Novel manganese-dependent transcriptional regulator mutant and method for producing L-glutamic acid using the same

Abstract

The present application relates to a novel manganese-dependent transcriptional regulator mutant polypeptide; a polynucleotide encoding the mutant polypeptide; a microorganism comprising the mutant polypeptide, a polynucleotide encoding the mutant polypeptide, or a vector comprising the polynucleotide; a method for producing L-glutamic acid, the method comprising culturing the microorganism in a medium; and use of the microorganism for producing L-glutamic acid.

Description

[Technical Field]

[0001] The present application relates to a novel manganese-dependent transcriptional regulator mutant polypeptide; a polynucleotide encoding the mutant polypeptide; a microorganism comprising the mutant polypeptide, a polynucleotide encoding the mutant polypeptide, or a vector comprising the polynucleotide; a method for producing L-glutamic acid, the method comprising culturing the microorganism in a medium; and use of the microorganism for producing L-glutamic acid. [Background technology]

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

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

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

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

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

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

[0007] An object of the present application is to provide a microorganism that is capable of culturing a microorganism containing a mutant polypeptide of a manganese-dependent transcriptional regulator and producing L-glutamic acid at a higher yield than microorganisms containing existing non-mutated polypeptides, and a method for producing L-glutamic acid using the microorganism. [Means for solving the problem]

[0008] One aspect of the present application provides a manganese-dependent transcriptional regulator mutant polypeptide in which the amino acid corresponding to position 160 of SEQ ID NO: 1 is substituted with another amino acid.

[0009] In one embodiment, the mutant polypeptide may have the amino acid corresponding to position 160 of SEQ ID NO: 1 substituted with aspartic acid.

[0010] In another embodiment, the variant polypeptide may consist of the amino acid sequence of SEQ ID NO:3.

[0011] Another aspect of the present application provides a polynucleotide encoding the mutant polypeptide.

[0012] Another aspect of the present application provides a microorganism comprising said mutant polypeptide, a polynucleotide encoding said mutant polypeptide, or a vector comprising said polynucleotide.

[0013] In one specific example, the microorganism may have an increased ability to produce L-glutamic acid compared to a microorganism containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same.

[0014] As a microorganism according to any one of the above-mentioned embodiments, the microorganism may be a Corynebacterium microorganism.

[0015] In any one of the above-mentioned embodiments, the Corynebacterium microorganism may be Corynebacterium glutamicum.

[0016] Another aspect of the present application provides a method for producing L-glutamic acid, comprising culturing the microorganism in a medium.

[0017] In one embodiment, the method may further comprise the step of recovering L-glutamic acid from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium.

[0018] Another aspect of the present application provides a composition for producing L-glutamic acid, comprising: the mutant polypeptide; a polynucleotide encoding the mutant polypeptide; a vector comprising the polynucleotide; or a microorganism comprising the mutant polypeptide, a polynucleotide encoding the mutant polypeptide, or a vector comprising the polynucleotide; a culture of the microorganism; or a combination of two or more of them.

[0019] Another aspect of the present application provides use of the microorganism for producing L-glutamic acid. [Effects of the Invention]

[0020] When a microorganism containing the mutant polypeptide of the manganese-dependent transcriptional regulator of the present application is cultured, it is possible to produce L-glutamic acid at a higher yield than a microorganism containing the existing non-modified polypeptide. DETAILED DESCRIPTION OF THE INVENTION

[0021] This will be explained in more detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the specific descriptions set forth below are not deemed to limit the category of this application. Furthermore, numerous papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the state of the art to which this application pertains and the contents of this application.

[0022] One aspect of the present application provides a manganese-dependent transcriptional regulator mutant polypeptide in which the amino acid corresponding to position 160 of SEQ ID NO: 1 is substituted with another amino acid.

[0023] As used herein, the term "manganese-dependent transcriptional regulator variant polypeptide" refers to a manganese-dependent transcriptional regulator variant polypeptide that contains one or more amino acid substitutions in the amino acid sequence of a manganese-dependent transcriptional regulator polypeptide; or a variant of a manganese-dependent transcriptional regulator polypeptide that contains one or more amino acid substitutions in the parent sequence, which is the amino acid sequence of a manganese-dependent transcriptional regulator polypeptide.

[0024] In the present application, the term "manganese-dependent transcriptional regulator (dtxR)" refers to a global regulatory factor involved in iron metabolism, which suppresses iron binding in diphtheria toxin gene expression. It may be used interchangeably with terms such as "diphtheria toxin repressor" and "toxin regulatory factor," and may refer to, but is not limited to, the manganese-dependent transcriptional regulator (dtxR) encoded by the dtxR gene.

[0025] The gene encoding the manganese-dependent transcriptional regulator may be derived from a microorganism of the genus Corynebacterium, specifically, dtxR derived from Corynebacterium glutamicum, but is not limited thereto.

[0026] Specifically, the manganese-dependent transcriptional regulatory protein may include, for example, the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60% or more homology or identity thereto, but is not limited thereto as long as it performs the manganese-dependent transcriptional regulatory function. Specifically, the amino acid sequence may include SEQ ID NO: 1 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. The sequence of SEQ ID NO: 1 can be obtained from publicly known databases such as NCBI's GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes). For example, the protein may be derived from the genus Corynebacterium or Corynebacterium glutamicum, and more specifically, the protein may be a polypeptide / protein comprising the amino acid sequence set forth in SEQ ID NO: 1, but is not limited thereto. Furthermore, it is obvious that auxiliary proteins having amino acid sequences in which a portion of the sequence has been deleted, modified, substituted or added are also included within the scope of this application, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the protein.

