Novel tellurium-resistant membrane protein transporter mutant and method for producing L-glutamic acid using the same

Abstract

The present application relates to a novel tellurium-resistant membrane protein transporter 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 tellurium-resistant membrane protein transporter mutant polypeptide; a polynucleotide encoding the mutant polypeptide; a microorganism comprising the mutant polypeptide, the polynucleotide encoding the mutant polypeptide or a vector comprising the polynucleotide; a method for producing L-glutamic acid comprising culturing the microorganism in a medium; and use of the microorganism for the production of L-glutamic acid.

Background Art

[0002] Glutamic acid is a typical amino acid produced by fermentation and has a unique and distinctive taste. Therefore, it is one of the important amino acids widely used in the food field, as well as in the pharmaceutical field and other animal feed fields.

[0003] In the conventional method for producing glutamic acid, it is mainly produced through fermentation using Coryneform bacteria including Brevibacterium, Corynebacterium genus and its mutant strains (Amino Acid Fermentation, Gakkai Shuppan Center: 195 - 215, 1986). In addition, methods using microorganisms such as Escherichia coli, Bacillus, Streptomyces, Penicillum genus, Klebsiella, Erwinia, Pantoea genus, etc. are also known (U.S. Patent Publication No. 3220929, U.S. Patent Registration No. 6682912).

[0004] Furthermore, various studies are being conducted to efficiently produce amino acids, such as efforts to develop highly efficient amino acid-producing microorganisms and fermentation process technologies. Specifically, target-specific approaches have been developed to increase the expression of genes encoding enzymes involved in amino acid biosynthesis in Corynebacterium strains, or to remove genes unnecessary for amino acid biosynthesis (Korean Patent Publication No. 10-0924065, Korean Patent Publication No. 10-1208480). In addition to these methods, methods for removing genes not involved in amino acid production or genes whose specific function in amino acid production is unknown are also being utilized. However, there is still a growing need for research on methods that can produce L-glutamic acid efficiently and in high yield. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] U.S. Patent Publication No. 3220929 [Patent Document 2] U.S. Patent Publication No. 6682912 [Patent Document 3] Korean Registered Patent Publication No. 10-0924065 [Patent Document 4] Korean Registered Patent Publication No. 10-1208480 [Patent Document 5] Korean Registered Patent Publication No. 10-0292299 [Patent Document 6] U.S. Registered Patent US 7662943 B2 [Patent Document 7] U.S. Registered Patent US 10584338 B2 [Patent Document 8] U.S. Registered Patent US 10273491 B2 [Patent Document 9] International Publication Patent No. 2008-033001 [Non-patent literature]

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

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Summary of the Invention

Problems to be Solved by the Invention

[0007] The problem of the present application is to culture a microorganism containing a tellurium-resistant membrane protein transporter mutant polypeptide, and to provide a microorganism capable of producing L-glutamic acid in a higher yield compared to a microorganism having an existing non-modified polypeptide, and an L-glutamic acid production method using the same.

Means for Solving the Problems

[0008] One aspect of the present application provides a tellurium-resistant membrane protein transporter mutant polypeptide in which the amino acid corresponding to the 231st position of SEQ ID NO: 1 is substituted with another amino acid.

[0009] In one specific example, the mutant polypeptide may be one in which the amino acid corresponding to the 231st position of SEQ ID NO: 1 is substituted with serine.

[0010] In another specific example, the mutant 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 this application provides a microorganism comprising the mutant polypeptide, a polynucleotide encoding the mutant polypeptide, or a vector containing the polynucleotide.

[0013] In one specific example, the microorganism may have increased L-glutamic acid production capacity compared to a microorganism containing the polypeptide of Sequence ID No. 1 or the polynucleotide encoding it.

[0014] As a microorganism based on any one of the specific examples mentioned above, the microorganism may be a microorganism of the genus Corynebacterium.

[0015] As a microorganism representing any one of the specific examples mentioned above, the Corynebacterium genus microorganism may also be Corynebacterium glutamicum.

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

[0017] In one specific example, the method may further include the step of recovering L-glutamic acid from the cultured microorganism, the culture of the microorganism, the fermented product of the microorganism, or the culture medium.

[0018] Another aspect of this application provides a composition for L-glutamic acid production comprising the mutant polypeptide; a polynucleotide encoding the mutant polypeptide; a vector containing the polynucleotide; or a microorganism containing the mutant polypeptide, the polynucleotide encoding the mutant polypeptide, or the vector containing the polynucleotide; a culture of the microorganism; or a combination of two or more of these.

