Variant polypeptide and method for producing l-glutamic acid using same

Abstract

The present application relates to: a novel variant polypeptide; a polynucleotide encoding the variant polypeptide; a microorganism comprising the variant polypeptide, a polynucleotide encoding the variant polypeptide, or a vector comprising the polynucleotide; a method for producing L-glutamic acid, comprising a step of culturing the microorganism in a culture medium; and the use of the variant polypeptide or microorganism for the production of L-glutamic acid.

Description

Technical Field

[0001] The present disclosure relates to novel variant polypeptides; polynucleotides encoding the variant polypeptides; microorganisms comprising the variant polypeptides, polynucleotides encoding the variant polypeptides, or vectors comprising the polynucleotides; a method for producing L-glutamic acid, comprising culturing the microorganism in a culture medium; and uses of the variant polypeptides or microorganisms for producing L-glutamic acid. Background Art

[0002] L-glutamic acid is a representative amino acid produced by fermentation and has a unique taste. Therefore, L-glutamic acid is one of the important amino acids widely used not only in the food industry but also in medicine, other animal feed and other fields.

[0003] Examples of conventional methods for producing L-glutamic acid include fermentation methods using coryneform bacteria including microorganisms of the genus Brevibacterium, Corynebacterium, and variants thereof ("AminoAcid Fermentation", Gakkai Shuppan Center, pp. 195-215, 1986); and methods using Escherichia coli and microorganisms belonging to the genus Bacillus, Streptomyces, Penicillium, Klebsiella, Erwinia, Pantoea, etc. (U.S. Application Publication No. 3220929, U.S. Patent No. 6682912).

[0004] In addition, various researches have been carried out to effectively produce amino acid; For example, efforts have been made to develop microorganisms or fermentation process technology for efficient production of amino acid. Especially, the approach method specific to target substance has been developed, such as the expression of the gene of the enzyme of amino acid biosynthesis in the enhanced coding participation Corynebacterium strain or the unwanted gene of disappearance amino acid biosynthesis (Korean Patent No. 10-0924065 and 1208480). In addition to these methods, the method for removing the gene that does not participate in amino acid production and the method for removing the unclear gene of its amino acid whose function is produced have also been used. However, the method for studying the effective production of L-Glutamic acid with high yield remains a growing demand. Summary of the Invention

[0005] Technical issues

[0006] The present inventors have developed a variant of a major facilitator superfamily transporter, a Corynebacterium glutamicum strain comprising the variant, and a method for producing L-glutamic acid using the strain, thereby completing the present disclosure.

[0007] Technical Solution

[0008] One object of the present disclosure is to provide a variant polypeptide, wherein the amino acid corresponding to position 351 of SEQ ID NO: 1 is substituted with another amino acid.

[0009] In one embodiment, the variant polypeptide may be one in which the amino acid corresponding to position 351 of SEQ ID NO: 1 is 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 another embodiment, the variant polypeptide may have the activity of a major facilitator superfamily transporter.

[0013] Another object of the present disclosure is to provide a polynucleotide encoding the variant polypeptide.

[0014] Another object of the present disclosure is to provide a microorganism comprising the variant polypeptide, a polynucleotide encoding the variant polypeptide, or a vector containing the polynucleotide.

[0015] As the microorganism according to any one of the above embodiments, the microorganism may further comprise: any one or more variant polypeptides selected from the group consisting of (a) and (b); a polynucleotide encoding the variant polypeptide; or a vector containing the polynucleotide:

[0016] Wherein (a) a variant polypeptide having the activity of a TetR family transcriptional regulatory factor, wherein the amino acid corresponding to position 105 of SEQ ID NO: 5 is replaced by another amino acid; and

[0017] (b) A variant polypeptide having isocitrate lyase activity, wherein the amino acid sequence corresponding to positions 336 to 346 of SEQ ID NO: 9 is deleted.

[0018] As a microorganism according to any one of the above embodiments, (a) the variant polypeptide having the activity of a TetR family transcriptional regulatory factor can be a variant polypeptide having the activity of a TetR family transcriptional regulatory factor in which the amino acid corresponding to position 105 of SEQ ID NO: 5 is replaced by leucine.

[0019] As a microorganism according to any one of the above embodiments, (a) the variant polypeptide having TetR family transcription regulatory factor activity can be composed of the amino acid sequence of SEQ ID NO: 7; and (b) the variant polypeptide having isocitrate lyase activity can be composed of the amino acid sequence of SEQ ID NO: 11.

[0020] As the microorganism according to any one of the above embodiments, the microorganism may have increased L-glutamic acid-producing ability compared to a microorganism comprising the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same.

[0021] As the microorganism according to any one of the above embodiments, the microorganism may be a microorganism of the genus Corynebacterium.

[0022] As the microorganism according to any one of the above embodiments, the Corynebacterium microorganism may be Corynebacterium glutamicum.

[0023] Another object of the present disclosure is to provide a method for producing L-glutamic acid, comprising culturing the microorganism in a culture medium.

[0024] In one embodiment, the method may further include recovering L-glutamic acid from the cultured microorganism, the culture product of the microorganism, the fermentation product of the microorganism, or the culture medium.

[0025] Another object of the present disclosure is to provide a composition for producing L-glutamic acid, comprising: the variant polypeptide; a polynucleotide encoding the variant polypeptide; a vector containing the polynucleotide; a microorganism comprising the variant polypeptide, the polynucleotide encoding the variant polypeptide, or the vector containing the polynucleotide; a culture product of the microorganism; or a combination of two or more thereof.

[0026] Another object of the present disclosure is to provide the variant polypeptide; a polynucleotide encoding the variant polypeptide; a vector containing the polynucleotide; a microorganism comprising the variant polypeptide, the polynucleotide encoding the variant polypeptide, or the vector containing the polynucleotide; a culture product of the microorganism; or a combination of two or more thereof for producing L-glutamic acid.

[0027] Beneficial effects

[0028] When a microorganism comprising the variant polypeptide of the present disclosure is cultured, L-glutamic acid can be produced at a high yield compared to a microorganism having the existing unmodified polypeptide. DETAILED DESCRIPTION

[0029] The present disclosure will be described in detail below. At the same time, each description and embodiment disclosed herein can be applied to other descriptions and embodiments, respectively. That is to say, all combinations of the various elements disclosed herein fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific description described below. In addition, many papers and patent documents are cited throughout the specification. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety, and will more clearly describe the level of the technical field to which the present disclosure belongs and the contents of the present disclosure.

[0030] One aspect of the present disclosure provides a variant polypeptide, wherein the amino acid corresponding to position 351 of SEQ ID NO: 1 is substituted with another amino acid.

[0031] As used herein, the term "major facilitator superfamily transporter (cgmA)" is a global transporter, may be used interchangeably with the term "cgmA", and may be a major facilitator superfamily transporter encoded by a cgmA gene, but is not particularly limited thereto.

[0032] The gene encoding the major facilitator superfamily transporter may be derived from a microorganism of the genus Corynebacterium, and may particularly be derived from cgmA of Corynebacterium glutamicum, but is not limited thereto.

[0033] Specifically, the major facilitator superfamily transporter can 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 the activity of a major facilitator superfamily transporter. Specifically, the amino acid sequence can include the amino acid sequence of 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. The sequence of SEQ ID NO: 1 can be obtained from known databases, such as GenBank of NCBI or the Kyoto Gene and Genome Database (KEGG). In one example, the amino acid sequence can be derived from a microorganism of the genus Corynebacterium or Corynebacterium glutamicum, more specifically, it can be a polypeptide / protein comprising the amino acid sequence shown in SEQ ID NO: 1, but is not limited thereto. In addition, it is obvious that any accessory protein having an amino acid sequence in which part of the sequence is deleted, modified, substituted or added can also fall within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits the corresponding efficacy of the protein.

[0034] In addition, the major facilitator superfamily transporter having the amino acid sequence of SEQ ID NO: 1 can be encoded by a polynucleotide having or including the 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 nucleotide sequence of SEQ ID NO: 2, or can consist of or essentially consist of the 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 nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

[0035] As used herein, the term "variant" refers to a polypeptide having one or more amino acids that are different from the amino acid sequence before the variant is mutated by conservative substitutions and / or modifications, wherein conservative substitutions and / or modifications retain the function and properties of the polypeptide. Such variants can generally be identified by modifying one or more amino acid sequences of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant can be enhanced, unchanged, or reduced relative to the polypeptide before mutation. In addition, some variants may include one or more regions, such as those in which an N-terminal leader sequence or a transmembrane domain has been removed. In addition, other variants may include those in which a certain region has been removed from the N- and / or C-terminus of the mature protein. The term "variant" can be used interchangeably with terms such as modification, modified protein, mutant, mutant protein, disproportionation (divergent), variant, etc. without limitation, as long as these terms are used to represent variation.

[0036] In addition, the variants may 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 may be conjugated to a signal (or leader) sequence involved in protein transfer at the N-terminus in a co-translational or post-translational manner. In addition, the polypeptide may also be conjugated to another sequence or linker to identify, purify or synthesize the polypeptide.

[0037] The variant polypeptide disclosed herein may be one in which the amino acid corresponding to position 351 of SEQ ID NO: 1 is substituted with another amino acid, but is not limited thereto.

[0038] In one embodiment, the variant polypeptide of the present disclosure may have 60% or more and less than 100% sequence homology with the amino acid sequence of SEQ ID NO: 1, specifically, 80% or more and less than 100% sequence homology, but is not limited thereto.

[0039] Specifically, the variant polypeptides of the present disclosure may include those in which the amino acid at position 351 from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid in an amino acid sequence having 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 with the amino acid sequence shown in SEQ ID NO: 1. In addition, it is obvious that any variant having an amino acid sequence in which a portion of the sequence is deleted, modified, substituted, conservatively substituted or added may also fall within the scope of the present disclosure, as long as it has such homology or identity and exhibits the efficacy corresponding to the variant of the present disclosure.

[0040] There is no limitation on the “another amino acid” as long as it is an amino acid other than the amino acid before substitution. Meanwhile, in the present disclosure, when it is expressed that “a specific amino acid has been substituted”, it is obvious that the amino acid has been substituted with an amino acid different from the amino acid before substitution, even if it is not specifically stated that the amino acid has been substituted with a different amino acid.

[0041] Amino acids can generally be classified based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0042] Amino acids can be divided into the following groups:

[0043] In one example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with non-polar side chains (non-polar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In another example, amino acids with charged side chains (charged amino acids) include arginine, lysine, histidine, glutamic acid, and aspartic acid; and amino acids with uncharged side chains (referred to as uncharged amino acids; neutral amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In another example, aromatic amino acids include phenylalanine, tryptophan, and tyrosine. In another example, branched-chain amino acids include valine, leucine, and isoleucine. In another example, the 20 amino acids can be divided into five groups based on their size, starting with the relatively small amino acid group, namely 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 the classification of amino acids is not limited thereto.

[0044] For example, when it is described as "the amino acid corresponding to position 351 of SEQ ID NO: 1 is substituted with another amino acid", it may mean that the amino acid is substituted with asparagine, valine, glycine, alanine, glutamic acid, phenylalanine, arginine, aspartic acid, cysteine, glutamine, histidine, proline, serine, tyrosine, lysine, tryptophan, methionine, threonine or leucine other than isoleucine, but is not limited thereto.