[0027] Furthermore, the manganese-dependent transcriptional regulatory factor protein having the amino acid sequence of SEQ ID NO: 1 may have or contain the sequence of SEQ ID NO: 2 or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 2, or may consist of or be encoded by a polynucleotide essentially consisting of the sequence of SEQ ID NO: 2 or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 2, but is not limited thereto.

[0028] As used herein, the term "variant" refers to a polypeptide in which one or more amino acids have been conservatively substituted and / or modified, resulting in a polypeptide that differs from the original amino acid sequence but maintains its functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the performance of the variant may be increased, unchanged, or decreased compared to the original polypeptide. Some variants may also include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been deleted. Other variants may include variants in which portions have been deleted from the N- and / or C-termini of the mature protein. The term "mutant" may be used interchangeably with terms such as mutant type, variant, mutant polypeptide, mutated protein, mutation, and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited thereto as long as it is a term used in the sense of mutation.

[0029] Variants can also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide can be conjugated to an N-terminal signal (or leader) sequence of a protein involved in co-translational or post-translational protein transfer. The polypeptide can also be conjugated to other sequences or linkers that allow the polypeptide to be identified, purified, or synthesized.

[0030] The manganese-dependent transcriptional regulatory factor mutant polypeptide of the present application may be, but is not limited to, a manganese-dependent transcriptional regulatory factor mutant polypeptide in which the amino acid corresponding to position 160 of SEQ ID NO: 1 is substituted with another amino acid.

[0031] In one embodiment, the manganese-dependent transcriptional regulator variant polypeptide of the present application may have a sequence identity of 60% or more and less than 100%, specifically, 80% or more and less than 100%, to the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0032] Specifically, the variants of the present application can include amino acid sequences that have at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity to the amino acid sequence set forth in SEQ ID NO: 1, in which the amino acid at position 160 from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid. It is also clear that variants having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also included within the scope of the present application, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the variants of the present application.

[0033] The "other amino acid" is not limited as long as it is an amino acid different from the amino acid before substitution. Meanwhile, when the expression "a specific amino acid is substituted" is used in the present application, it is obvious that the amino acid is substituted with an amino acid different from the amino acid before substitution, even if it is not specifically stated that the amino acid is substituted with another amino acid.

[0034] Amino acids can generally be classified based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0035] Examples of such classifications include positively charged (basic) amino acids such as arginine, lysine, and histidine; negatively charged (acidic) amino acids such as glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) such as serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, amino acids can be classified into electrically charged amino acids (arginine, lysine, histidine, glutamic acid, and aspartic acid) and uncharged amino acids (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). Phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. Valine, leucine, and isoleucine can be classified as branched-chain amino acids. As another example, the 20 amino acids can be classified according to size into five groups, starting from the amino acid group with a relatively small volume: glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, and tyrosine, but are not necessarily limited thereto.

[0036] For example, when it is stated that "the amino acid corresponding to position 160 in SEQ ID NO: 1 is substituted with another amino acid," it may mean that the amino acid is substituted with aspartic acid, valine, alanine, isoleucine, glutamic acid, phenylalanine, arginine, cysteine, asparagine, glutamine, histidine, proline, serine, tyrosine, lysine, tryptophan, methionine, threonine, or leucine, excluding glycine, but is not limited thereto.

[0037] Even if the present application describes a "protein having an amino acid sequence set forth in a particular SEQ ID NO," it is clear that proteins having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also used in the present application, so long as they have the same or corresponding activity as a protein consisting of the amino acid sequence of that SEQ ID NO. For example, if they have the same or corresponding activity as the mutant protein, this does not exclude addition of sequences before or after the amino acid sequence that do not change the function of the protein, naturally occurring mutations, silent mutations, or conservative substitutions thereof, and it is clear that even cases in which such additions or mutations of sequences exist fall within the scope of the present application.

[0038] The "Nth position" in the present application can include the Nth position and an amino acid position corresponding to the Nth position. Specifically, it can include an amino acid position corresponding to any amino acid residue in a mature polypeptide disclosed in a specific amino acid sequence. The specific amino acid sequence may be the amino acid sequence of SEQ ID NO: 1.

[0039] As used herein, the term "corresponding to" refers to the amino acid residue at the recited position in the polypeptide, or an amino acid residue that is similar, identical, or homologous to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position may be determining the particular amino acid in a sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0040] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, based on which each amino acid residue in the amino acid sequence can be numbered with reference to the numeric position of the corresponding amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described herein can identify amino acid positions or positions where variations such as substitutions, insertions or deletions occur compared to a query sequence (also referred to as a "reference sequence").

[0041] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) or the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) can be used, but is not limited thereto. Sequence alignment programs, pairwise sequence comparison algorithms, and the like known in the art can also be used appropriately.

[0042] In one specific example, the mutant polypeptide may have the amino acid corresponding to position 160 of SEQ ID NO: 1 substituted with an amino acid selected from the group consisting of aspartic acid, valine, alanine, isoleucine, arginine, leucine, methionine, threonine, asparagine, glutamine, proline, serine, tryptophan, phenylalanine, histidine, cysteine, tyrosine, lysine, and glutamic acid, but is not limited thereto.

[0043] In one embodiment of the above-mentioned embodiments, the mutant polypeptide provided in the present application may have the amino acid corresponding to the 160th position from the N-terminus of SEQ ID NO: 1 substituted with an amino acid selected from the negatively charged (acidic) amino acids glutamic acid and aspartic acid.

[0044] In any one of the above-mentioned embodiments, the variant polypeptide provided herein may have the amino acid corresponding to the 160th position from the N-terminus of SEQ ID NO: 1 substituted with an amino acid having an electrically charged side chain selected from arginine, lysine, histidine, glutamic acid, and aspartic acid.