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

[0020] When culturing microorganisms containing the tellurium-resistant membrane protein transporter mutant polypeptide of this application, it is possible to produce L-glutamic acid in a higher yield compared to microorganisms with existing non-deformed polypeptides. [Modes for carrying out the invention]

[0021] This can be explained in more detail as follows: On the other hand, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the categories of this application are not limited by the specific descriptions described below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated as a whole into this specification by reference to more clearly explain the level of the technical field to which this application belongs and the content of this application.

[0022] One aspect of this application provides a tellurium-resistant membrane protein transporter mutant polypeptide in which the amino acid corresponding to the 231st position of SEQ ID NO: 1 is substituted with another amino acid.

[0023] In this application, the term "tellurium-resistant membrane protein transporter mutant polypeptide" means a tellurium-resistant membrane protein transporter mutant polypeptide that includes one or more amino acid substitutions in the amino acid sequence of the tellurium-resistant membrane protein transporter polypeptide; or a mutant of the tellurium-resistant membrane protein transporter polypeptide that includes one or more amino acid substitutions in the parent sequence, which is the amino acid sequence of the tellurium-resistant membrane protein transporter polypeptide.

[0024] In this application, the term "Tellurium resistance membrane protein transporter (terC)" refers to a global transporter that has activity involved in the efflux of tellurium ions, but is not limited to this, and may also refer to a tellurium resistance membrane protein transporter (terC) encoded by the terC gene. Tellurium resistance membrane protein transporters are known to be associated with tellurium resistance, phage suppression, colisin resistance, or pathogenicity, but are not limited to these.

[0025] The gene encoding the tellurium-resistant membrane protein transporter may be derived from a microorganism of the genus Corynebacterium, and specifically, it may be terC derived from Corynebacterium glutamicum, but is not limited thereto.

[0026] Specifically, the tellurium-resistant membrane protein transporter protein may include, for example, the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60% or more homology or identity thereto, but is not limited thereto as long as it has tellurium-resistant membrane protein transporter protein activity. Specifically, the amino acid sequence may include SEQ ID NO: 1 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with SEQ ID NO: 1. The sequence of SEQ ID NO: 1 can be obtained from known databases such as NCBI's GenBank or KEGG (Kyoto Encyclopedia of Genes and Genomes). For example, it may be derived from the genus Corynebacterium or Corynebacterium glutamicum, and more specifically, it may be a polypeptide / protein containing the amino acid sequence described in SEQ ID NO: 1, but is not limited thereto. Furthermore, it is obvious that accessory proteins having amino acid sequences in which some sequences are deleted, modified, substituted, or added are also included within the scope of this application, as long as they have such homology or identity and exhibit the efficacy corresponding to the aforementioned protein.

[0027] Furthermore, the tellurium-resistant membrane protein transporter protein having the amino acid sequence of SEQ ID NO: 1 may have, contain, or consist of 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 sequence of SEQ ID NO: 2, or may be encoded by a polynucleotide consisting of 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 sequence of SEQ ID NO: 2, or is not limited to this.

[0028] In this application, the term "variant" refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, resulting in a sequence that differs from the amino acid sequence of the original polypeptide, but in which the functions or properties are maintained. Such variants may 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 capabilities of the variant may be increased, unchanged, or decreased compared to the original polypeptide. Some variants may include those in which one or more parts, such as the N-terminal leader sequence or the transmembrane domain, are removed. Other variants may include those in which parts of the N- and / or C-terminus of a mature protein are removed. The aforementioned term "mutant" may be used interchangeably with terms such as variant, modified, mutant polypeptide, mutated protein, mutation, and divergent (in English, these may include modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited to these terms as long as they are used in the sense of a mutated form.

[0029] Furthermore, the variants may include the deletion or addition of amino acids that have minimal impact on the polypeptide's properties and secondary structure. For example, the polypeptide can be conjugated with a protein N-terminal signal (or leader) sequence involved in protein transfer co-translationally or post-translationally. The polypeptide can also be conjugated with other sequences or linkers to enable the polypeptide to be identified, purified, or synthesized.

[0030] The tellurium-resistant membrane protein transporter mutant polypeptide of this application may be, but is not limited to, a tellurium-resistant membrane protein transporter mutant polypeptide in which the amino acid corresponding to position 231 of SEQ ID NO: 1 is substituted with another amino acid.

[0031] As one example, the tellurium-resistant membrane protein transporter mutant polypeptide of this application may have sequence homology of 60% or more and less than 100% with the amino acid sequence of SEQ ID NO: 1, specifically, 80% or more and less than 100%.

[0032] Specifically, the variants of this application include amino acid sequences 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 described in Sequence ID No. 1, wherein the amino acid at position 231 from the N-terminus of Sequence ID No. 1 is substituted with another amino acid. Furthermore, it is obvious that variants having amino acid sequences in which some sequences are deleted, modified, substituted, conservedly substituted, or added are also included within the scope of this application, as long as they have such homology or identity and exhibit the efficacy corresponding to the variants of this application.