[0045] Although described in the present disclosure as "a protein having an amino acid sequence described by a specific sequence number," it is apparent that any protein having an amino acid sequence in which a portion of the sequence is deleted, modified, substituted, conservatively substituted, or added can also be used in the present disclosure, as long as it has the same or corresponding activity as a protein consisting of an amino acid sequence with a corresponding sequence number. For example, sequence additions, naturally occurring mutations, silent mutations, or conservative substitutions upstream or downstream of an amino acid sequence that do not alter the function of the protein are not excluded, as long as the protein has the same or corresponding activity as the variant protein, and it is apparent to those skilled in the art that such sequence additions or mutations fall within the scope of the present disclosure.

[0046] The "Nth position" of the present disclosure may include the Nth position and the amino acid position corresponding to the Nth position. Specifically, it may include the amino acid position corresponding to any amino acid residue in the mature polypeptide disclosed in the specific amino acid sequence. The specific amino acid sequence may be the amino acid sequence of SEQ ID NO: 1.

[0047] As used herein, the term "corresponding to" refers to an amino acid residue at a listed position in a peptide, or an amino acid residue that is similar, identical, or homologous to a listed residue in a peptide. Identifying an amino acid at a corresponding position can be a specific amino acid in a sequence that determines a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or a reference protein.

[0048] For example, for any amino acid sequence aligned with SEQ ID NO: 1, each amino acid residue in the amino acid sequence can be numbered, based on the alignment, with reference to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, sequence alignment algorithms as described herein can identify positions of amino acids or positions where modifications, such as substitutions, insertions, or deletions occur compared to a query sequence (also referred to as a "reference sequence").

[0049] Examples of alignment can be determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) and the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), etc., but are not limited thereto, and sequence alignment programs known in the art, such as pairwise sequence comparison algorithms, etc., can be appropriately used.

[0050] In one embodiment, the variant polypeptide may be a variant polypeptide in which the amino acid corresponding to position 351 of SEQ ID NO: 1 is 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, aspartic acid and glutamic acid, but is not limited thereto.

[0051] In any of the above embodiments, the variant polypeptide provided herein can be a variant polypeptide in which the amino acid corresponding to position 351 of SEQ ID NO: 1 is replaced by an amino acid having a polar or hydrophilic side chain (polar amino acid), i.e., an amino acid selected from serine, threonine, cysteine, tyrosine, asparagine and glutamine.

[0052] In any of the above embodiments, the variant polypeptide provided herein can be a variant polypeptide in which the amino acid corresponding to position 351 of SEQ ID NO: 1 is replaced by an amino acid having an uncharged side chain (referred to as an uncharged amino acid; a neutral amino acid), i.e., an amino acid selected from the group consisting of glycine, alanine, valine, leucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine and glutamine.

[0053] In any of the above embodiments, the variant polypeptide provided herein can be a variant polypeptide in which the amino acid corresponding to position 351 of SEQ ID NO: 1 is substituted with an amino acid selected from cysteine, proline, threonine, aspartic acid and asparagine.

[0054] In any of the above embodiments, the variant polypeptide of the present disclosure may be a polypeptide in which the amino acid corresponding to position 351 of SEQ ID NO: 1 is substituted with asparagine, but is not limited thereto.

[0055] For example, the variant polypeptides of the present disclosure may include an amino acid sequence having 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 with SEQ ID NO: 1, while the amino acid asparagine corresponding to position 351 in the amino acid sequence set forth in SEQ ID NO: 1 is fixed. In addition, it is obvious that any variant polypeptide having an amino acid sequence in which part of the sequence is deleted, modified, substituted or added may also fall within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the variant polypeptides of the present disclosure.

[0056] At the same time, those skilled in the art can determine the amino acid corresponding to position 351 of the amino acid sequence of SEQ ID NO: 1 of the present disclosure in any amino acid sequence by sequence alignment known in the art, and even if not separately described herein, it is obvious that the phrase "the amino acid at a specific position in a specific sequence number" obviously even includes the "amino acid at the corresponding position" in any polynucleotide sequence.

[0057] In another embodiment, the variant polypeptide may consist of the amino acid sequence of SEQ ID NO:3.

[0058] Specifically, the variant polypeptides of the present disclosure may have or include the amino acid sequence 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 thereto, may consist of the above amino acid sequence, or may consist essentially of the above amino acid sequence.

[0059] For example, this may be the case with sequence additions or deletions, naturally occurring mutations, silent mutations or conservative substitutions thereof at the N-terminus, C-terminus and / or within the amino acid sequence that do not alter the function of the variant polypeptides of the present disclosure.

[0060] As used herein, the term "conservative substitution" refers to the replacement of an amino acid with another amino acid of similar structure and / or chemical properties. Such amino acid substitutions can generally occur based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic properties of the residues. For example, positively charged (basic) amino acids include arginine, lysine and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan and tyrosine; hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan. In addition, amino acids can be divided into amino acids with charged side chains and amino acids with uncharged side chains, and amino acids with charged side chains include aspartic acid, glutamic acid, lysine, arginine and histidine, and amino acids with uncharged side chains can be further divided into non-polar amino acids or polar amino acids. Non-polar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Generally, conservative substitutions have little or no effect on the activity of the resulting polypeptide. Generally, conservative substitutions have little or no effect on the activity of the protein or polypeptide. In one embodiment, the variant polypeptide can have the activity of a major facilitator superfamily transporter.

[0061] Another aspect of the present disclosure provides a polynucleotide encoding the variant polypeptide.

[0062] As used herein, the term "polynucleotide" is a nucleotide polymer composed of nucleotide monomers linked to form a long chain by covalent bonds, and is a DNA or RNA chain of at least a certain length. More specifically, it can refer to a polynucleotide fragment encoding the variant.

[0063] The polynucleotide encoding the variant polypeptide of the present invention may include any polynucleotide sequence encoding the variant polypeptide of the present invention without limitation. For example, the polynucleotide encoding the variant polypeptide of the present invention may be a polynucleotide sequence encoding the amino acid sequence of the variant polypeptide of the present invention, but is not limited thereto.

[0064] For example, it may include a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 3. In one example, the polynucleotide of the present disclosure may have or include SEQ ID NO: 4. In addition, the polynucleotide of the present disclosure may consist of or consist essentially of SEQ ID NO: 4.

[0065] Due to codon degeneracy or in consideration of preferred codons in the organism in which the disclosed variant is to be expressed, the polynucleotides of the present disclosure may be modified in various ways in the coding region without changing the amino acid sequence of the disclosed variant. Thus, it is apparent that polynucleotides that can be translated into a polypeptide consisting of the amino acid sequence of the disclosed variant or a polypeptide having homology or identity thereto through codon degeneracy may also be included. For example, the polynucleotide of the present disclosure may be SEQ ID NO: 4 or a degenerate sequence thereof.

[0066] For example, the polynucleotides of the present disclosure may include, but are not limited to, those polynucleotides in which the codon encoding isoleucine (corresponding to the amino acid at position 1052 of SEQ ID NO: 2) is substituted with a codon encoding another amino acid other than isoleucine (e.g., asparagine) in 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 with the sequence of SEQ ID NO: 2. In addition, it is obvious that any variant having a polynucleotide sequence in which a portion of the sequence is deleted, modified, substituted, conservatively substituted or added can also fall within the scope of the present disclosure, as long as the polynucleotide sequence has such homology or identity and encodes the amino acid sequence of the variant polypeptide of the present disclosure.

[0067] In another example, the polynucleotide of the present disclosure may have or include 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: 4, or may consist of 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: 4, but is not limited thereto. In addition, a sequence having such homology or identity may be a sequence in which the codon corresponding to position 351 of SEQ ID NO: 3 encoded by SEQ ID NO: 4 is fixed to a codon encoding asparagine.

[0068] Furthermore, the polynucleotides of the present disclosure may include probes that can be prepared from known gene sequences, for example, any polynucleotide sequence that can hybridize under stringent conditions to a complementary sequence of all or part of a polynucleotide sequence of the present disclosure, without limitation.

[0069] "Stringent conditions" refer to conditions that allow specific hybridization between polynucleotides. Such conditions are disclosed in detail in the literature (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, stringent conditions may include conditions under which polynucleotides with high homology or identity, i.e., polynucleotides with 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 to each other, while polynucleotides with lower homology or identity do not hybridize to each other; or may include conventional Southern hybridization washing conditions, i.e., washing once, particularly 2 or 3 times, at a salt concentration and temperature corresponding to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0070] Hybridization requires that the two nucleic acids have complementary sequences, although 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 each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present disclosure may also include isolated nucleic acid fragments that are complementary to the entire sequence, as well as nucleic acid sequences that are substantially similar thereto.

[0071] Specifically, one can use a m The hybridization conditions of the hybridization step are 55°C. Polynucleotides having homology or identity with the polynucleotides disclosed herein are detected under the above conditions. m The value may be 60° C., 63° C. or 65° C., but is not limited thereto, and those skilled in the art may appropriately adjust it according to their purpose.

[0072] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, and these variables are well known in the art (see J. Sambrook et al., supra).

[0073] As used herein, the term "homology" or "identity" refers to the degree of similarity between two given amino acid sequences or nucleotide sequences and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0074] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard alignment algorithms and can be used together with default gap penalties established by the program used. Basically, homologous or identical sequences are generally expected to hybridize to all or part of the sequence under moderate or high stringency conditions. Obviously, hybridization to polynucleotides containing universal codons or degenerate codons in the hybridizing polynucleotide is also included.

[0075] Whether any two polynucleotide or polypeptide sequences share homology, similarity or identity can be determined by known computer algorithms, such as the "FASTA" program, using the default parameters of Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444. Alternatively, it can be determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), which is implemented using the Needleman program (version 5.0.0 or later) in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (GCG program package (Devereux, J. et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF et al., J MOLEC BIOL 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and CARILLO et al. (1988) SIAM J Applied Math 48:1073). For example, homology, similarity or identity can be determined by using BLAST or ClustalW from the National Center for Biotechnology Information (NCBI).

[0076] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, the GAP computer program, as disclosed in Needleman et al. (1970), J Mol Biol. 48: 443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2: 482. In summary, the GAP program defines homology, similarity or identity as the value obtained by dividing the number of similar aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program may include: (1) a binary comparison matrix (including a value of 1 for identity and a value of 0 for non-identity), and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix), as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, the terms "homology" or "identity" as used herein refer to a measure of relatedness between sequences.

[0077] Another aspect of the present disclosure provides a vector containing the polynucleotide of the present disclosure. The vector may be an expression vector for expressing the polynucleotide in a microorganism, but is not limited thereto.

[0078] As used herein, the term "vector" may include a DNA construct comprising a nucleotide sequence of a polynucleotide encoding a target polypeptide, the polynucleotide being operably linked to a suitable expression regulatory region (expression regulatory sequence) so that the target polypeptide can be expressed in a suitable host cell. The expression regulatory sequence may include a promoter capable of initiating transcription, any operator sequence regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating transcription and translation termination. Once the vector is transformed into a suitable microorganism, it may replicate or function independently of the host genome, or it may be integrated into its genome.