[0045] As an example of any of the above-mentioned embodiments, the mutant polypeptide provided in the present application may have the amino acid corresponding to the 160th position from the N-terminus of SEQ ID NO: 1 substituted with an amino acid selected from cysteine, proline, threonine, aspartic acid, and asparagine.

[0046] As an example of any of the above-mentioned embodiments, the manganese-dependent transcriptional regulator mutant polypeptide of the present application may be, but is not limited to, a polypeptide in which the amino acid corresponding to position 160 of SEQ ID NO: 1 is substituted with aspartic acid.

[0047] For example, the variant polypeptide of the present application may include an amino acid sequence in which the aspartic acid corresponding to position 160 in the amino acid sequence set forth in SEQ ID NO: 1 is fixed and which has at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity to SEQ ID NO: 1. It is also clear that variant polypeptides having amino acid sequences with partial deletions, modifications, substitutions, conservative substitutions, or additions are also within the scope of the present application, so long as the amino acid sequence has such homology or identity and exhibits efficacy corresponding to the variant polypeptide of the present application.

[0048] Meanwhile, a person skilled in the art can identify the amino acid corresponding to the 160th position of the amino acid sequence of SEQ ID NO: 1 in the present application in any amino acid sequence through sequence alignment known in the art, and it is obvious that when an "amino acid at a specific position in a particular SEQ ID NO" is mentioned in the present application, it also includes the "amino acid at the corresponding position" in any amino acid sequence, even if not otherwise specified.

[0049] In another embodiment, the variant polypeptide may consist of the amino acid sequence of SEQ ID NO:3.

[0050] Specifically, the variant polypeptides of the present application may have, comprise, consist of, or essentially consist of SEQ ID NO:3 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to said SEQ ID NO:3.

[0051] For example, the amino acid sequence may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally that do not alter the function of the variant of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0052] As used herein, the term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions generally occur on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Amino acids can also be classified as those with electrically charged side chains or those with uncharged side chains. Charged amino acids include aspartic acid, glutamic acid, lysine, arginine, and histidine. Uncharged amino acids can again be classified as nonpolar or polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Conservative substitutions typically have little or no effect on the activity of the resulting polypeptide. Conservative substitutions typically have little or no effect on the activity of the resulting protein or polypeptide.

[0053] In one embodiment, the manganese-dependent transcriptional regulator variant polypeptide of the present application may have enhanced manganese-dependent electron regulator activity, but is not limited thereto. Furthermore, the manganese-dependent transcriptional regulator variant polypeptide of the present application may have, but is not limited to, an activity that increases L-glutamate production ability compared to a wild-type manganese-dependent transcriptional regulator polypeptide.

[0054] Another aspect of the present application provides a polynucleotide encoding the mutant polypeptide.

[0055] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide units are linked in a long chain by covalent bonds, and a DNA or RNA chain of a certain length or more, and more specifically, a polynucleotide fragment encoding the above-mentioned variant.

[0056] The polynucleotide encoding the manganese-dependent transcriptional regulator mutant polypeptide of the present application may include, without limitation, any polynucleotide sequence that encodes the manganese-dependent transcriptional regulator mutant polypeptide of the present application. For example, the polynucleotide encoding the manganese-dependent transcriptional regulator mutant polypeptide of the present application may be, but is not limited to, a polynucleotide sequence that encodes the amino acid sequence of the manganese-dependent transcriptional regulator mutant polypeptide of the present application. In the present application, the polynucleotide may be, but is not limited to, one that enhances the activity of the manganese-dependent transcriptional regulator or one that increases the amount of glutamate produced.

[0057] For example, it may include a nucleic acid sequence that encodes the amino acid sequence set forth in SEQ ID NO: 3. As an example of the present application, the polynucleotide of the present application may have or include SEQ ID NO: 4. The polynucleotide of the present application may also consist of or consist essentially of SEQ ID NO: 4.

[0058] The polynucleotides of the present application may be modified in various ways in their coding regions, taking into account codon degeneracy or the codons preferred in the organism in which the variants of the present application are to be expressed, without changing the amino acid sequence of the variants of the present application. Therefore, it is clear that polynucleotides that are translated into polypeptides consisting of the amino acid sequence of the variants of the present application or polypeptides having homology or identity thereto due to codon degeneracy are also included. For example, the polynucleotides of the present application may be SEQ ID NO: 4 or a degenerated sequence thereof.

[0059] For example, the polynucleotide of the present application may include, but is not limited to, a base sequence having 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity to the sequence of SEQ ID NO: 2, in which the codon encoding glycine, the amino acid corresponding to position 479 of SEQ ID NO: 2, is replaced with a codon encoding an amino acid other than glycine, such as aspartic acid. It is also clear that the scope of the present application also includes variants having a polynucleotide sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, as long as the polynucleotide sequence has such homology or identity and encodes the amino acid sequence of the manganese-dependent transcriptional regulator mutant polypeptide of the present application.

[0060] As another example, the polynucleotide of the present application may have, comprise, or consist essentially of a nucleic acid sequence that is 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to SEQ ID NO: 4, but is not limited thereto. Alternatively, the sequence having the homology or identity may be one in which the codon corresponding to position 160 of SEQ ID NO: 3, which is encoded by SEQ ID NO: 4, is fixed to a codon encoding aspartic acid.

[0061] Furthermore, the polynucleotides of the present application may include, without limitation, probes prepared from known gene sequences, for example, sequences that can hybridize under stringent conditions to a complementary sequence to all or part of the polynucleotide sequence of the present application.

[0062] The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, pp. 9.50-9.51, 11.7-11.8). For example, conditions include conditions under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, but polynucleotides with lower homology or identity do not hybridize with each other; or conditions under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization, such as 60°C, 1X SSC, and 0.1% SDS, specifically 60°C, 0.1X SSC, and 0.1% SDS, more specifically 68°C, 0.1X SSC, and 0.1% SDS.