[0033] The aforementioned "other amino acids" are not limited to any amino acid different from the amino acid before substitution. On the other hand, when the expression "a specific amino acid is substituted" is used in this application, it is self-evident that the substitution is made with an amino acid different from the amino acid before substitution, even without specifically stating that it is substituted with another amino acid.

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

[0035] Examples of this classification 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, aspartic acid) and uncharged amino acids (also called neutral amino acids) (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). As yet another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. As yet another example, valine, leucine, and isoleucine can be classified as branched amino acids. As another example, the 20 amino acids can be classified according to their size and divided into five groups based on their 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, tyrosine. However, this classification is not necessarily limited to these groups.

[0036] For example, when stating that "the amino acid corresponding to position 231 in sequence number 1 has been substituted with another amino acid," it may mean, but is not limited to, that the substitution is with serine, valine, glycine, isoleucine, glutamate, phenylalanine, arginine, aspartate, cysteine, asparagine, glutamine, histidine, alanine, tyrosine, lysine, tryptophan, methionine, threonine, or leucine, excluding proline.

[0037] Even if this application states "a protein having an amino acid sequence described by a specific sequence number," it is obvious that proteins having amino acid sequences in which some sequences are deleted, modified, substituted, conservedly substituted, or added are also used in this application, as long as they have the same or corresponding activity as the protein consisting of the amino acid sequence of said sequence number. For example, if they have the same or corresponding activity as the mutant protein, this does not exclude the 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, and it is obvious that even if such sequences are added or mutated, they fall within the scope of this application.

[0038] The "position N" in this application may include the position N and the position of an amino acid corresponding to (correspoding with) the position N. Specifically, it may include the position of an amino acid corresponding to any amino acid residue in a mature polypeptide disclosed in a particular amino acid sequence. The particular amino acid sequence may be the amino acid sequence of SEQ ID NO: 1.

[0039] In this application, the term "corresponding to" refers to an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar, identical, or homologous to a residue listed in the polypeptide. Identifying the amino acid at the corresponding position may also mean determining a specific amino acid in a sequence that references a particular sequence. As used in this application, "corresponding region" generally refers to a similar or corresponding position in a related protein or reference protein.

[0040] For example, any amino acid sequence can be aligned with sequence number 1, and based on this, each amino acid residue in the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in sequence number 1. For example, a sequence alignment algorithm such as the one described in this application can be used to verify the position of amino acids, or the position where deformations such as substitutions, insertions, or deletions occur, by comparing them with a query sequence (also called a "reference sequence").

[0041] For such sorting, one can use, 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), but is not limited to these. Any sequence sorting program or pairwise sequence comparison algorithm known in this field can be used appropriately.

[0042] In one specific example, the mutant polypeptide may be in which the amino acid corresponding to position 231 of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of serine, valine, alanine, glycine, isoleucine, arginine, leucine, methionine, threonine, asparagine, glutamine, tryptophan, phenylalanine, histidine, cysteine, tyrosine, lysine, aspartate, and glutamic acid, but is not limited thereto.

[0043] As one example of any of the aforementioned embodiments, the mutant polypeptide provided in this application may be substituted with an amino acid selected from serine, threonine, cysteine, tyrosine, asparagine, and glutamine, in which the amino acid corresponding to the 231st position from the N-terminus of SEQ ID NO: 1 is a polar amino acid having a polar or hydrophilic side chain.

[0044] As one example of any of the aforementioned embodiments, the mutant polypeptide provided in this application may be one in which the amino acid corresponding to the 231st position from the N-terminus of SEQ ID NO: 1 is substituted with an amino acid selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, which are uncharged amino acids (also called neutral amino acids).

[0045] As one example of any of the aforementioned embodiments, the mutant polypeptide provided in this application may be one in which the amino acid corresponding to the 231st position from the N-terminus of SEQ ID NO: 1 is replaced with an amino acid selected from glycine, alanine, and serine.

[0046] As one example of any of the aforementioned embodiments, the tellurium-resistant membrane protein transporter mutant polypeptide of this application may be, but is not limited to, a polypeptide in which the amino acid corresponding to position 231 of SEQ ID NO: 1 is substituted with serine.

[0047] For example, the mutant polypeptides of this application may include amino acid sequences in which serine, the amino acid corresponding to position 231 in the amino acid sequence described in Sequence ID No. 1, is fixed, and which have 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 Sequence ID No. 1. Furthermore, it is obvious that mutant polypeptides having amino acid sequences in which some sequences are deleted, modified, substituted, conservedly substituted, or added are also included within the scope of this application, as long as they have such homology or identity and exhibit the efficacy corresponding to the mutant polypeptides of this application.