[0079] The vectors used in the present disclosure are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors may include natural 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 vectors or cosmid vectors; those based on 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.

[0080] In one example, the polynucleotide encoding target protein can be inserted into the chromosome by the vector for chromosome insertion in the cell. The polynucleotide being inserted into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. The carrier can also include a selective marker to confirm that it is inserted into the chromosome. The selective marker is used to select the cell transformed with the carrier, i.e., for confirming whether the target nucleic acid molecule has been inserted, and the marker providing a selectable phenotype (such as drug resistance, auxotrophic type, cytotoxic agent resistance or surface polypeptide expression) can be used. Only the cell expressing the selective marker can survive or show different phenotypes under the environment of processing with the selection agent, so the cell transformed can be selected.

[0081] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the microorganism. As long as the transformed polynucleotide can be expressed in the microorganism, it does not matter whether the transformed polynucleotide is integrated into the chromosome of the microorganism and located therein or is located outside the chromosome, and both situations can be included. In addition, the polynucleotide can include DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form, as long as it can be introduced into the microorganism and expressed therein. For example, the polynucleotide can be introduced into the microorganism in the form of an expression cassette, which is a genetic construct containing all the elements required for its autonomous expression. The expression cassette can generally contain a promoter, a transcription terminator, a ribosome binding site, or a translation terminator operably linked to the polynucleotide. The expression cassette can be in the form of a self-replicating expression vector. In addition, the polynucleotide can be introduced into the microorganism as is and operably linked to the sequence required for expression in the microorganism, but is not limited thereto.

[0082] Furthermore, as used herein, the term "operably linked" means that a polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the variant of interest of the present disclosure.

[0083] Another aspect of the present disclosure provides a microorganism comprising the variant polypeptide, a polynucleotide encoding the variant polypeptide, or a vector containing the polynucleotide.

[0084] In one embodiment, the microorganism may be a microorganism having L-glutamic acid-producing ability.

[0085] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms or natural or artificial genetically modified microorganisms, and it can be a microorganism in which a specific mechanism is weakened or enhanced due to the insertion of exogenous genes, or the enhancement or inactivation of endogenous gene activity, and can be a microorganism comprising a genetic modification that produces a desired polypeptide, protein or product. In the present disclosure, "microorganism" and "strain" have the same meaning and can be used interchangeably without limitation.

[0086] As used herein, the term "L-glutamic acid (L-glutamate)" refers to an amino acid and is classified as a non-essential amino acid. L-glutamic acid is known to be the most common excitatory neurotransmitter in the central nervous system. Because L-glutamic acid has an umami taste, monosodium glutamate (MSG) has been developed from it and is widely used as a flavor enhancer.

[0087] As used herein, the term "L-glutamic acid-producing microorganism" refers to a prokaryotic or eukaryotic microbial strain capable of producing L-glutamic acid in vivo, and may include microorganisms in which the ability to produce L-glutamic acid is imparted to a parent strain that does not have the ability to produce L-glutamic acid, or microorganisms that endogenously have the ability to produce L-glutamic acid. The ability to produce L-glutamic acid can be imparted or enhanced through species improvement.

[0088] In one embodiment, the microorganism of the present disclosure may be a microorganism that naturally has the variant polypeptide of the present disclosure or the ability to produce L-glutamic acid; or a microorganism in which the variant of the present disclosure or the polynucleotide encoding the same (or a vector containing the polynucleotide) has been introduced into a parent strain that does not have the variant polypeptide of the present disclosure or the ability to produce L-glutamic acid and / or in which the ability to produce L-glutamic acid has been imparted, but is not limited thereto.

[0089] In one embodiment, the microorganisms of the present disclosure may include: microorganisms comprising the variant polypeptide sequences of the present disclosure due to mutations in genes encoding the variant polypeptides of the present disclosure on chromosomes, and / or microorganisms comprising the variant polypeptides of the present disclosure by introducing vectors containing polynucleotides encoding the variant polypeptides of the present disclosure, but are not limited thereto.

[0090] As used herein, the term "unmodified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, and can refer to wild-type strains or natural strains themselves, or strains before their properties have been altered due to genetic modification caused by natural or artificial factors. For example, an unmodified microorganism can refer to a strain that has not been introduced into a variant polypeptide disclosed herein, or a strain before it has been introduced. "Unmodified microorganism" can be used interchangeably with "strain before modification," "microorganism before modification," "unmutated strain," "unmodified strain," "unmutated microorganism," or "reference microorganism."

[0091] The microorganism having L-glutamic acid-producing ability disclosed herein may be a microorganism comprising any one or more of the variant disclosed herein, the polynucleotide disclosed herein, and a vector containing the polynucleotide disclosed herein; a microorganism modified to express the variant disclosed herein or the polynucleotide disclosed herein; a microorganism (e.g., a recombinant strain) expressing the variant disclosed herein or the polynucleotide disclosed herein; or a microorganism (e.g., a recombinant strain) having variant activity disclosed herein, but is not limited thereto.

[0092] In one example, the strain of the present invention, which is a cell or microorganism transformed with a polynucleotide of the present invention or a vector containing a polynucleotide encoding a variant of the present invention to express a variant of the present invention, can include all microorganisms capable of producing L-glutamic acid, which contain variants of the present invention. For example, the microorganism of the present invention can be a recombinant strain that increases L-glutamic acid production capacity by introducing a polynucleotide encoding a variant of the present invention into a natural wild-type microorganism or a microorganism with L-glutamic acid production capacity to express a variant polypeptide. The recombinant strain with increased L-glutamic acid production capacity can be a microorganism with increased L-glutamic acid production capacity compared to a natural wild-type microorganism or an unmodified microorganism of a major facilitator superfamily transporter (e.g., a microorganism expressing a wild-type major facilitator superfamily transporter or a microorganism that does not express a variant of the present invention), but is not limited thereto. In one example, the microorganism with increased L-glutamic acid production capacity can be a microorganism with increased L-glutamic acid production capacity compared to a microorganism containing a polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same, but is not limited thereto. In one example, the unmodified microorganism as the target strain for comparison of L-glutamic acid productivity increase may be ATCC13869 or ATCC13032, which are wild-type Corynebacterium glutamicum strains; or KFCC11074 strain (KR 10-0292299B1), which is a glutamic acid-producing strain, but is not limited thereto.

[0093] In addition to introducing nucleic acids or vectors, the microorganisms may also include all microorganisms capable of expressing the variant polypeptides of the present disclosure by various known methods.

[0094] In one example, the microorganism with increased L-glutamic acid-producing ability can have an increase of 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, compared to the L-glutamic acid-producing ability of the parent strain or unmodified microorganism before modification. The L-glutamic acid-producing capacity of the modified microorganism may be about 49% or more, about 50% or more, about 51% or more, or about 52% or more (the upper limit is not particularly limited, 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 with a positive (+) value compared to the production capacity of the parent strain before modification or an unmodified microorganism. In another example, the recombinant strain having an increased L-glutamic acid-producing ability may have an L-glutamic acid-producing ability increased by about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, about 1.05 times or more, about 1.1 times or more, about 1.15 times or more, about 1.20 times or more, about 1.25 times or more, about 1.30 times or more, about 1.35 times or more, about 1.40 times or more, about 1.45 times or more, about 1.46 times or more, about 1.47 times or more, about 1.48 times or more, about 1.49 times or more, about 1.50 times or more, about 1.51 times or more, or about 1.52 times or more (the upper limit is not particularly limited, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, or about 1.53 times or less), compared to the L-glutamic acid-producing ability of the parent strain or unmodified microorganism before modification, but is not limited thereto. As used herein, the term "about" is meant to include all ranges of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values ​​equivalent to the value immediately following the term "about" or values ​​within a similar range, but the range is not limited thereto.

[0095] As a microorganism according to any of the above embodiments, the microorganism of the present disclosure may be a microorganism belonging to the genus Corynebacteria sp., Escherichia sp., Erwinia sp., Serratia sp., Providencia sp., Pseudomonas sp., Leptospira sp., Salmonella sp., Brevibacteria sp., Hypomononas sp., Chromobacterium sp. and Norcardia sp., or a fungus or yeast, in particular, a microorganism belonging to the genus Corynebacterium, but not limited thereto.

[0096] In one example, the microorganism of the present disclosure can be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganism of the present disclosure may be a microorganism belonging to the genus Corynebacterium, more specifically, Corynebacterium glutamicum, but is not limited thereto.

[0097] The microorganism having L-glutamic acid production ability disclosed herein may further comprise: any one or more variant polypeptides selected from the group consisting of the following (a) and (b); a polynucleotide encoding the variant polypeptide; or a vector containing the polynucleotide, but is not limited thereto:

[0098] Wherein (a) a variant polypeptide having the activity of a TetR family transcriptional regulatory factor, wherein the amino acid corresponding to position 105 of SEQ ID NO: 5 is replaced by another amino acid; and

[0099] (b) A variant polypeptide having isocitrate lyase activity, wherein the amino acid sequence corresponding to positions 336 to 346 of SEQ ID NO: 9 is deleted.

[0100] In one example, the microorganism having L-glutamic acid production ability disclosed herein may further comprise: a variant polypeptide having TetR family transcriptional regulatory factor activity, wherein the amino acid corresponding to position 105 of SEQ ID NO: 5 is replaced by another amino acid; a polynucleotide encoding the variant polypeptide; or a vector containing the polynucleotide, but is not limited thereto.

[0101] As used herein, the term "variant polypeptide having TetR family transcriptional regulatory factor activity" may refer to a variant polypeptide having TetR family transcriptional regulatory factor activity comprising one or more amino acid substitutions in the amino acid sequence of a polypeptide having TetR family transcriptional regulatory factor activity; or a variant of a polypeptide having TetR family transcriptional regulatory factor activity comprising one or more amino acid substitutions in the parent sequence (the amino acid sequence of a polypeptide having TetR family transcriptional regulatory factor activity).

[0102] As used herein, the term "TetR family transcriptional regulator (mmpLR)" is a global regulatory factor with activities involved in secretion, cell division and stress response, and can be used interchangeably with the term "mmpLR" and can be a TetR family transcriptional regulator encoded by the mmpLR gene, but is not particularly limited thereto.

[0103] The gene encoding the TetR family transcriptional regulatory factor may be derived from a microorganism of the genus Corynebacterium, and may particularly be derived from mmpLR of Corynebacterium glutamicum, but is not limited thereto.

[0104] Specifically, the TetR family transcriptional regulatory factor 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 the activity of a TetR family transcriptional regulatory factor. Specifically, the amino acid sequence may include the amino acid sequence of 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. The sequence of SEQ ID NO: 5 can be obtained from known databases, such as GenBank of NCBI or the Kyoto Gene and Genome Database (KEGG). In one example, the amino acid sequence may be derived from a microorganism of the genus Corynebacterium or Corynebacterium glutamicum, more specifically, it may be a polypeptide / protein comprising the amino acid sequence shown in SEQ ID NO: 5, but is not limited thereto. In addition, it is obvious that any auxiliary protein having an amino acid sequence in which part of the sequence is deleted, modified, substituted or added can also fall within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits the corresponding efficacy of the protein.