[0063] Hybridization requires that two nucleic acids have complementary sequences, even if the stringency of the hybridization allows for mismatches between bases. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can also include isolated nucleic acid fragments that are complementary to entire sequences, as well as substantially similar nucleic acid sequences.

[0064] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the above-mentioned hybridization conditions, including a hybridization step at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and may be appropriately adjusted by those skilled in the art depending on the purpose.

[0065] The appropriate stringency for hybridizing such polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables well known in the art (eg, J. Sambrook et al., supra).

[0066] As used herein, the term "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0067] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences can generally hybridize to all or part of the sequence under moderately or highly stringent conditions. Hybridization obviously includes hybridization to polynucleotides containing common codons in polynucleotides or codons that take codon degeneracy into account.

[0068] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example, as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later), the GCG program package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.][F.,][ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [Ed.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48:1073. For example, BLAST or ClustalW from the National Database Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0069] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that of Needleman et al. (1970), J Mol Biol. 48:443, as known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines a match as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a binary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, as used herein, the terms "homology" or "identity" indicate the relevance between sequences.

[0070] Another aspect of the present application provides a vector comprising the polynucleotide of the present application. The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a microorganism.

[0071] In this application, the term "vector" may also include a DNA construct containing a base sequence of a polynucleotide encoding a polypeptide of interest operably linked to a suitable expression control region (or expression control sequence) so as to enable the expression of the polypeptide of interest in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable microorganism, the vector can replicate or function independently of the host genome, or it can be integrated into the genome itself.

[0072] The vectors used in the present application are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0073] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a vector for chromosomal integration in a cell. The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of insertion into the chromosome may also be included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of insertion of the target nucleic acid molecule. Markers that confer selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0074] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a microorganism or microorganisms, thereby enabling the expression of the polypeptide encoded by the polynucleotide in the microorganism. A transformed polynucleotide may include any polynucleotide, regardless of whether it is located within the chromosome of the microorganism or extrachromosomally, as long as it is expressible in the microorganism. The polynucleotide may also include DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form that can be introduced and expressed in the microorganism. For example, the polynucleotide may be introduced into the microorganism in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the microorganism in its own form and operably linked to sequences necessary for its expression in the microorganism, but is not limited thereto.

[0075] In addition, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant of the present application.

[0076] Another aspect of the present application provides a microorganism comprising said mutant polypeptide, a polynucleotide encoding said mutant polypeptide, or a vector comprising said polynucleotide.

[0077] In one embodiment, the microorganism of the present application may be a microorganism capable of producing L-glutamic acid.

[0078] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, and may be a microorganism in which a specific mechanism has been weakened or enhanced by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and may also be a microorganism that has been genetically modified for the production of a desired polypeptide, protein, or product. In this application, the terms "microorganism" and "strain" may be used interchangeably without restriction and have the same meaning.

[0079] In this application, the term "microorganism capable of producing L-glutamic acid" refers to a prokaryotic or eukaryotic microbial strain capable of producing L-glutamic acid in vivo, and includes both microorganisms in which the ability to produce L-glutamic acid has been imparted to a parent strain that does not have the ability to produce L-glutamic acid, and microorganisms that have the ability to produce L-glutamic acid endogenously. The ability to produce L-glutamic acid can be imparted or enhanced by species improvement.

[0080] In one embodiment, the microorganism of the present application may be, but is not limited to, a microorganism that naturally has the ability to produce a manganese-dependent transcriptional regulator mutant polypeptide or L-glutamic acid; or a microorganism into which the mutant of the present application or a polynucleotide encoding it (or a vector containing the polynucleotide) has been introduced into a parent strain that does not have the ability to produce a manganese-dependent transcriptional regulator mutant polypeptide or L-glutamic acid, and / or which has been conferred the ability to produce L-glutamic acid.

[0081] In one embodiment, the microorganism of the present application includes, but is not limited to, a microorganism in which a chromosomal gene encoding a variant manganese-dependent transcriptional regulator polypeptide is mutated to contain the variant manganese-dependent transcriptional regulator polypeptide sequence of the present application and / or a microorganism in which a vector containing a polynucleotide encoding the variant manganese-dependent transcriptional regulator polypeptide of the present application is introduced to contain the variant manganese-dependent transcriptional regulator polypeptide of the present application.

[0082] In the present application, the term "non-transformed microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type or naturally occurring strain itself, or a strain before its traits are changed due to genetic mutations caused by natural or artificial factors. For example, the non-transformed microorganism refers to a strain into which the manganese-dependent transcriptional regulator mutant polypeptide described herein has not been introduced or before it has been introduced. The term "non-transformed microorganism" may be used interchangeably with "pre-transformed strain," "pre-transformed microorganism," "non-mutated strain," "non-transformed strain," "non-mutated microorganism," or "reference microorganism."

[0083] The term "L-glutamic acid (L-glutamate)" as used herein refers to an amino acid, classified as a non-essential amino acid, and generally produced through the fermentation of glutamic acid-producing microorganisms, but is not limited thereto. L-glutamic acid is known as the most common excitatory neurotransmitter in the central nervous system, and monosodium glutamate (MSG) has been developed and widely used as a seasoning due to its rich flavor.

[0084] The microorganism capable of producing L-glutamic acid of the present application may be, but is not limited to, a microorganism comprising one or more of the mutant of the present application, the polynucleotide of the present application, and the vector comprising the polynucleotide of the present application; a microorganism that has been modified to express the mutant of the present application or the polynucleotide of the present application; a microorganism (e.g., a recombinant strain) that expresses the mutant of the present application or the polynucleotide of the present application; or a microorganism (e.g., a recombinant strain) that has the activity of the mutant of the present application.