[0048] On the other hand, a person skilled in the art can identify the amino acid corresponding to the 231st position of the amino acid sequence of Sequence ID No. 1 of this application in any amino acid sequence through sequence alignment known in the industry, and it is self-evident that when "amino acids at a specific position in a particular Sequence ID No. 1" is described in this application, it also includes "amino acids at the corresponding positions" in any amino acid sequence, even without further specification.

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

[0050] Specifically, the variant polypeptide of this application may have, contain, consist of, or be essentially composed of, 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: 3.

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

[0052] In this application, the term "conservative substitution" means the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartate; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Furthermore, amino acids can be classified into those with electrically charged side chains and those with uncharged side chains. Amino acids with electrically charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino acids with uncharged side chains can be further classified into nonpolar amino acids and 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. Typically, conservative substitutions have little to no effect on the activity of the resulting polypeptide. Typically, conservative substitutions have little to no effect on the activity of a protein or polypeptide.

[0053] Another aspect of this application provides a polynucleotide encoding the mutant polypeptide.

[0054] In this application, the term "polynucleotide" means a polymer of nucleotides in which nucleotide units (monomers) are covalently linked together in a long chain, and is a DNA or RNA chain of a certain length or longer. More specifically, it means a polynucleotide fragment that encodes the aforementioned variant.

[0055] The polynucleotide encoding the tellurium-resistant membrane protein transporter variant polypeptide of this application may include, without limitation, any polynucleotide sequence encoding the tellurium-resistant membrane protein transporter variant polypeptide of this application. For example, the polynucleotide encoding the tellurium-resistant membrane protein transporter variant polypeptide of this application may be, but is not limited to, a polynucleotide sequence encoding the amino acid sequence of the tellurium-resistant membrane protein transporter variant polypeptide of this application.

[0056] For example, it may include a nucleic acid sequence encoding the amino acid sequence described in Sequence ID No. 3. As an example of this application, the polynucleotide of this application may have or include Sequence ID No. 4. Alternatively, the polynucleotide of this application may consist of or be essentially composed of Sequence ID No. 4.

[0057] The polynucleotides of this application may be subjected to various modifications to the coding region, taking into consideration codon degeneracy or preferred codons in organisms that intend to express the variants of this application, as long as these modifications do not alter the amino acid sequence of the variants. Therefore, it is obvious that polynucleotides that are translated by codon degeneracy into polypeptides consisting of the amino acid sequence of the variants of this application or polypeptides homologous or identical thereto are also included. For example, the polynucleotides of this application may be Sequence ID No. 4 or its degenerated sequence.

[0058] For example, the polynucleotides of this application may include, but are 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 with the sequence of SEQ ID NO: 2, in which the codon encoding proline, the amino acid corresponding to position 691 of SEQ ID NO: 2, is substituted with an amino acid other than proline, such as a codon encoding serine. It is also obvious that any polynucleotide sequence having such homology or identity and encoding the amino acid sequence of the tellurium-resistant membrane protein transporter mutant polypeptide of this application, including any variants having deletions, alterations, substitutions, conservative substitutions, or additions to some sequences, is also within the scope of this application.

[0059] As another example, the polynucleotides of this application may have, contain, or consist of nucleic acid sequences 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: 4, or may be required to consist of, but are not limited to, nucleic acid sequences 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: 4. Alternatively, the homologous or identical sequence may be fixed with a codon that codes for serine, corresponding to the 231st position of SEQ ID NO: 3 coded by SEQ ID NO: 4.

[0060] Furthermore, the polynucleotides of this application may include, without limitation, any probes produced from known gene sequences, such as sequences that can be hybridized under stringent conditions with complementary sequences to all or part of the polynucleotide sequences of this application.

[0061] The aforementioned "stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, we can list conditions in which polynucleotides with high homology or identity hybridize with each other, with homology or identity levels 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, but do not hybridize with polynucleotides with lower homology or identity levels. Alternatively, we can list conditions in which the polynucleotides are washed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of a normal Southern hybridization: 60°C, 1×SSC, 0.1% SDS, more specifically 60°C, 0.1×SSC, 0.1% SDS, or more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0062] Hybridization requires that two nucleic acids have complementary sequences, even if hybridization stringency allows for mismatches between bases. The term “complementary” is used to describe the relationships between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of this application may also include isolated nucleic acid fragments that are complementary to the overall sequence as well as substantially similar nucleic acid sequences.

[0063] Specifically, polynucleotides homologous or identical to the polynucleotides of this application can be detected using hybridization conditions that include a hybridization step at a Tm value of 55°C, and under the conditions described above. The Tm value may also be 60°C, 63°C, or 65°C, but is not limited thereto and may be appropriately adjusted by those skilled in the art depending on the purpose.

[0064] The appropriate stringency for hybridizing the aforementioned polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (e.g., J. Sambrook et al., ibid.).