[0105] In addition, the TetR family transcriptional regulatory factor having the amino acid sequence of SEQ ID NO: 5 can be encoded by a polynucleotide, which can have or include the nucleotide sequence of SEQ ID NO: 6, 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 with the nucleotide sequence of SEQ ID NO: 6, or can consist of or essentially consist of the nucleotide sequence of SEQ ID NO: 6, 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 with the nucleotide sequence of SEQ ID NO: 6, but is not limited thereto.

[0106] The variant polypeptide having TetR family transcriptional regulatory factor activity disclosed herein may be a variant polypeptide having TetR family transcriptional regulatory factor activity in which the amino acid corresponding to position 105 of SEQ ID NO: 5 is substituted with another amino acid, but is not limited thereto.

[0107] In one embodiment, the variant polypeptide having TetR family transcriptional regulatory factor activity disclosed herein may have a sequence homology of 60% or more and less than 100% with the amino acid sequence of SEQ ID NO: 5, specifically, 80% or more and less than 100% sequence homology, but is not limited thereto.

[0108] Specifically, the variant polypeptides of the present disclosure having the activity of a TetR family transcriptional regulator may include those in which the amino acid at position 105 from the N-terminus of SEQ ID NO: 5 is substituted with another amino acid in an amino acid sequence having 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 with the amino acid sequence shown in SEQ ID NO: 5. In addition, it is obvious that any variant having an amino acid sequence in which a portion of the sequence is deleted, modified, substituted, conservatively substituted or added may also fall within the scope of the present disclosure, as long as it has such homology or identity and exhibits the efficacy corresponding to the variant of the present disclosure.

[0109] In one embodiment, the variant polypeptide having the activity of a TetR family transcriptional regulator disclosed herein may be a polypeptide in which the amino acid corresponding to position 105 of SEQ ID NO: 5 is substituted with leucine, but is not limited thereto.

[0110] For example, the variant polypeptides of the present disclosure having the activity of a TetR family transcriptional regulator may include an amino acid sequence having 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 with SEQ ID NO: 5, and the amino acid leucine at position 105 of the amino acid sequence shown in SEQ ID NO: 5 is fixed. In addition, it is obvious that any variant polypeptide having an amino acid sequence in which part of the sequence is deleted, modified, substituted or added may also fall within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the variant polypeptide of the present disclosure.

[0111] In another embodiment, the variant polypeptide having the activity of a TetR family transcriptional regulatory factor may consist of the amino acid sequence of SEQ ID NO:7.

[0112] Specifically, the variant polypeptide having TetR family transcriptional regulatory factor activity disclosed herein may have or include the amino acid sequence 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 with SEQ ID NO:7, may consist of the above amino acid sequence, or may consist essentially of the above amino acid sequence.

[0113] For example, this may be the case with sequence additions or deletions, naturally occurring mutations, silent mutations or conservative substitutions thereof at the N-terminus, C-terminus and / or within the amino acid sequence that do not alter the function of the variant polypeptides of the present disclosure.

[0114] The polynucleotides encoding the variant polypeptides having the activity of the TetR family transcriptional regulator of the present invention may include those in which the codon encoding the amino acid proline at position 314 of SEQ ID NO: 6 is replaced by a codon encoding another amino acid (e.g., leucine) in 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 with the sequence of SEQ ID NO: 6, but are not limited thereto. In addition, it is obvious that any variant having a polynucleotide sequence in which a portion of the sequence is deleted, modified, substituted, conservatively substituted or added can also fall within the scope of the present disclosure, as long as the polynucleotide sequence has such homology or identity and encodes the amino acid sequence of the variant polypeptide having the activity of the TetR family transcriptional regulator of the present invention.

[0115] In another example, the polynucleotide encoding the variant polypeptide having the activity of a TetR family transcriptional regulator of the present invention may have or include 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 with SEQ ID NO: 8, or may consist of 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 with SEQ ID NO: 8, but is not limited thereto. In addition, the sequence having such homology or identity may be a sequence 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.

[0116] In one example, the microorganism having L-glutamic acid-producing ability disclosed herein may further comprise: a variant polypeptide having isocitrate lyase activity, wherein the amino acid sequence corresponding to positions 336 to 346 of SEQ ID NO: 9 is deleted; a polynucleotide encoding the variant polypeptide; or a vector containing the polynucleotide, but is not limited thereto.

[0117] As used herein, the term "variant polypeptide having isocitrate lyase activity" may refer to a variant polypeptide having isocitrate lyase activity comprising one or more amino acid substitutions in the amino acid sequence of a polypeptide having isocitrate lyase activity; or a variant of a polypeptide having isocitrate lyase activity comprising one or more amino acid substitutions in the parent sequence (the amino acid sequence of a polypeptide having isocitrate lyase activity).

[0118] As used herein, the term "isocitrate lyase (aceA)" is an enzyme that produces succinate using isocitrate as a substrate, may be used interchangeably with the term "aceA", and may be isocitrate lyase encoded by the aceA gene, but is not particularly limited thereto.

[0119] The gene encoding isocitrate lyase may be derived from a microorganism belonging to the genus Corynebacterium, and may particularly be derived from aceA of Corynebacterium glutamicum, but is not limited thereto.

[0120] Isocitrate lyase can include, for example, the amino acid sequence of SEQ ID NO:9, or an amino acid sequence with 60% or more homology or identity thereto, but is not limited thereto, as long as it has the activity of isocitrate lyase. Specifically, the amino acid sequence can include the amino acid sequence of SEQ ID NO:9 or an amino acid sequence with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology or identity thereto with SEQ ID NO:9. The sequence of SEQ ID NO:9 can be obtained from known databases, such as GenBank or Kyoto gene and genome database (KEGG) of NCBI. In an example, the amino acid sequence can be derived from a microorganism of the genus Corynebacterium or Corynebacterium glutamicum, more specifically, it can be a polypeptide / protein comprising the amino acid sequence shown in SEQ ID NO:9, but is not limited thereto. In addition, it is obvious that any auxiliary protein having an amino acid sequence in which part of the sequence is deleted, modified, substituted or added can also fall within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits the corresponding efficacy of the protein.

[0121] In addition, the isocitrate lyase having the amino acid sequence of SEQ ID NO: 9 can be encoded by a polynucleotide having or including the 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 nucleotide sequence of SEQ ID NO: 10, or can consist of or essentially consist of the 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 nucleotide sequence of SEQ ID NO: 10, but is not limited thereto.

[0122] The variant polypeptide having isocitrate lyase activity disclosed herein may be a variant polypeptide having isocitrate lyase activity in which the amino acid sequence corresponding to positions 336 to 346 of SEQ ID NO: 9 is deleted, but is not limited thereto.

[0123] In one embodiment, the variant polypeptide having isocitrate lyase activity of the present disclosure may have a sequence identity of 60% or more and less than 97% with the amino acid sequence of SEQ ID NO: 9, specifically, 80% or more and less than 97% sequence identity, but is not limited thereto.

[0124] Specifically, the variant polypeptides having isocitrate lyase activity of the present disclosure may include those in which, in an amino acid sequence having 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 with the amino acid sequence shown in SEQ ID NO: 9, the amino acid sequence corresponding to positions 336 to 346 from the N-terminus of SEQ ID NO: 9 is deleted. In addition, it is obvious that any variant having an amino acid sequence in which a portion of the sequence is deleted, modified, substituted, conservatively substituted or added may also fall within the scope of the present disclosure, as long as it has such homology or identity and exhibits the efficacy corresponding to the variant of the present disclosure.

[0125] In another embodiment, the variant polypeptide having isocitrate lyase activity may consist of the amino acid sequence of SEQ ID NO:11.

[0126] Specifically, the variant polypeptide having isocitrate lyase activity of the present disclosure can have or include the amino acid sequence 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 with SEQ ID NO: 11, can consist of the above amino acid sequence, or can consist essentially of the above amino acid sequence.

[0127] For example, this may be the case with sequence additions or deletions, naturally occurring mutations, silent mutations or conservative substitutions thereof at the N-terminus, C-terminus and / or within the amino acid sequence that do not alter the function of the variant polypeptides of the present disclosure.

[0128] The polynucleotides encoding the variant polypeptides having isocitrate lyase activity of the present disclosure may include those in which the nucleotide sequence corresponding to positions 1006 to 1038 of SEQ ID NO: 10 is deleted in 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 with the sequence of SEQ ID NO: 10, but are not limited thereto. In addition, it is obvious that any variant having a polynucleotide sequence in which a portion of the sequence is deleted, modified, substituted, conservatively substituted or added can also fall within the scope of the present disclosure, as long as the polynucleotide sequence has such homology or identity and encodes the amino acid sequence of the variant polypeptide having isocitrate lyase activity of the present disclosure.

[0129] In another example, the polynucleotide encoding the variant polypeptide having isocitrate lyase activity of the present disclosure can have or include 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: 12, or can consist of 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: 12, but is not limited thereto.

[0130] As a microorganism according to any one of the above embodiments, the microorganism having L-glutamic acid-producing ability of the present disclosure may be a microorganism having increased L-glutamic acid-producing ability by comprising: a variant polypeptide in which the amino acid corresponding to position 351 of SEQ ID NO: 1 is substituted with another amino acid; a variant polypeptide having TetR family transcriptional regulatory factor activity in which the amino acid corresponding to position 105 of SEQ ID NO: 5 is substituted with another amino acid; and a variant polypeptide having isocitrate lyase activity in which the amino acid sequence corresponding to positions 336 to 346 of SEQ ID NO: 9 is deleted, but is not limited thereto.

[0131] As a microorganism according to any one of the above embodiments, the microorganism having L-glutamic acid-producing ability of the present disclosure may be a microorganism having increased L-glutamic acid-producing ability by comprising: a variant polypeptide in which the amino acid corresponding to position 351 of SEQ ID NO: 1 is substituted with asparagine; a variant polypeptide having TetR family transcriptional regulatory factor activity in which the amino acid corresponding to position 105 of SEQ ID NO: 5 is substituted with leucine; and a variant polypeptide having isocitrate lyase activity in which the amino acid sequence corresponding to positions 336 to 346 of SEQ ID NO: 9 is deleted, but is not limited thereto.

[0132] Meanwhile, the microorganisms having L-glutamic acid-producing ability disclosed herein may include all of the following: natural wild-type microorganisms themselves; microorganisms in which the activity of a polypeptide involved in the L-glutamic acid production mechanism is enhanced or weakened, thereby having improved L-glutamic acid-producing ability; or microorganisms in which the activity of an exogenous polypeptide is introduced or enhanced, thereby having improved L-glutamic acid-producing ability.

[0133] Meanwhile, although it is known that Corynebacterium microorganisms can produce L-glutamic acid, they have very low L-glutamic acid production capacity, and the genes that act on this production mechanism or the principle of this mechanism have not yet been fully discovered. Therefore, the Corynebacterium microorganisms having L-glutamic acid production capacity disclosed herein can include all of the following: naturally occurring wild-type microorganisms themselves; Corynebacterium microorganisms in which the activity of a polypeptide involved in the L-glutamic acid production mechanism is enhanced or weakened, thereby having improved L-glutamic acid production capacity; or Corynebacterium microorganisms in which the activity of an exogenous polypeptide is introduced or enhanced, thereby having improved L-glutamic acid production capacity.