[0085] For example, the strain of the present application is a cell or microorganism that has been transformed with a vector containing a polynucleotide encoding the polynucleotide of the present application or a polynucleotide encoding the variant of the present application and expresses the variant of the present application. The strain of the present application can include all microorganisms capable of producing L-glutamic acid, including the variant of the present application. For example, the microorganism of the present application may be a recombinant strain having increased L-glutamic acid production ability, as a result of introducing a polynucleotide encoding the variant of the present application into a naturally occurring wild-type microorganism or a microorganism capable of producing L-glutamic acid, thereby expressing a mutant polypeptide of a manganese-dependent transcriptional regulator. The recombinant strain having increased L-glutamic acid production ability may be, but is not limited to, a naturally occurring wild-type microorganism or a microorganism not modified with a manganese-dependent transcriptional regulator (e.g., a microorganism expressing a wild-type manganese-dependent transcriptional regulator or a microorganism not expressing the variant of the present application). For example, the microorganism of the present application having increased L-glutamic acid production ability may be, but is not limited to, a microorganism having increased L-glutamic acid production ability compared to a microorganism containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same. For example, the untransformed microorganism, which is the subject strain for comparing the increase in L-glutamic acid production ability, may be, but is not limited to, the wild-type Corynebacterium glutamicum strain ATCC13869 or ATCC13032, or the glutamic acid-producing strain KFCC11074 (KR 10-0292299 B1).

[0086] The microorganisms of the present application may include all microorganisms that can express the mutant manganese-dependent transcriptional regulator polypeptide of the present application by various known methods other than the introduction of the nucleic acid or vector.

[0087] For example, the microorganism having increased L-glutamic acid production ability may 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 50% or more, about 55% or more, about 60% or more, about 61% or more, about 62% or more, about 63% or more, or about 64% or more (there is no particular upper limit, and it may be, for example, about 200% or less, about 150% or less, about 100% or less, about 90% or less, about 80% or less, about 70% or less, or about 65% or less), compared to the L-glutamic acid production ability of the parent strain or unmodified microorganism before mutation, but is not limited thereto as long as there is an increase in the + value compared to the parent strain or unmodified microorganism. In another example, the recombinant strain having increased L-glutamic acid producing ability may have an increased L-glutamic acid producing ability of about 1.01-fold or more, about 1.02-fold or more, about 1.03-fold or more, about 1.05-fold or more, about 1.1-fold or more, about 1.15-fold or more, about 1.20-fold or more, about 1.25-fold or more, about 1.30-fold or more, about 1.35-fold or more, about 1.40-fold or more, about 1.45-fold or more, about 1.50-fold or more, about 1.55-fold or more, about 1.60-fold or more, about 1.61-fold or more, about 1.62-fold or more, about 1.63-fold or more, or about 1.64-fold or more (there is no particular upper limit, and the increase may be, for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, about 2-fold or less, or about 1.7-fold or less) compared to the parent strain or untransformed microorganism before mutation, but is not limited thereto. The term "about" refers to a range that includes, but is not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​in a range that is equal to or similar to the numerical value following the term "about."

[0088] As a microorganism according to any one of the above-mentioned specific examples, the microorganism of the present application may be a microorganism belonging to the genus Corynebacteria, Escherichia, Erwinia, Serratia, Providencia, Pseudomonas, Leptospira, Salmonella, Brevibacteria, Hypomonas, Chromobacterium, or Norcardia, or a microorganism belonging to fungi or yeast, specifically, a microorganism of the genus Corynebacterium, but is not limited thereto.

[0089] As an example of the present application, the microorganism of the present application is Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganism of the present application may be a Corynebacterium microorganism, more specifically, Corynebacterium glutamicum, but is not limited thereto.

[0090] Meanwhile, the microorganisms capable of producing L-glutamic acid in the present application include all of the following: natural wild-type microorganisms themselves; microorganisms that have improved L-glutamic acid production ability by strengthening or weakening the activity of genes related to the L-glutamic acid production mechanism; and microorganisms that have improved L-glutamic acid production ability by introducing or strengthening the activity of exogenous genes.

[0091] While it has been known that microorganisms of the genus Corynebacterium can produce L-glutamic acid, their productivity is extremely low, and the genes and mechanisms involved in the production mechanism have not yet been fully elucidated. Therefore, the Corynebacterium microorganisms of the present application that are capable of producing L-glutamic acid may include natural wild-type microorganisms themselves, Corynebacterium microorganisms that have been improved in their ability to produce L-glutamic acid by strengthening or weakening the activity of genes involved in the L-glutamic acid production mechanism, or Corynebacterium microorganisms that have been improved in their ability to produce L-glutamic acid by introducing or strengthening the activity of exogenous genes.

[0092] Furthermore, the microorganism of the present application may have enhanced activity of the manganese-dependent transcriptional regulator protein compared to the parent strain.

[0093] In this application, the term "protein activity" of a manganese-dependent transcriptional regulator may be used interchangeably with terms such as polypeptide activity and protein activity.

[0094] In the present application, the term "enhancement" of manganese-dependent transcriptional regulator protein activity may be used interchangeably with terms such as activation, up-regulation, overexpression, and increase. Here, activation, enhancement, up-regulation, overexpression, and increase can all include exhibiting an activity that was not originally possessed, or exhibiting an activity that is improved compared to the endogenous activity or the activity before modification.

[0095] The term "endogenous activity" refers to the activity of a specific polypeptide that was originally possessed by a parent strain or non-transformed microorganism before the trait change, when the trait is changed due to genetic mutation caused by natural or artificial factors. This term may be used interchangeably with "activity before transformation." When the activity of a polypeptide is "enhanced," "up-regulated," "overexpressed," or "increased" compared to the endogenous activity, it means that the activity and / or concentration (expression level) of the specific polypeptide is improved compared to the activity and / or concentration (expression level) that was originally possessed by a parent strain or non-transformed microorganism before the trait change.

