ ID NO: 7, The microorganism according to claim 6, wherein the mutant polypeptide (b) having isocitrate decomposition enzyme activity consists of the amino acid sequence of SEQ ID NO:

11.

9. 6. The microorganism according to claim 5, wherein the microorganism has an increased ability to produce L-glutamic acid compared to a microorganism containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the polypeptide.

10. The microorganism according to claim 5 , wherein the microorganism is a Corynebacterium microorganism.

11. The microorganism according to claim 10, wherein the Corynebacterium microorganism is Corynebacterium glutamicum.

12. A method for producing L-glutamic acid, comprising culturing the microorganism of claim 5 in a medium.

13. 13. The method of claim 12, further comprising recovering L-glutamic acid from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium.

14. A composition for producing L-glutamic acid, comprising: a mutant polypeptide according to any one of claims 1 to 3; a polynucleotide encoding said mutant polypeptide; a vector comprising said polynucleotide; or a microorganism comprising said mutant polypeptide, a polynucleotide encoding said mutant polypeptide, or a vector comprising said polynucleotide; a culture of said microorganism; or a combination of two or more of these.

15. Use of the mutant polypeptide according to any one of claims 1 to 3; a polynucleotide encoding said mutant polypeptide; a vector comprising said polynucleotide; or a microorganism comprising said mutant polypeptide, a polynucleotide encoding said mutant polypeptide, or a vector comprising said polynucleotide; a culture of said microorganism; or a combination of two or more of these, for the production of L-glutamic acid.

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