[0134] As used herein, the term "increase" of polypeptide activity refers to an increase in polypeptide activity compared to its endogenous activity. Increase can be used interchangeably with terms such as activation, upregulation, overexpression, enhancement, etc.

[0135] Increase may include both cases where an activity that was not originally present is expressed, or where the activity is enhanced compared to the endogenous activity or the activity before modification.

[0136] For example, the phrase "exhibits an activity not originally possessed" can refer to "introduction of a protein," but is not limited thereto. Protein introduction means that a gene not originally possessed by the microorganism is expressed in the microorganism, resulting in the microorganism exhibiting the activity of a specific protein or an increase or enhancement of the activity of the corresponding protein compared to the endogenous activity or the activity before modification. For example, the phrase can mean that a polynucleotide encoding a specific protein is introduced into the chromosome of the microorganism, or that a vector containing a polynucleotide encoding a specific protein is introduced into the microorganism, thereby exhibiting the activity of the specific protein.

[0137] "Endogenous activity" refers to the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation, when the trait is altered due to genetic variation caused by natural or artificial factors. Endogenous activity can be used interchangeably with "activity before modification."

[0138] Increased polypeptide activity compared to endogenous activity means that the polypeptide activity is enhanced compared to the activity and / or concentration (expression level) of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before transformation.

[0139] In one example, increase can mean that an activity of the corresponding protein is exhibited that was not originally possessed, or its activity or concentration is generally increased by about 1%, about 10%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, up to about 1000%, or about 2000% or more based on the activity or concentration of the wild-type protein or the initial microbial strain, but is not limited thereto.

[0140] Increased polypeptide activity can be achieved by introducing an exogenous polypeptide or increasing the activity of an endogenous polypeptide. Whether the activity of a polypeptide is increased can be confirmed by an increase in the activity level of the corresponding polypeptide, its expression level, or the amount of product produced by the corresponding polypeptide.

[0141] The increase in polypeptide activity can be achieved by various methods well known in the art, and is not limited thereto, as long as it can increase the activity of the target polypeptide compared to the microorganism before modification. Specifically, genetic engineering and / or protein engineering well known to those skilled in the art can be used, which are conventional methods in molecular biology, but the method 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.).

[0142] Specifically, the increase in the activity of the polypeptide of the present disclosure can be achieved by:

[0143] 1) increasing the copy number of the polynucleotide encoding the polypeptide in the cell;

[0144] 2) Modifying the expression regulatory region of a gene encoding a polypeptide on a chromosome (e.g., inducing modification within the expression regulatory region, replacing it with a sequence having greater activity, or inserting a sequence having greater activity);

[0145] 3) modifying the nucleotide sequence encoding the start codon or 5'-UTR of the gene transcript encoding the polypeptide;

[0146] 4) Modifying the amino acid sequence of a polypeptide to increase its activity;

[0147] 5) Modifying a polynucleotide sequence encoding a polypeptide so that the activity of the polypeptide is increased (e.g., modifying the polynucleotide sequence of a polypeptide gene to encode a modified polypeptide to increase the activity of the polypeptide);

[0148] 6) introducing an exogenous polypeptide that exhibits polypeptide activity or an exogenous polynucleotide encoding the polypeptide;

[0149] 7) Codon optimization of polynucleotides encoding polypeptides;

[0150] 8) Analyze the tertiary structure of the polypeptide and select and modify exposed sites or perform chemical modifications thereon;

[0151] 9) Regulate the cellular localization of proteins (peptides); or

[0152] 10) A combination of two or more selected from the above 1) to 9), but not particularly limited thereto.

[0153] For example,

[0154] 1) The method of increasing the copy number of the polynucleotide encoding the polypeptide in the cell can be achieved by introducing a vector into the host cell, the vector being operably linked to the polynucleotide encoding the polypeptide and capable of replicating and functioning independently of the host cell. Alternatively, the method can be achieved by introducing one or two copies of the polynucleotide encoding the polypeptide into the chromosome of the host cell. Introduction into the chromosome can be carried out by introducing a vector capable of inserting the polynucleotide into the host cell chromosome into the host cell, but is not limited thereto. The vector is as described above.

[0155] 2) The method of replacing the expression control region (or expression control sequence) of the gene encoding the polypeptide on the chromosome with a sequence having strong activity can be achieved by, for example, introducing modifications to the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof to further increase the activity of the expression control region, or by replacing the sequence with a sequence having stronger activity. The expression control region may include but is not particularly limited to a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating transcription and translation termination, and the like. In one example, the method may include replacing the original promoter with a strong promoter, but is not limited thereto.

[0156] Examples of known strong promoters include cj1 to cj7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US10584338 B2), O2 promoter (US10273491 B2), tkt promoter, yccA promoter, etc., but strong promoters are not limited thereto.

[0157] 3) The method of modifying the nucleotide sequence encoding the start codon or 5'-UTR of the gene encoding the polypeptide can be achieved by, for example, replacing the start codon with another start codon having a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0158] 4) and 5) methods for modifying amino acid sequences or polynucleotide sequences can be achieved by: inducing sequence modifications by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to increase the activity of the polypeptide; or by replacing the sequence with an amino acid sequence or polynucleotide sequence that has been modified to have stronger activity, or an amino acid sequence or polynucleotide sequence that has been modified to increase activity, but is not limited thereto. Specifically, the polynucleotide can be inserted into the chromosome for replacement by homologous recombination, but is not limited thereto. The vector used herein may also contain a selection marker to confirm insertion into the chromosome. The selection marker is as described above.

[0159] 6) Introduction of an exogenous polynucleotide exhibiting polypeptide activity can be achieved by introducing an exogenous polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide into the host cell. Any exogenous polynucleotide can be used without limitation, regardless of its source or sequence, as long as it exhibits the same / similar activity as the polypeptide. Introduction can be performed using a transformation method known in the art, appropriately selected by one of ordinary skill in the art. Expression of the introduced polynucleotide in the host cell can produce the polypeptide, thereby enhancing its activity.

[0160] 7) The method of codon optimization of a polynucleotide encoding a polypeptide can be achieved by codon optimization of an endogenous polynucleotide to increase transcription or translation in a host cell, or by optimizing codons to enable optimized transcription and translation of an exogenous polynucleotide in a host cell.

[0161] 8) The method of analyzing the tertiary structure of a polypeptide and thereby selecting and modifying an exposed site or chemically modifying it can be achieved by, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins to determine template protein candidates based on the degree of sequence similarity, and thus confirming the structure based on this information, thereby selecting and transforming or modifying the exposed site to be modified or chemically modified.

[0162] 9) Methods for regulating the cellular localization of proteins (polypeptides) can be achieved by targeting the protein (polypeptide) to a specific organelle or specific space within the cell. For example, this can be achieved by adding or removing a leader sequence that plays a role in targeting the protein (polypeptide) to the periplasm or cytoplasm, but is not limited thereto.

[0163] Such enhancement of polypeptide activity may mean that the activity or concentration of the corresponding polypeptide is increased relative to the activity or concentration of the polypeptide expressed in a wild-type strain or a microbial strain before modification, or the amount of the product produced by the corresponding polypeptide is increased, but is not limited thereto.

[0164] As used herein, the term "reduction" of polypeptide activity is a comprehensive concept, including that the activity is reduced or inactive compared to its endogenous activity. Reduction can be used interchangeably with terms such as lack, inactivation, deletion, destruction, downregulation, reduction, attenuation, inhibition, and reduction.

[0165] For example, attenuation may refer to a state in which a protein exhibits activity but is not completely inactivated due to deletion, and may refer to a state in which the activity of a protein is attenuated compared to an unmodified microorganism, a wild-type strain, or a parent strain, but is not limited thereto.

[0166] For example, attenuation may be, but is not limited to, inactivation. Inactivation may mean that the protein is not expressed at all, or even if the protein is expressed, it does not show activity or has reduced activity, compared to the parent strain or unmodified strain.

[0167] Attenuation may also include: due to mutations in the polynucleotide encoding the polypeptide, the polypeptide activity itself is weakened or eliminated compared to the polypeptide activity originally possessed by the microorganism; due to inhibition of expression of genes encoding the polypeptide or inhibition of translation into polypeptides, the overall level of polypeptide activity in the cell is reduced compared to natural strains; the gene is not expressed at all; and even when the gene is expressed, no polypeptide activity is exhibited.

[0168] The activity of a polypeptide is weakened compared to its endogenous activity, meaning that the activity of the polypeptide is reduced compared to the activity of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before modification. Whether the activity of a polypeptide is weakened can be confirmed by a decrease in the activity level of the corresponding polypeptide, its expression level, or the amount of product produced by the corresponding polypeptide.

[0169] In one example, attenuation can refer to a protein activity of about 100% or less, 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% relative to the protein activity of the parent strain or unmodified microorganism before modification, but is not limited thereto.

[0170] For example, inactivation may mean that the protein is not expressed at all, or even if the protein is expressed, it does not show activity or exhibits reduced activity compared to an unmodified microorganism.

[0171] The attenuation of polypeptide activity can be performed by any method known in the art, but the method is not limited thereto, and can be achieved by applying various methods well known in the art (for example, Nakashima N et al., Bacterialcellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014; 15(2): 2773-2793; Sambrook et al., Molecular Cloning 2012, etc.).

[0172] Specifically, the attenuation of the activity of the polypeptide of the present disclosure can be achieved by:

[0173] 1) Delete all or part of the gene encoding the polypeptide;

[0174] 2) Modifying the expression regulatory region (expression regulatory sequence) to reduce the expression of the gene encoding the polypeptide;

[0175] 3) Modifying the amino acid sequence of a polypeptide so that the activity of the polypeptide is eliminated or weakened (for example, deleting / substituting / adding one or more amino acids in the amino acid sequence);

[0176] 4) Modifying a polynucleotide sequence encoding a polypeptide so that the activity of the polypeptide is eliminated or weakened (for example, deleting / replacing / adding one or more nucleotides in the nucleotide sequence of the polypeptide gene to encode a modified polypeptide to eliminate or weaken the activity of the polypeptide);

[0177] 5) modifying the nucleotide sequence encoding the start codon or 5'-UTR of a gene encoding a polypeptide;

[0178] 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the gene transcript encoding the polypeptide;

[0179] 7) adding a sequence complementary to the Shine-Dalgarno (SD) sequence of the gene encoding the polypeptide to the front end of the SD sequence to form a secondary structure, thereby inhibiting ribosome attachment;

[0180] 8) Reverse transcription engineering (RTE), which adds a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the polynucleotide sequence encoding the polypeptide;

[0181] 9) Regulate the cellular localization of proteins (peptides); or

[0182] 10) A combination of two or more selected from the above methods 1) to 9), but not particularly limited thereto.