[0096] The enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. The enhancement of the activity of the polypeptide can be confirmed by an increase in the activity, expression level, or amount of a product secreted from the polypeptide.

[0097] The activity of the polypeptide can be enhanced by various methods well known in the art, and is not limited thereto, as long as the activity of the polypeptide of interest can be enhanced compared to that of the microorganism before transformation. Specifically, the enhancement may be achieved by using genetic engineering and / or protein engineering, which are routine methods in molecular biology and well known to those skilled in the art, but is not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2, 1-16; Sambrook et al., Molecular Cloning 2012, etc.).

[0098] Specifically, the enhancement of the polypeptide of the present application is 1) an increase in the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the expression regulatory region of a gene on a chromosome that encodes a polypeptide with a sequence with strong activity; 3) a modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the polypeptide activity is enhanced; 5) modifying a polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide's activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide's activity); 6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same; 7) codon optimization of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide and selectively deforming or chemically modifying exposed sites; or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0099] More specifically, The 1) increase in the intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a host cell a vector capable of replicating and functioning independently of the host, to which a polynucleotide encoding the polypeptide is operably linked. Alternatively, the increase may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into a chromosome in the host cell. The introduction into a chromosome can be achieved by, but is not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell. The vector is as described above.

[0100] The replacement of the expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide with a sequence with stronger activity may involve, for example, mutation of the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or replacement with a sequence with stronger activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. For example, the original promoter may be replaced with a strong promoter, but this is not limited thereto.

[0101] Examples of known strong promoters include, but are not limited to, the cj1 to cj7 promoters (U.S. Patent No. 7,662,943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (U.S. Patent No. 10,584,338 B2), the O2 promoter (U.S. Patent No. 10,273,491 B2), the tkt promoter, and the yccA promoter.

[0102] The modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, but is not limited to, substituting a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon.

[0103] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above may be, but is not limited to, a sequence mutation such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, or an amino acid sequence or polynucleotide sequence modified to have stronger activity or to have increased activity. Specifically, the modification can be achieved by, but is not limited to, inserting the polynucleotide into a chromosome via homologous recombination. The vector used in this case may further contain a selection marker for detecting the presence or absence of insertion into the chromosome. The selection marker is as described above.

[0104] The introduction of an exogenous polynucleotide that exhibits the activity of a polypeptide (6) may be the introduction of an exogenous polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide into a host cell. The exogenous polynucleotide is not limited in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be any known transformation method appropriately selected by those skilled in the art, and the introduced polynucleotide may be expressed in a host cell to produce a polypeptide, and its activity may be increased.

[0105] The codon optimization of the polynucleotide encoding the polypeptide (7) may be that of an endogenous polynucleotide that has been codon-optimized to increase transcription or translation in a host cell, or that of an exogenous polynucleotide that has been codon-optimized to optimize transcription or translation in a host cell.

[0106] 8) Analyzing the tertiary structure of a polypeptide and selecting and deforming or chemically modifying exposed sites may involve, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and deforming or chemically modifying exposed sites.

[0107] Such enhancement of polypeptide activity may be, but is not limited to, an increase in the activity or expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in a wild-type or untransformed microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0108] Modification of a portion or the entire polynucleotide in the microorganism of the present application may be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal insertion in the microorganism or genome editing using engineered nucleases (e.g., CRISPR-Cas9) and / or (b) treatment with light and / or chemicals, such as ultraviolet light and radiation. Methods for modifying a portion or the entire gene include DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene may be injected into the microorganism to cause homologous recombination, thereby deleting a portion or the entire gene. The injected nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

[0109] Another aspect of the present application provides a method for producing L-glutamic acid, comprising culturing the microorganism of the present application in a medium.

[0110] Specifically, the method for producing L-glutamic acid of the present application may include, but is not limited to, culturing a microorganism containing a mutant of the present application, a polynucleotide of the present application, or a vector of the present application in a medium.

[0111] In the present application, the term "culturing" means growing the microorganism of the present application under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using appropriate media and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be, but is not limited to, a batch, continuous, and / or fed-batch culture.

[0112] As used herein, the term "culture medium" refers to a mixture of nutrients, primarily those required for culturing the microorganisms of the present application, and provides nutrients, including water, and growth factors essential for survival and growth. Specifically, the culture medium and other culture conditions used to culture the microorganisms of the present application can be any medium commonly used for culturing microorganisms, without any particular limitations. The microorganisms of the present application can be cultured under aerobic conditions in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while adjusting the temperature, pH, and other parameters. For example, culture media for Corynebacterium strains can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0113] In the present application, examples of the carbon source include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; amino acids such as glutamic acid, methionine, and lysine; and glycerol and propanediol. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can be used. A variety of other suitable carbon sources can also be used without limitation. These carbon sources can be used alone or in combination, and are not limited thereto.

[0114] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; or an organic nitrogen source such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0115] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or the corresponding sodium-containing salts. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and other compounds, including amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, the present invention is not limited to these.

[0116] During the cultivation of the microorganism of the present application, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner. Furthermore, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, to maintain an aerobic state in the medium, oxygen or an oxygen-containing gas can be injected into the medium, and to maintain an anaerobic or microaerobic state, no gas can be injected or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limiting.

[0117] In the culture of the present application, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and the culture time can be continued until the desired amount of the target substance is produced, which can be approximately 10 to 160 hours, but is not limited thereto.

[0118] The L-glutamic acid produced by the culture of the present application is either secreted into the medium or remains intracellularly.

[0119] In one embodiment, the method for producing L-glutamic acid of the present application may further include a step of preparing the microorganism of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order), for example, before the culturing step.