[0065] In this application, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or base sequences, and may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0066] The sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequencing algorithms, and a default gap penalty established by the program used may be applied. Substantially homologous or identical sequences can generally be hybridized with whole or partial sequences under moderate to high stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing codons, or codons considering codon degeneracy in general, in polynucleotides.

[0067] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms such as the "FASTA" program with default parameters, for example, as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.][F.,][ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San (Including Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Databases.

[0068] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443, as is publicly known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In summary, the GAP program can be defined as the total number of symbols in the shorter of two sequences divided by the number of similarly sequenced symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program may include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14:6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Thus, the terms “homology” or “identity” as used in this application refer to the relevance between sequences.

[0069] Another aspect of this application provides a vector comprising the polynucleotide of this application. The vector may, but is not limited to, be an expression vector for expressing the polynucleotide in a microorganism.

[0070] In this application, the term “vector” may include a DNA product comprising a polynucleotide sequence encoding the target polypeptide, operably linked to a suitable regulatory region (or regulatory sequence) so as to enable the expression of the target polypeptide in a suitable host. The regulatory region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences regulating the termination of transcription and decoding. The vector, after being transformed into a suitable microorganism, can replicate or function independently of the host genome and integrate into the genome itself.

[0071] The vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0072] As an example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a chromosome insertion vector within a cell. The insertion of the polynucleotide into the chromosome may be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker may further be included to confirm the presence or absence of the chromosome insertion. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the target nucleic acid molecule insertion, and markers conferring selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of surface polypeptides can be used. Transformed cells can be selected because, in an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes.

[0073] In this application, the term "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a microorganism or into a microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the microorganism. The transformed polynucleotide may include all of them, regardless of whether they are inserted into or outside the chromosome of the microorganism, as long as they can be expressed in the microorganism. The polynucleotide also includes DNA and / or RNA encoding the polypeptide of interest. The polynucleotide may be introduced into a microorganism in any form that can be introduced and expressed in the microorganism. For example, the polynucleotide may be introduced into a microorganism in the form of an expression cassette, which is a gene structure containing all the elements necessary for its expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into a microorganism in its own form and operably linked to the sequence necessary for expression in the microorganism, but is not limited thereto.

[0074] Furthermore, the term "operably linked" in the foregoing means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target variant of this application.

[0075] Another aspect of this application provides a microorganism comprising the mutant polypeptide, a polynucleotide encoding the mutant polypeptide, or a vector containing the polynucleotide.

[0076] In one specific example, the microorganism of this application may be a microorganism capable of producing L-glutamic acid.

[0077] In this application, the terms "microorganism (or strain)" include all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and are microorganisms in which a particular mechanism has been weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of the activity of endogenous genes, and may include microorganisms that undergo genetic modification for the production of a target polypeptide, protein, or product. In this application, "microorganism" and "strain" may be used interchangeably without limitation as they have the same meaning.

[0078] In this application, the term "microorganism producing L-glutamic acid" refers to a prokaryotic or eukaryotic microbial strain capable of producing L-glutamic acid within its body, and may include all microorganisms in which the ability to produce L-glutamic acid has been conferred to a parent strain that lacks the ability to produce L-glutamic acid, or microorganisms that inherently possess the ability to produce L-glutamic acid. The ability to produce L-glutamic acid can be conferred or enhanced through selective breeding.

[0079] As one example, the microorganisms of this application may be, but are not limited to, microorganisms that naturally possess the ability to produce tellurium-resistant membrane protein transporter mutant polypeptides or L-glutamate; or microorganisms in which the mutant of this application or a polynucleotide encoding it (or a vector containing said polynucleotide) is introduced into a parent strain that does not possess the ability to produce tellurium-resistant membrane protein transporter mutant polypeptides or L-glutamate, and / or into which the ability to produce L-glutamate is conferred.

[0080] As one example, the microorganisms of this application include, but are not limited to, all microorganisms in which a gene on a chromosome encoding the tellurium-resistant membrane protein transporter mutant polypeptide is mutated and contains the tellurium-resistant membrane protein transporter mutant polypeptide sequence of this application, and / or microorganisms containing the tellurium-resistant membrane protein transporter mutant polypeptide of this application by introducing a vector containing a polynucleotide encoding the tellurium-resistant membrane protein transporter mutant polypeptide of this application.

[0081] In this application, the term "non-myxoid microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but rather means the wild-type or native strain itself, or a strain before its characteristics are altered by genetic mutations due to natural or artificial factors. For example, the non-myxoid microorganism means a strain in which the tellurium-resistant membrane protein transporter mutant polypeptide described herein has not been introduced, or before it has been introduced. The term "non-myxoid microorganism" may be used interchangeably with "pre-deformation strain," "pre-deformation microorganism," "non-mutant strain," "non-myxoid strain," "non-mutant microorganism," or "reference microorganism."