[0183] For example,

[0184] 1) The method of deleting part or all of the gene encoding the polypeptide can be achieved by deleting all polynucleotides encoding the endogenous target polypeptide in the chromosome, or by replacing the polynucleotide with a polynucleotide partially deleted of nucleotides or with a marker gene.

[0185] The method of deleting part or all of a polynucleotide can be achieved by: deleting the polynucleotide by homologous recombination through a vector for insertion into the chromosome of a microorganism, or a method in which light such as UV light or chemicals can be used to induce mutations, thereby selecting a strain in which the target gene is deleted from the resulting mutants, but is not limited thereto. The method of deleting part or all of a gene can include a method using DNA recombination technology. For example, by injecting a nucleotide sequence or a vector containing a nucleotide sequence homologous to the target gene into a microorganism to induce homologous recombination, part or all of the gene can be deleted. The injected nucleotide sequence or vector can contain a dominant selection marker, but is not limited thereto.

[0186] 2) Modification of the expression regulatory region can be achieved by: inducing modification of the expression regulatory region (expression regulatory sequence) through deletion, insertion, non-conservative substitution, conservative substitution, or a combination thereof; or by replacing the sequence with a sequence with less active 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 sequences that regulate transcription and translation.

[0187] Methods 3) and 4) for modifying amino acid sequences or polynucleotide sequences can be achieved by: inducing sequence modification through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to reduce the activity of the polypeptide; or by replacing the sequence with a modified amino acid sequence or polynucleotide sequence having weaker activity or a modified amino acid sequence or polynucleotide sequence having no activity, but are not limited thereto. For example, gene expression can be inhibited or reduced by introducing a modification into the polynucleotide sequence to form a stop codon, but is not limited thereto.

[0188] 5) Modification of the nucleotide sequence encoding the start codon or 5'-UTR of a gene encoding a polypeptide can be achieved, for example, by replacing the start codon with another start codon having a lower polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0189] 6) Methods for introducing antisense oligonucleotides (e.g., antisense RNA) that complementarily bind to gene transcripts encoding polypeptides can be found in the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol. 1(1) 1986].

[0190] 7) A method of adding a sequence complementary to the Shine-Dalgarno (SD) sequence of a polypeptide-encoding gene to the front end of the SD sequence to form a secondary structure, thereby inhibiting ribosome attachment can be achieved by inhibiting mRNA translation or reducing its speed.

[0191] 8) Reverse transcription engineering (RTE), which adds a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide, and can be achieved by forming antisense nucleotides complementary to the gene transcript encoding the polypeptide to weaken the activity.

[0192] 9) Methods for regulating the cellular localization of proteins (polypeptides) can be achieved by targeting the protein (polypeptide) to a specific organelle or specific space within the cell. For example, this can be achieved by adding or removing a leader sequence that plays a role in targeting the protein (polypeptide) to the periplasm or cytoplasm, but is not limited thereto.

[0193] Such reduction in polypeptide activity may mean that the activity or concentration of the corresponding polypeptide is reduced relative to the activity or concentration of the polypeptide expressed in a wild-type strain or a microbial strain before modification, or that the amount of the product produced by the corresponding polypeptide is increased, but is not limited thereto.

[0194] Another aspect of the present disclosure provides a method for producing L-glutamic acid, comprising culturing the microorganism of the present disclosure in a culture medium.

[0195] Specifically, the method for producing L-glutamic acid disclosed herein may include culturing a microorganism comprising the variant disclosed herein, the polynucleotide disclosed herein, or the vector disclosed herein in a culture medium, but is not limited thereto.

[0196] As used herein, the term "culturing" refers to growing the microorganisms of the present disclosure under appropriately controlled environmental conditions. The culturing process of the present disclosure can be carried out under suitable culture media and culture conditions known in the art. Such culturing processes can be easily adjusted for use by those skilled in the art based on the strain to be selected. Specifically, the culturing can be batch culture, continuous culture, and / or fed-batch culture, but is not limited thereto.

[0197] As used herein, term " culture medium " refers to the material mixture containing the required nutrients for cultivating microorganism of the present disclosure as main component, and it provides nutrients and growth factors, and the necessary water for survival and growth. Particularly, the culture medium and other culture conditions for cultivating microorganism of the present disclosure can be any culture medium for routine cultivation of microorganisms, without any particular restrictions. However, microorganism of the present disclosure can be cultivated under aerobic conditions in a conventional culture medium containing suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin etc., while regulating temperature, pH etc. For example, the culture medium for Corynebacterium strain can be found in document [" Manual of Methods for General Bacteriology " (Washington DC, USA, 1981) of American Society for Bacteriology).

[0198] In the present disclosure, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc.; and glycerol, propylene glycol, etc. In addition, the carbon source may include natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, sugarcane molasses, and corn steep liquor, etc. Specifically, carbohydrates such as glucose and sterile pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and in addition, various other carbon sources may be used in appropriate amounts without restriction. These carbon sources may be used alone or in combinations of two or more, but are not limited thereto.

[0199] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; amino acids such as glutamic acid, methionine, glutamine, etc.; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0200] The phosphorus source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate or corresponding sodium salts. Examples of inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins and / or suitable precursors may be included. These components or precursors may be added to the culture medium in a batch or continuous manner, but these phosphorus sources are not limited thereto.

[0201] In addition, during the process of culturing the microorganisms of the present disclosure in an appropriate manner, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid. In addition, defoaming agents such as fatty acid polyethylene glycol esters can be used during the culture process to prevent bubble formation. In addition, oxygen or oxygen-containing gas can be injected into the culture medium to maintain aerobic conditions of the culture medium; or nitrogen, hydrogen, or carbon dioxide can be injected or no gas can be injected to maintain anaerobic or microaerobic conditions, but the gas is not limited thereto.

[0202] The temperature during the culturing of the present disclosure may be in the range of 20° C. to 45° C., specifically 25° C. to 40° C., and the culturing may last for about 10 to 160 hours, but is not limited thereto.

[0203] L-glutamic acid produced by the culture of the present disclosure may be released into the culture medium or retained in the cells.

[0204] In one embodiment, the method for producing L-glutamic acid of the present disclosure may further include a step of preparing the microorganism of the present disclosure, a step of preparing a culture medium for culturing the strain, or a combination thereof (regardless of the order, in any order), for example, before the culturing step.

[0205] In one embodiment, the method for producing L-glutamic acid of the present disclosure may further include a step of recovering L-glutamic acid from the culture medium (culture medium therein) or from the microorganism of the present disclosure. A recovery step may also be included after the culture step.

[0206] In the recovery step, the method for cultivating the microorganism of the present disclosure can be used, for example, according to batch culture, continuous culture or fed-batch culture method, using suitable methods known in the art to collect L-glutamic acid. For example, methods such as centrifugation, filtration, treatment with a protein crystallization precipitant (salting out), extraction, ultrasonication, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC or a combination thereof can be used, and suitable methods known in the art can be used to recover L-glutamic acid from the culture medium or microorganism.

[0207] In addition, the method for producing L-glutamic acid disclosed herein may further include a purification step, which may be performed using a suitable method known in the art. In one example, when the method for producing L-glutamic acid disclosed herein includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or intermittently without regard to the order, or may be performed simultaneously, or may be integrated into one step, but the method is not limited thereto.

[0208] In the methods disclosed herein, the variant polypeptides, polynucleotides, and L-glutamic acid are as described above in other aspects.

[0209] Another aspect of the present disclosure provides a composition for producing L-glutamic acid, comprising: a variant polypeptide of the present disclosure; a polynucleotide encoding the variant polypeptide; a vector containing the polynucleotide; a microorganism comprising the variant polypeptide of the present disclosure, a polynucleotide encoding the variant polypeptide, or a vector containing the polynucleotide; a culture product of the microorganism; or a combination of two or more thereof.

[0210] The composition of the present disclosure may further comprise any suitable excipient commonly used in compositions for producing L-glutamic acid, and such excipients may include, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents, but are not limited thereto.

[0211] In one embodiment, the various components present in the compositions of the present disclosure may be included in microbiologically effective amounts, or in amounts that may be suitably present in the composition for manufacture.

[0212] In the composition of the present disclosure, the variant polypeptide, polynucleotide and L-glutamic acid are as described in other aspects above.

[0213] Another aspect of the present disclosure provides use of the variant polypeptide of the present disclosure for producing L-glutamic acid.

[0214] The variant polypeptides, L-glutamic acid, etc. disclosed herein are as described above in other aspects.

[0215] Another aspect of the present disclosure provides use of the microorganism of the present disclosure for producing L-glutamic acid.

[0216] The variant polypeptides, L-glutamic acid, etc. disclosed herein are as described above in other aspects.

[0217] Mode for Carrying Out the Invention

[0218] The present disclosure will be described in detail below by way of examples. However, these examples are preferred embodiments provided for illustrative purposes only, and the scope of the present disclosure is not intended to be limited to or by these examples. Furthermore, those skilled in the art of the present disclosure or similar technical fields can fully understand and easily implement technical features not described herein.

[0219] Example 1. Screening of mutant strains for increasing L-glutamic acid production capacity by artificial mutation

[0220] Example 1-1. Inducing artificial mutations by UV irradiation

[0221] In order to screen for mutant strains with improved L-glutamic acid (the target product of fermentation) production capacity, wild-type Corynebacterium glutamicum (ATCC13869) was first plated on a nutrient medium containing agar and cultured for 16 hours at 30° C. Hundreds of colonies thus obtained were irradiated with UV light at room temperature to induce random mutations in the strain genome.

[0222] Example 1-2. Fermentation titer experiment of mutagenized strains and screening strains

[0223] A fermentation titer experiment was conducted on the mutant strain in which random mutations had been induced in Example 1-1.

[0224] Each colony was subcultured in a nutrient medium and then cultured in a fermentation medium for 5 hours. Thereafter, 25% tween 40 was added to each culture medium at a concentration of 0.4%, and each colony was cultured for an additional 32 hours.

[0225] <Nutrient medium>

[0226] 1% glucose, 0.5% meat juice, 1% polypeptone, 0.25% sodium chloride, 0.5% yeast extract, 2% agar, 0.2% urea, pH 7.2

[0227] <Fermentation medium>

[0228] 6% glucose, 5% calcium carbonate, 2.25% ammonium sulfate, 0.1% potassium monophosphate, 0.04% magnesium sulfate, 10mg / L ferrous sulfate, 0.3mg / L biotin, 0.2mg / L thiamine hydrochloride

[0229] Under these conditions, cultivate each bacterium colony, then screen the mutant strain that the amount of producing L-Glutamic acid is equal to or greater than the amount of wild-type Corynebacterium glutamicum (ATCC13869) producing L-Glutamic acid.After this, for selected mutant strain, measure the concentration of L-Glutamic acid by HPLC.The L-Glutamic acid concentration measured is as shown in table 1 below.

[0230] [Table 1]

[0231]

[0232]

[0233] Based on Table 1 above, "ATCC13869-C4" and "ATCC13869-C7" were selected as mutant strains in which the amount of L-glutamic acid production was increased compared to the wild-type strain.