[0120] In one embodiment, the method for producing L-glutamic acid of the present application may further include a step of recovering L-glutamic acid from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium. The recovering step may be further performed after the culturing step.

[0121] The recovery may involve collecting L-glutamic acid using a suitable method known in the art based on the microbial culture method of the present application, such as a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination of these methods can be used to recover L-glutamic acid from the medium or the microorganism using a suitable method known in the art.

[0122] The method for producing L-glutamic acid of the present application may further include a purification step. The purification can be performed using a suitable method known in the art. For example, when the method for producing L-glutamic acid of the present application includes both a recovery step and a purification step, the recovery step and the purification step can be performed continuously or discontinuously, in any order, simultaneously, or integrated into one step, but are not limited thereto.

[0123] In the methods of the present application, the mutant polypeptide, polynucleotide, L-glutamic acid, etc. are as described above in other aspects.

[0124] Another aspect of the present application provides a composition for producing L-glutamic acid, comprising: a mutant polypeptide of the present application; a polynucleotide encoding the mutant polypeptide; a vector comprising the polynucleotide; or a microorganism comprising the mutant polypeptide of the present application, a polynucleotide encoding the mutant polypeptide, or a vector comprising the polynucleotide; a culture of the microorganism; or a combination of two or more of them.

[0125] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing L-glutamic acid, and such excipients may be, for example, but are not limited to, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, or an isotonic agent.

[0126] In one embodiment, each component present in the compositions of the present application can be included in a microbiologically effective amount, or in an amount that can be suitably present in a production composition.

[0127] In the composition of the present application, the variant polypeptide, polynucleotide, L-glutamic acid, etc. are as described above in other aspects.

[0128] Another aspect of the present application provides use of a microorganism comprising a mutant polypeptide of the present application, a polynucleotide encoding the mutant polypeptide, or a vector comprising the polynucleotide in the production of L-glutamic acid.

[0129] The mutant polypeptide, polynucleotide, vector, microorganism, and L-glutamic acid of the present application are as described above in other aspects.

[0130] The present application will be described in more detail below through experimental examples. However, the following examples are merely preferred embodiments for illustrating the present application, and are not intended to limit the scope of the present application. Meanwhile, technical matters not described in this specification can be fully understood and easily performed by those of ordinary skill in the technical field of the present application or a similar technical field.

[0131] Example 1. Screening of mutants with increased glutamic acid production through artificial mutagenesis Example 1-1. Induction of artificial mutations through UV irradiation To select mutant strains with improved glutamic acid production, the target product of fermentation, wild-type Corynebacterium glutamicum (ATCC 13869) was first spread onto a nutrient medium containing agar and cultured at 30°C for 16 hours. Several hundred colonies thus obtained were then irradiated with UV light at room temperature to induce random mutations in the genome of the strain.

[0132] Example 1-2. Fermentation titer test of mutagenized strains and selection of strains Fermentation titer was tested using the mutant strains randomly mutated in Example 1-1.

[0133] Each colony was subcultured in nutrient medium and then cultured in fermentation medium for 5 hours. Then, 25% Tween 40 was added to each medium at a concentration of 0.4%, and each colony was cultured for an additional 32 hours.

[0134] <Nutrient medium> Glucose 1%, meat juice 0.5%, polypeptone 1%, sodium chloride 0.25%, yeast extract 0.5%, agar 2%, urea 0.2%, pH 7.2

[0135] <Fermentation medium> Glucose 6%, calcium carbonate 5%, ammonium sulfate 2.25%, monopotassium phosphate 0.1%, magnesium sulfate 0.04%, ferrous sulfate 10 mg / L, biotin 0.3 mg / L, thiamine hydrochloride 0.2 mg / L

[0136] Each colony was cultured under the above conditions, and mutant strains that produced L-glutamic acid equivalent to or greater than that of wild-type Corynebacterium glutamicum (ATCC 13869) were selected. The L-glutamic acid concentrations of the selected mutant strains were then measured using HPLC. The measured L-glutamic acid concentrations are shown in Table 1 below.

[0137] [Table 1]

[0138] Referring to Table 1 above, "ATCC13869-y1" and "ATCC13869-y11" were selected as mutant strains that exhibited increased glutamic acid production compared to the wild-type strain.

[0139] Example 2. Confirmation of mutations through gene sequencing To confirm the genetic mutations of the mutant strains, the genes of the ATCC13869-y1 and ATCC13869-y11 strains selected in Example 1-2 were compared with those of the wild-type strain.

[0140] As a result, it was confirmed that the ATCC13869-y1 and ATCC13869-y11 strains contain the same mutation (substitution of A at the 479th nucleotide of the polynucleotide sequence shown in SEQ ID NO: 2) at a specific position in the dtxR gene (SEQ ID NO: 2), which encodes a manganese-dependent transcriptional regulator.

[0141] Therefore, in Examples 3 and 4 below, it was determined whether the mutations affect the amount of glutamic acid produced by Corynebacterium microorganisms.

[0142] Example 3. Construction of mutant strains and confirmation of glutamic acid production Example 3-1. Construction of a strain with introduced mutations A mutant strain was constructed incorporating the mutation identified in Example 2. Specifically, to introduce the mutation (substitution of A at nucleotide 479 of the polynucleotide sequence shown in SEQ ID NO: 2) into wild-type Corynebacterium glutamicum (ATCC13869 and ATCC13032), a gene replacement vector was constructed to replace glycine at nucleotide 160 of the manganese-dependent transcriptional regulator shown in SEQ ID NO: 1 with aspartic acid. The gene fragment for constructing the vector was obtained by PCR using ATCC13869 genomic DNA as a template. Primers containing polynucleotides shown in SEQ ID NOs: 5 to 8 were constructed based on information on the Corynebacterium glutamicum (ATCC13869) gene and surrounding nucleotide sequence registered in the National Institutes of Health GenBank.