[0082] In this application, the term "L-glutamic acid (L-glutamate)" refers to a type of amino acid, classified as a non-essential amino acid, and is generally produced through fermentation by microorganisms that produce glutamic acid, but is not limited to this. It is known as the most common excitatory neurotransmitter in the central nervous system, and its monosodium glutamate (MSG) has been developed and widely used as a seasoning due to its rich flavor.

[0083] The microorganism having L-glutamic acid production ability of this application may be, but not limited to, a microorganism comprising one or more of the mutants of this application, the polynucleotides of this application, and vectors comprising the polynucleotides of this application; a microorganism modified to express the mutants or polynucleotides of this application; a microorganism expressing the mutants or polynucleotides of this application (e.g., recombinant strains); or a microorganism having mutant activity of this application (e.g., recombinant strains).

[0084] For example, the strains of this application are cells or microorganisms transformed with a vector containing the polynucleotide or the variant of the present application, expressing the variant of the present application, and the strains of this application may include all microorganisms capable of producing L-glutamic acid, including the variant of the present application. For example, the microorganisms of this application may be recombinant strains in which a tellurium-resistant membrane protein transporter mutant polypeptide is expressed and L-glutamic acid production capacity is increased by introducing the polynucleotide encoding the variant of the present application into a naturally occurring wild-type microorganism or a microorganism capable of producing L-glutamic acid. The recombinant strains with increased L-glutamic acid production capacity may be, but are not limited to, naturally occurring wild-type microorganisms or microorganisms that do not undergo tellurium-resistant membrane protein transporter mutation (e.g., microorganisms expressing the wild-type tellurium-resistant membrane protein transporter or microorganisms that do not express the variant of the present application). For example, the microorganisms with increased L-glutamic acid production capacity in this application may be, but are not limited to, microorganisms with increased L-glutamic acid production capacity compared to microorganisms containing the polypeptide of Sequence ID No. 1 or the polynucleotide encoding it. For example, the non-mutated microorganisms used as the comparison strains for the presence or absence of increased L-glutamic acid production capacity may be, but are not limited to, wild-type Corynebacterium glutamicum strains ATCC13869 or ATCC13032; or glutamic acid-producing strain KFCC11074 (KR 10-0292299 B1).

[0085] The microorganisms of this application may include all microorganisms that can express the tellurium-resistant membrane protein transporter mutant polypeptide of this application by various known methods other than the introduction of the nucleic acid or vector described above.

[0086] For example, the microorganism with increased L-glutamic acid production capacity may have an increase of approximately 1% or more compared to the L-glutamic acid production capacity of the parent strain or non-mutant microorganism before mutation. Specifically, this could be approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 5% or more, approximately 10% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, approximately 45% or more, approximately 46% or more, approximately 47% or more, or approximately 48% or more (there are no special restrictions on the upper limit; for example, it may be approximately 200% or less, approximately 150% or less, approximately 100% or less, approximately 90% or less, approximately 80% or less, approximately 70% or less, approximately 65% ​​or less, approximately 60% or less, approximately 55% or less, or approximately 50% or less). However, it is not limited to this as long as it has a positive increase compared to the production capacity of the parent strain or non-mutant microorganism before mutation. In other examples, the recombinant strain with increased L-glutamic acid production capacity may be, but is not limited to, a strain with increased L-glutamic acid production capacity of approximately 1.01 times or more, approximately 1.02 times or more, approximately 1.03 times or more, approximately 1.05 times or more, approximately 1.15 times or more, approximately 1.20 times or more, approximately 1.25 times or more, approximately 1.30 times or more, approximately 1.35 times or more, approximately 1.40 times or more, approximately 1.45 times or more, approximately 1.46 times or more, approximately 1.47 times or more, or approximately 1.48 times or more (there is no special limit on the upper limit; for example, it may be approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, approximately 2 times or less, or approximately 1.5 times or less). The term "about" includes all ranges such as ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, and includes, but is not limited to, all numerical values ​​within a range equivalent to or similar to the numerical value following the term "about".

[0087] As a microorganism representing any one of the specific examples mentioned above, the microorganism of this application may be, but is not limited to, a microorganism belonging to the genera Corynebacterium 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. Specifically, it may be, but is not limited to, a microorganism of the genus Corynebacterium.

[0088] As an example of this application, the microorganisms covered in this application are Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, and Corynebacterium ammoniagenes. The microorganisms may be Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganisms of this application may be, but are not limited to, microorganisms of the genus Corynebacterium, more specifically Corynebacterium glutamicum.