[0234] Example 2. Confirmation of mutations by gene sequencing

[0235] In order to confirm the gene mutation of the mutant strains, the genes of the ATCC13869-C4 and ATCC13869-C7 strains selected in Example 1-2 were compared with the genes of the wild-type strain.

[0236] As a result, it was found that the ATCC13869-C4 and ATCC13869-C9 strains contained the same mutation at a specific position of the cgmA gene (SEQ ID NO: 2) encoding the major cofactor superfamily transporter (wherein the nucleotide at position 1052 of the polynucleotide sequence shown by SEQ ID NO: 2 was substituted by A).

[0237] Therefore, in Examples 3 and 4, it was attempted to confirm whether the above mutations have an effect on the production of L-glutamic acid in microorganisms of the genus Corynebacterium.

[0238] Example 3. Preparation of mutant-introduced strains and confirmation of L-glutamic acid production-1

[0239] Example 3-1. Preparation of mutant-introduced strain-1

[0240] Attempt to prepare the mutant strain that has been introduced the sudden change that confirmed among the embodiment 2.Particularly, preparation is used for the carrier of gene replacement, by sudden change (wherein the 1052nd Nucleotide of the polynucleotide sequence shown in SEQ ID NO:2 is replaced by A) is introduced among the wild-type Corynebacterium glutamicum (ATCC13869 and ATCC13032), so that be included in the 351st isoleucine of the major facilitator superfamily transporter shown in the SEQ ID NO:1 in the bacterial strain with asparagine replacement.Use ATCC13869 genomic DNA as template, obtain the gene fragment that is used to prepare carrier by PCR.Based on the gene of Corynebacterium glutamicum (ATCC13869) that registers in the National Institutes of Health GenBank (NIH GenBank) and the information of adjacent sequence, preparation comprises the primer of the polynucleotide of SEQ ID NO:13,14,15 and 16.

[0241] PCR was performed under the following conditions: denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 20 seconds, annealing at 55°C for 20 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. More specifically, polynucleotides (500 bp) amplified using primers of SEQ ID NOs: 13 and 14, and polynucleotides (500 bp) amplified using primers of SEQ ID NOs: 15 and 16 were obtained. The two DNA fragments obtained were ligated to a vector pDZ (Korean Patent No. 10-0924065 and International Publication No. 2008-033001) that had been digested with restriction enzymes BamHI and SalI using a fusion enzyme to prepare a vector for gene replacement. The prepared vector was named "pDZ-cgmA (I351N)". The information of the primer sequences used to prepare the vector is shown in Table 2 below.

[0242] [Table 2]

[0243]

[0244] Subsequently, the vector for gene replacement is transformed into the wild-type strain by homologous recombination on the chromosome ( van der Rest et al., Appl Microbiol Biotechnol 52: 541-545, 1999 ).

[0245] Strains that had inserted the vector into their chromosomes via homologous recombination were screened in a medium containing 25 mg / L kanamycin. Subsequently, gene sequencing analysis of the transformed C. glutamicum strains that had completed secondary recombination confirmed the introduction of the target mutation. The resulting strains were named "ATCC13869::cgmA(I351N)" and "ATCC13032::cgmA(I351N)."

[0246] Example 3-2. Confirmation of L-glutamic acid production-1

[0247] The ATCC13869::cgmA(I351N) and ATCC13032::cgmA(I351N) mutant strains and their wild-type Corynebacterium glutamicum (ATCC13869 and ATCC13032) strains prepared in Example 3-1 were cultured in the same manner as in Example 1-2.

[0248] After the culture was completed, the L-glutamic acid concentration in each culture medium was measured. The measured L-glutamic acid concentrations are shown in Table 3 below.

[0249] [Table 3]

[0250] strains L-glutamic acid (g / L) ATCC13869 7.0 ATCC13869::cgmA(I351N) 8.5 ATCC13032 3.6 ATCC13032::cgmA(I351N) 4.4

[0251] As shown in Table 3, it was confirmed that the concentration of L-glutamic acid produced by the mutation-introduced Corynebacterium glutamicum ATCC13869::cgmA(I351N) strain was about 1.5 g / L (about 21%) higher than that produced by the wild-type Corynebacterium glutamicum ATCC13869.

[0252] Furthermore, it was confirmed that the concentration of L-glutamic acid produced by the mutation-introduced Corynebacterium glutamicum ATCC13032::cgmA(I351N) strain was about 0.8 g / L (about 22%) higher than that produced by the wild-type Corynebacterium glutamicum ATCC13032.

[0253] Therefore, it was confirmed that the variant of the present disclosure increased the L-glutamic acid-producing ability of the microorganism.

[0254] Example 4. Preparation of strains into which mutations were introduced and confirmation of L-glutamic acid production-2

[0255] Example 4-1. Preparation of mutant-introduced strain-2

[0256] An attempt was further made to introduce variants of the TetR family transcriptional regulatory factor (mmpLR) into the ATCC13869::cgmA(I351N) and ATCC13032::cgmA(I351N) mutant strains prepared in Example 3-1.

[0257] Specifically, the carrier for gene replacement is prepared by introducing mmpLR mutation (wherein the 314th nucleotide of the polynucleotide sequence shown in SEQ ID NO:6 is replaced by T) into wild-type Corynebacterium glutamicum (ATCC13869 and ATCC13032) to replace the 105th proline of the TetR family transcriptional regulatory factor shown in the SEQ ID NO:5 included in the bacterial strain with leucine.Use ATCC13869 genomic DNA as template, obtain the gene fragment for preparing the carrier by PCR.Based on the information of the gene and adjacent sequence of Corynebacterium glutamicum (ATCC13869) registered in the National Institutes of Health GenBank (NIH GenBank), the primers of the polynucleotide comprising SEQ ID NO:17, 18, 19 and 20 are prepared.

[0258] PCR was performed under the following conditions: denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 20 seconds, annealing at 55°C for 20 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. More specifically, polynucleotides (500 bp) amplified using primers of SEQ ID NOs: 17 and 18, and polynucleotides (500 bp) amplified using primers of SEQ ID NOs: 19 and 20 were obtained. The two DNA fragments thus obtained were ligated to a vector pDZ (Korean Patent No. 10-0924065 and International Publication No. 2008-033001) that had been digested with restriction enzymes BamHI and SalI using a fusion enzyme to prepare a vector for gene replacement. The prepared vector was named "pDZ-mmpLR (P105L)". The information on the primer sequences used to prepare the vector is shown in Table 4 below.

[0259] [Table 4]

[0260]

[0261] The chromosomal vector of the present invention is transformed into ATCC13869::cgmA (I351N) and ATCC13032::cgmA (I351N) bacterial strains by the homologous recombination on the chromosome.From the substratum that contains 25mg / L kanamycin, screening is carried out the bacterial strain that the vector is inserted into the chromosome by homologous sequence recombination.After this, the Corynebacterium glutamicum bacterial strain of the conversion that has completed secondary recombination is carried out gene sequencing analysis, the result confirms that the target mutation is introduced into bacterial strain, and with the gained bacterial strain named as " ATCC13869::cgmA (I351N)_mmpLR (P105L)) " and " ATCC13032::cgmA (I351N)_mmpLR (P105L) ".

[0262] Example 4-2. Confirmation of L-glutamic acid production-2

[0263] The ATCC13869::cgmA(I351N)_mmpLR(P105L) and ATCC13032::cgmA(I351N)_mmpLR(P105L) mutant strains and their wild-type Corynebacterium glutamicum (ATCC13869 and ATCC13032) strains prepared in Example 4-1 were cultured in the same manner as in Example 1-2.

[0264] After the culture was completed, the L-glutamic acid concentration in each culture medium was measured. The measured L-glutamic acid concentrations are shown in Table 5 below.

[0265] [Table 5]

[0266] strains L-glutamic acid (g / L) ATCC13869 7.0 ATCC13869::cgmA(I351N)_mmpLR(P105L) 9.4 ATCC13032 3.6 ATCC13032::cgmA(I351N)_mmpLR(P105L) 4.7

[0267] As shown in Table 5, it was confirmed that the concentration of L-glutamic acid produced by the mutation-introduced Corynebacterium glutamicum ATCC13869::cgmA(I351N)_mmpLR(P105L) strain was about 2.4 g / L (about 34%) higher than that produced by the wild-type Corynebacterium glutamicum ATCC13869.

[0268] Furthermore, it was confirmed that the concentration of L-glutamic acid produced by the mutation-introduced Corynebacterium glutamicum ATCC13032::cgmA(I351N)_mmpLR(P105L) strain was about 1.1 g / L (about 31%) higher than that produced by the wild-type Corynebacterium glutamicum ATCC13032.

[0269] That is, it was confirmed that when a variant polypeptide having the activity of a TetR family transcriptional regulatory factor was additionally introduced into a microorganism comprising the variant polypeptide of the present disclosure, the L-glutamic acid-producing ability was further increased.

[0270] Example 5. Preparation of mutant-introduced strains and confirmation of L-glutamic acid production-3

[0271] Example 5-1. Preparation of mutant-introduced strain-3

[0272] An attempt was further made to introduce variants of isocitrate lyase (aceA) into the ATCC13869::cgmA(I351N)_mmpLR(P105L) and ATCC13032::cgmA(I351N)_mmpLR(P105L) mutant strains prepared in Example 4-1.

[0273] Specifically, preparation is used for the carrier of gene replacement, by aceA sudden change (wherein corresponding to the nucleotide sequence of position 1006 to position 1038 of the polynucleotide sequence shown in SEQ ID NO:10 is deleted) is introduced in wild-type Corynebacterium glutamicum (ATCC13869 and ATCC13032), so that the lysine corresponding to position 336 of the isocitrate lyase shown in the SEQ ID NO:9 that deletion is included in the bacterial strain is to the glutamine corresponding to position 346.Use ATCC13869 genomic DNA as template, obtain the gene fragment for the preparation of carrier by PCR.Based on the gene of Corynebacterium glutamicum (ATCC13869) registered in National Institutes of Health GenBank (NIH GenBank) and the information of adjacent sequences, preparation comprises the primer of the polynucleotide of SEQ ID NO:21,22,23 and 24.

[0274] PCR was performed under the following conditions: denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 20 seconds, annealing at 55°C for 20 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. More specifically, polynucleotides (500 bp) amplified using primers of SEQ ID NOs: 21 and 22 were obtained, and polynucleotides (500 bp) amplified using primers of SEQ ID NOs: 23 and 24 were obtained. The two DNA fragments thus obtained were ligated to a vector pDZ (Korean Patent No. 10-0924065 and International Publication No. 2008-033001) that had been digested with restriction enzymes BamHI and SalI using a fusion enzyme to prepare a vector for gene replacement. The prepared vector was named "pDZ-aceA_deletion". The information of the primer sequences used to prepare the vector is shown in Table 6 below.

[0275] [Table 6]

[0276]

[0277]

[0278] The chromosomal expression vector of the present invention is transformed into ATCC13869::cgmA (I351N)_mmpLR (P105L) and ATCC13032::cgmA (I351N)_mmpLR (P105L) bacterial strains by the homologous recombination on the chromosome.From the substratum that contains 25mg / L kanamycin, screening is carried out the bacterial strain that carrier is inserted into chromosome by homologous sequence recombination.After this, the Corynebacterium glutamicum bacterial strain of the conversion that has completed secondary recombination is carried out gene sequencing analysis, result confirms that target mutation is introduced into bacterial strain, and with gained bacterial strain named as " ATCC13869::cgmA (I351N)_mmpLR (P105L)_aceA_deletion " and " ATCC13032::cgmA (I351N)_mmpLR (P105L)_aceA_deletion ".