[0143] PCR was performed by denaturing at 95°C for 5 minutes, followed by 30 cycles of denaturing at 95°C for 20 seconds, annealing at 55°C for 20 seconds, and polymerizing at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. More specifically, a 500-bp polynucleotide was obtained by amplified using primers with SEQ ID NOs: 5 and 6, and another 500-bp polynucleotide was obtained by amplified using primers with SEQ ID NOs: 7 and 8. The two resulting gene fragments were ligated using infusion enzyme into pDZ vector (Korean Patent Registration No. 10-0924065 and International Patent Publication No. 2008-033001) digested with restriction enzymes BamHI and SalI to construct a gene replacement vector, designated "pDZ-dtxR(G160D)." The primer sequences used to construct the vector are listed in Table 2 below.

[0144] [Table 2]

[0145] The gene replacement vector was then transformed into a wild-type strain by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector had been integrated into the chromosome through recombination of the homologous sequences were selected on a medium containing 25 mg / L of kanamycin. Gene sequence analysis of the Corynebacterium glutamicum transformants after the secondary recombination confirmed that the targeted mutation had been introduced into the strains. The mutated strains were designated "ATCC13869::dtxR(G160D)" and "ATCC13032::dtxR(G160D)."

[0146] Example 3-2. Confirmation of glutamic acid production amount The mutant strains ATCC13869::dtxR(G160D) and ATCC13032::dtxR(G160D) prepared in Example 3-1 and their wild-type Corynebacterium glutamicum strains (ATCC13869 and ATCC13032) were cultured in the same manner as in Example 1-2.

[0147] After the cultivation was completed, the concentration of L-glutamic acid in each medium was measured. The measured L-glutamic acid concentrations are shown in Table 3 below.

[0148] [Table 3]

[0149] As shown in Table 3, the concentration of L-glutamic acid produced by the mutant Corynebacterium glutamicum ATCC13869::dtxR(G160D) strain was approximately 2.1 g / L (approximately 29%) higher than that produced by wild-type Corynebacterium glutamicum ATCC13869.

[0150] Furthermore, the concentration of L-glutamic acid produced by the mutant Corynebacterium glutamicum ATCC13032::dtxR(G160D) strain was approximately 0.9 g / L (approximately 27%) higher than that produced by wild-type Corynebacterium glutamicum ATCC13032.

[0151] That is, it was confirmed that the mutant of the present application increases the L-glutamic acid-producing ability of a microorganism.

[0152] Example 4. Confirmation of glutamic acid production amount of the mutant KFCC11074 strain Example 4-1. Construction of a strain with introduced mutations To confirm whether the mutation has the same effect on strains with increased glutamic acid production, other than the wild-type strain, the mutation was introduced into the KFCC11074 strain (Korean Patent Registered Publication No. 10-0292299), which is known as a glutamic acid-producing strain.

[0153] Specifically, to replace the glycine at position 160 of SEQ ID NO: 1 with aspartic acid, the pDZ-dtxR(G160D) vector prepared in Example 3-1 was transformed into the KFCC11074 strain by homologous recombination (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector had been integrated into the chromosome through homologous recombination were selected in a medium containing 25 mg / L of kanamycin. Gene sequence analysis of the Corynebacterium glutamicum transformant after the secondary recombination confirmed that the targeted mutation had been introduced into the strain. The mutated strain was designated "KFCC11074_dtxR(G160D)."

[0154] Example 4-2. Confirmation of glutamic acid production amount The unmutated Corynebacterium glutamicum KFCC11074 and the mutant KFCC11074_dtxR(G160D) strain introduced in Example 4-1 were cultured in the same manner as in Example 1-2.

[0155] After the cultivation was completed, the L-glutamic acid concentration in each medium was measured. The measured L-glutamic acid concentrations are shown in Table 4 below.

[0156] [Table 4]

[0157] As shown in Table 4, the concentration of L-glutamic acid produced by the mutant Corynebacterium glutamicum KFCC11074_dtxR(G160D) strain was approximately 3.5 g / L (approximately 64%) higher than that produced by the unmutated Corynebacterium glutamicum KFCC11074.

[0158] That is, it was confirmed that the mutations of the present application increase the L-glutamic acid productivity of microorganisms even in strains with increased glutamic acid productivity.

[0159] Taking all of the above results into consideration, the mutants of the present application can enhance the activity of manganese-dependent transcriptional regulators in wild-type or glutamic acid-producing strains through mutated gene activity, thereby increasing the productivity of glutamic acid, the target product of fermentation. Therefore, they can be useful in various industrial fields that require high-yield production of glutamic acid.

[0160] From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including within the meaning and scope of the claims below, and any modifications or variations derived from the equivalent concepts thereof, rather than the above detailed description.

Claims

1. A manganese-dependent transcriptional regulator mutant polypeptide in which the amino acid corresponding to position 160 of SEQ ID NO: 1 is substituted with another amino acid.

2. 2. The mutant polypeptide of claim 1, wherein the amino acid corresponding to position 160 of SEQ ID NO: 1 is substituted with aspartic acid.

3. The mutant polypeptide of claim 1 , wherein the mutant polypeptide consists of the amino acid sequence of SEQ ID NO:

3.

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

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

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

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

8. The microorganism according to claim 7, wherein the Corynebacterium microorganism is Corynebacterium glutamicum.

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

10. 10. The method for producing L-glutamic acid according to claim 9, further comprising the step of recovering L-glutamic acid from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium.

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

12. 4. Use of a microorganism comprising the mutant polypeptide according to any one of claims 1 to 3, a polynucleotide encoding said mutant polypeptide, or a vector comprising said polynucleotide in the production of L-glutamic acid.

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