[0089] On the other hand, the microorganisms having L-glutamic acid production ability of this application include all naturally occurring wild-type microorganisms themselves, microorganisms that have acquired improved L-glutamic acid production ability by enhancing or weakening the activity of polypeptides related to the L-glutamic acid production mechanism, and microorganisms that have acquired improved L-glutamic acid production ability by introducing or enhancing the activity of external polypeptides.

[0090] On the other hand, while it was already known that microorganisms of the genus Corynebacterium can produce L-glutamic acid, their production capacity is remarkably low, and the genes and mechanisms involved in the production process have not yet been fully elucidated. Therefore, the Corynebacterium microorganisms possessing L-glutamic acid production ability described in this application can include all of the following: the natural wild-type microorganisms themselves, Corynebacterium microorganisms that have acquired improved L-glutamic acid production ability by enhancing or weakening the activity of polypeptides related to the L-glutamic acid production mechanism, or Corynebacterium microorganisms that have acquired improved L-glutamic acid production ability by introducing or enhancing the activity of external polypeptides.

[0091] In this application, the term "increase" of polypeptide activity means that the activity of the polypeptide is enhanced compared to its endogenous activity. This enhancement may be used interchangeably with terms such as activation, up-regulation, overexpression, and enhancement.

[0092] The aforementioned enhancement may include exhibiting activity that was not originally present, or exhibiting improved activity compared to the intrinsic activity or the activity before the transformation.

[0093] For example, "exhibiting activity that was not originally present" may, but is not limited to, "introduction of a protein." The introduction of a protein means that a gene that was not originally present in the microorganism is expressed within the microorganism, thereby exhibiting activity of a specific protein, or exhibiting enhanced or improved activity compared to the intrinsic activity or pre-modification activity of the protein. For example, this may involve introducing a polynucleotide encoding a specific protein into a chromosome within the microorganism, or introducing a vector containing a polynucleotide encoding a specific protein into the microorganism, and exhibiting its activity.

[0094] The aforementioned "intrinsic activity" refers to the activity of a specific polypeptide that was originally present in the parent strain or non-mutant microorganism before the trait change due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before deformation."

[0095] Enhanced polypeptide activity compared to endogenous activity means that the activity and / or concentration (expression level) of a specific polypeptide has improved compared to what was originally present in the parental strain or non-mutant microorganism before the trait change.

[0096] For example, the enhancement may be, but is not limited to, an enhancement of the activity or concentration of the corresponding protein, where the activity or concentration was previously absent, or an enhancement of approximately 1%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, or 500%, up to approximately 1000% or 2000% or more, relative to the activity or concentration of the wild-type protein or the initial microbial strain.

[0097] The enhancement of the polypeptide's activity can be achieved by introducing an exogenous polypeptide or by enhancing the activity of an endogenous polypeptide. Whether or not the polypeptide's activity has been enhanced can be confirmed by the enhancement of its activity level, expression level, or the amount of product excreted from the polypeptide.

[0098] The enhancement of the activity of the polypeptide can be achieved by applying various methods well known in the field, and is not limited as long as it can enhance the activity of the target polypeptide compared to the microorganism before deformation. Specifically, this may involve, but is not limited to, the use of gene engineering and / or protein engineering, which are routine methods in molecular biology and are well known to ordinary technicians in the field (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol.2.1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0099] Specifically, the enhancement of the activity of the polypeptide in this application is 1) Enhancement of intracellular copy number of polynucleotides encoding polypeptides; 2) Modification of gene expression regulatory regions on chromosomes that encode polypeptides (e.g., mutation within the regulatory region, replacement with a more active sequence, or insertion of a more active sequence); 3) Modifications of the nucleotide sequence encoding the start codon or 5'-UTR region of a polypeptide-encoding gene transcript; 4) Modification of the amino acid sequence of the polypeptide so as to enhance polypeptide activity; 5) Modification of the polynucleotide sequence encoding the polypeptide so as to enhance polypeptide activity (for example, modification of the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified to enhance polypeptide activity); 6) Introduction of a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding such activity; 7) Codon optimization of polynucleotides encoding polypeptides; 8) Analyze the tertiary structure of the polypeptide, select exposed sites to deform or chemically modify; or 9) A combination of two or more selected from items 1) to 8) above is also acceptable, but is not particularly limited thereto.

[0100] for example, The enhancement of the intracellular copy number of the polynucleotide encoding the polypeptide described in 1) above may be achieved by introducing a vector into the host cell that is operablely linked to the polynucleotide encoding the polypeptide and capable of replicating and functioning independently of the host. Alternatively, one or more copies of the polynucleotide encoding the polypeptide may be introduced into the chromosomes within the host cell. The introduction into the chromosomes can be achieved by introducing a vector into the host cell that can insert the polynucleotide into the chromosomes within the host cell, but is not limited to these methods. The vector is as described above.