[0279] Example 5-2. Confirmation of L-glutamic acid production-3

[0280] The ATCC13869::cgmA(I351N)_mmpLR(P105L)_aceA_deletion and ATCC13032::cgmA(I351N)_mmpLR(P105L)_aceA_deletion mutant strains and their wild-type Corynebacterium glutamicum (ATCC13869 and ATCC13032) strains prepared in Example 5-1 were cultured in the same manner as in Example 1-2.

[0281] After the culture was completed, the L-glutamic acid concentration in each culture medium was measured. The measured L-glutamic acid concentrations are shown in Table 7 below.

[0282] [Table 7]

[0283]

[0284]

[0285] As shown in Table 7, it was confirmed that the concentration of L-glutamic acid produced by the mutation-introduced Corynebacterium glutamicum ATCC13869::cgmA(I351N)_mmpLR(P105L)_aceA_deletion strain was about 3.3 g / L (about 47%) higher than that produced by the wild-type Corynebacterium glutamicum ATCC13869.

[0286] Furthermore, it was confirmed that the concentration of L-glutamic acid produced by the mutation-introduced Corynebacterium glutamicum ATCC13032::cgmA(I351N)_mmpLR(P105L)_aceA_deletion strain was about 1.6 g / L (about 44%) higher than that produced by the wild-type Corynebacterium glutamicum ATCC13032.

[0287] That is, it was confirmed that when a variant polypeptide having TetR family transcriptional regulatory factor activity and a variant polypeptide having isocitrate lyase activity were additionally introduced into a microorganism comprising the variant polypeptide of the present disclosure, L-glutamic acid-producing ability was further increased.

[0288] Example 6. Confirmation of L-glutamic acid production in KFCC11074 strain with introduced mutation

[0289] Example 6-1. Preparation of mutant-introduced strain-1

[0290] To confirm whether this mutation has the same effect in strains having increased L-glutamic acid production ability in addition to the wild type strain, attempts were made to introduce this mutation into KFCC11074 strain (Korean Patent Publication No. 10-0292299), which is well known as an L-glutamic acid-producing strain.

[0291] Particularly, by the homologous recombination on the karyomit(e)y, pDZ-cgmA (I351N) carrier of preparation among the embodiment 3-1 is transformed into the KFCC11074 bacterial strain (people such as van der Rest, Appl Microbiol Biotechnol 52:541-545,1999).From the substratum that contains 25mg / L kanamycin, screening by homologous sequence recombination carrier is inserted into the chromosomal bacterial strain.After this, the Corynebacterium glutamicum bacterial strain of the conversion that has completed secondary recombination is carried out gene sequencing analysis, the result confirms that target sudden change is introduced into bacterial strain, and with gained bacterial strain named " KFCC11074_cgmA (I351N) ".

[0292] Example 6-2. Preparation of mutant-introduced strain-2

[0293] An attempt was further made to introduce a variant of the TetR family transcriptional regulatory factor (mmpLR) into the KFCC11074_cgmA(I351N) mutant strain prepared in Example 6-1.

[0294] Particularly, by homologous recombination on karyomit(e)y, pDZ-mmpLR (P105L) carrier of preparation among the embodiment 4-1 is transformed into KFCC11074_cgmA (I351N) bacterial strain (people such as van der Rest, Appl Microbiol Biotechnol 52:541-545,1999).From the substratum that contains 25mg / L kanamycin, screening is carried out the bacterial strain that carrier is inserted into karyomit(e)y by homologous sequence recombination.After this, the Corynebacterium glutamicum bacterial strain of the conversion that has completed secondary recombination is carried out gene sequencing analysis, and result confirms that target mutation is introduced into bacterial strain, and with gained bacterial strain named as " KFCC11074_cgmA (I351N)_mmpLR (P105L) ".

[0295] Example 6-3. Preparation of mutant-introduced strain-3

[0296] An attempt was further made to introduce a variant of isocitrate lyase (aceA) into the KFCC11074_cgmA(I351N)_mmpLR(P105L) mutant strain prepared in Example 6-2.

[0297] The pDZ-aceA_deletion vector prepared in embodiment 5-1 is transformed into KFCC11074_cgmA (I351N)_mmpLR (P105L) bacterial strain by homologous recombination on chromosome (people such as van der Rest, Appl Microbiol Biotechnol 52:541-545,1999).From the substratum that contains 25mg / L kanamycin, screening is carried out the bacterial strain that carrier is inserted into chromosome by homologous sequence recombination.After this, the Corynebacterium glutamicum bacterial strain of the conversion that has completed secondary recombination is carried out gene sequencing analysis, result confirms that target mutation is introduced into bacterial strain, and with gained bacterial strain named as " KFCC11074_cgmA (I351N)_mmpLR (P105L)_aceA_deletion ".

[0298] Example 7. Confirmation of L-glutamic acid production in KFCC11074 strain with introduced mutation

[0299] Example 7-1. Confirmation of L-glutamic acid production-1

[0300] The KFCC11074_cgmA(I351N) mutant strain prepared in Example 6-1 and its wild-type KFCC11074 strain were cultured in the same manner as in Example 1-2.

[0301] After the culture was completed, the L-glutamic acid concentration in each culture medium was measured. The measured L-glutamic acid concentrations are shown in Table 8 below.

[0302] [Table 8]

[0303] strains L-glutamic acid (g / L) KFCC11074 5.6 KFCC11074_cgmA(I351N) 6.9

[0304] As shown in Table 8, it was confirmed that the concentration of L-glutamic acid produced by the mutation-introduced Corynebacterium glutamicum KFCC11074_cgmA(I351N) strain was about 1.3 g / L (about 23%) higher than that produced by Corynebacterium glutamicum KFCC11074 without mutation.

[0305] That is, it was confirmed that the mutation of the present disclosure increased the L-glutamic acid-producing ability of the microorganism, even in a strain having increased L-glutamic acid-producing ability.

[0306] Example 7-2. Confirmation of L-glutamic acid production-2

[0307] The KFCC11074_cgmA(I351N) and KFCC11074_cgmA(I351N)_mmpLR(P105L) mutant strains and their wild-type KFCC11074 strain prepared in Examples 6-1 and 6-2, respectively, were cultured in the same manner as in Example 1-2.

[0308] After the culture was completed, the L-glutamic acid concentration in each culture medium was measured. The measured L-glutamic acid concentrations are shown in Table 9 below.

[0309] [Table 9]

[0310]

[0311] As shown in Table 9, it was confirmed that the concentration of L-glutamic acid produced by the mutation-introduced Corynebacterium glutamicum KFCC11074_cgmA(I351N)_mmpLR(P105L) strain was about 2.2 g / L (about 39%) higher than that produced by the non-mutation-introduced Corynebacterium glutamicum KFCC11074 strain.

[0312] That is, it was confirmed that when a variant polypeptide having TetR family transcriptional regulatory factor activity was additionally introduced into a microorganism comprising the variant polypeptide of the present disclosure, the L-glutamic acid-producing ability of the microorganism was further increased, even in a strain having increased L-glutamic acid-producing ability.

[0313] Example 7-3. Confirmation of L-glutamic acid production-3

[0314] The KFCC11074_cgmA(I351N), KFCC11074_cgmA(I351N)_mmpLR(P105L) and KFCC11074_cgmA(I351N)_mmpLR(P105L)_aceA_deletion mutant strains and their wild-type KFCC11074 strain prepared in Examples 6-1, 6-2 and 6-3, respectively, were cultured in the same manner as in Example 1-2.

[0315] After the culture was completed, the L-glutamic acid concentration in each culture medium was measured. The measured L-glutamic acid concentrations are shown in Table 10 below.

[0316] [Table 10]

[0317]

[0318] As shown in Table 10, it was confirmed that the concentration of L-glutamic acid produced by the mutation-introduced Corynebacterium glutamicum KFCC11074_cgmA(I351N)_mmpLR(P105L)_aceA_deletion strain was about 3.1 g / L (about 52%) higher than the concentration of L-glutamic acid produced by the non-mutation-introduced Corynebacterium glutamicum KFCC11074 strain.

[0319] That is, it was confirmed that when a variant polypeptide having the activity of a TetR family transcriptional regulatory factor and a variant polypeptide having the activity of isocitrate lyase were additionally introduced into a microorganism comprising the variant polypeptide of the present disclosure, the L-glutamic acid-producing ability of the microorganism was further increased, even in a strain having increased L-glutamic acid-producing ability.

[0320] Based on the foregoing, those skilled in the art to which the present disclosure pertains will appreciate that the present disclosure may be implemented in other specific forms without modifying the technical concepts or essential features of the present disclosure. In this regard, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of the present disclosure. On the contrary, the present disclosure is intended to cover not only the exemplary embodiments, but also various alternatives, modifications, equivalent replacements, and other embodiments that may be included within the spirit and scope of the present disclosure as defined by the appended claims.

Claims

1. A variant polypeptide, wherein the amino acid corresponding to position 351 of SEQ ID NO: 1 is substituted by another amino acid.

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

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

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

6. The microorganism according to claim 5, wherein the microorganism further comprises: any one or more variant polypeptides selected from the group consisting of (a) and (b); a polynucleotide encoding the variant polypeptide; or a vector comprising the polynucleotide: Wherein (a) a variant polypeptide having the activity of a TetR family transcriptional regulatory factor, wherein the amino acid corresponding to position 105 of SEQ ID NO: 5 is replaced by another amino acid; and (b) A variant polypeptide having isocitrate lyase activity, wherein the amino acid sequence corresponding to positions 336 to 346 of SEQ ID NO: 9 is deleted.

7. The microorganism according to claim 6, wherein (a) the variant polypeptide having the activity of a TetR family transcriptional regulatory factor is a variant polypeptide having the activity of a TetR family transcriptional regulatory factor in which the amino acid corresponding to position 105 of SEQ ID NO: 5 is substituted by leucine.

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

11.

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

10. The microorganism according to claim 5, wherein the microorganism is a microorganism of the genus Corynebacterium. 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 according to claim 5 in a culture medium.

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

14. A composition for the production of L-Glutamic acid, comprising: a variant polypeptide according to any one of claims 1 to 3; a polynucleotide encoding the variant polypeptide; a vector comprising the polynucleotide; a microorganism comprising the variant polypeptide, the polynucleotide encoding the variant polypeptide, or the vector comprising the polynucleotide; a culture product of the microorganism; or a combination of two or more thereof.

15. The variant polypeptide of any one of claims 1 to 3; a polynucleotide encoding the variant polypeptide; a vector comprising the polynucleotide; a microorganism comprising the variant polypeptide, the polynucleotide encoding the variant polypeptide, or the vector comprising the polynucleotide; a culture product of the microorganism; or a combination of two or more thereof for the production of L-glutamic acid.

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