[0101] The replacement of a gene expression regulatory region (or regulatory sequence) on a chromosome encoding a polypeptide with a more potent sequence may, for example, involve the generation of a sequence mutation by deletion, insertion, non-conservative or conservative substitution or a combination thereof, or replacement with a sequence having stronger activity, in order to further enhance the activity of the regulatory region. The regulatory region may include, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences that regulate transcription and decoding termination. For example, the original promoter may be replaced with a potent promoter, but is not limited to these.

[0102] Examples of well-known strong promoters include, but are not limited to, the cj1-cj7 promoter (US Registered Patent US 7662943 B2), the lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Registered Patent US 10584338 B2), O2 promoter (US Registered Patent US 10273491 B2), tkt promoter, and yccA promoter.

[0103] The sequence modification of the start codon or 5'-UTR region of the polypeptide-encoding gene described in 3) above may, but is not limited to, substitution with another start codon that has a higher polypeptide expression rate compared to the endogenous start codon.

[0104] The modifications of the amino acid sequence or polynucleotide sequence described in 4) and 5) above may be, but are not limited to, deletion, insertion, non-conservative or conservative substitution or combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, thereby causing a sequence mutation, or replacement with an improved amino acid sequence or polynucleotide sequence that has stronger activity or an improved amino acid sequence or polynucleotide sequence that enhances activity, in order to enhance the activity of the polypeptide. Specifically, such replacement can be carried out by inserting a polynucleotide into the chromosome by homologous recombination, but is not limited to these methods. The vector used in this case may further include a selection marker for confirming the presence or absence of chromosomal insertion. The selection marker is as described above.

[0105] The introduction of a foreign polynucleotide exhibiting polypeptide activity (6) above may be the introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same or similar activity as the polypeptide into the host cell. The foreign polynucleotide is not 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 appropriately selected by those skilled in the art from known transformation methods, and the polypeptide may be generated and its activity enhanced by the expression of the introduced polynucleotide in the host cell.

[0106] The codon optimization of the polynucleotide encoding the polypeptide described in 7) above may be codon optimization of the endogenous polynucleotide so that transcription or translation is enhanced within the host cell, or the codon of the exogenous polynucleotide may be optimized so that optimized transcription or translation occurs within the host cell.

[0107] The 8) analysis of the tertiary structure of the polypeptide and the selection of exposed sites for deformation or chemical modification may, for example, involve comparing the sequence information of the polypeptide to be analyzed with a database containing sequence information of known proteins to determine candidate template proteins according to the degree of sequence similarity, confirming the structure based on that, and selecting exposed sites to be deformed or chemically modified for deformation or modification.

[0108] Such enhancement of polypeptide activity may be achieved by increasing the activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type or pre-deformation microbial strain, or by increasing the amount of product produced from the polypeptide, but is not limited to these methods.

[0109] In this application, the term "weakening" of polypeptide activity encompasses all concepts of reduced activity compared to endogenous activity or complete absence of activity. This weakening may be used interchangeably with terms such as deficiency, inactivation, deletion, disruption, down-regulation, decline, attenuation, repression, and reduction.

[0110] For example, the aforementioned weakening means, but is not limited to, a state in which the protein is active but not completely inactivated by deletion, and the protein's activity is weakened compared to that of a non-myxoid microorganism, wild-type strain, or parent strain.

[0111] For example, the weakening may be inactivation, but is not limited to this. Inactivation means that the protein is not expressed at all compared to the parent strain or the non-mutated strain, or if it is expressed, its activity is absent or weakened.

[0112] The aforementioned weakening may include cases where the activity of the polypeptide itself is weakened or eliminated compared to the polypeptide activity originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide, cases where the overall polypeptide activity within the cell is lower than that of the natural strain due to inhibition of the expression of the gene encoding it or inhibition of translation into the polypeptide, cases where the gene is not expressed at all, and cases where the gene is expressed but the polypeptide is not active.

[0113] A weakening of polypeptide activity compared to its endogenous activity means that the activity of the specific polypeptide is lower than that originally possessed by the parent strain or non-mutant microorganism before the trait change. Whether or not the polypeptide activity has weakened can be confirmed by a decrease in the activity level, expression level, or amount of product excreted from the polypeptide.

[0114] For example, the weakening may be, but is not limited to, a protein activity of less than 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 0% of the protein activity of the parent strain or non-mutant microorganism before the trait change.

[0115] For example, the inactivation means that the protein is not expressed at all compared to non-mutant microorganisms, or if it is expressed, its activity is absent or weakened.

[0116] Such weakening of polypeptide activity can be achieved by any method known in the art, but is not limited to this method, and can be achieved by applying a variety of methods well known in the field (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

[0117] Specifically, the weakening of polypeptide activity in this application is, 1) Deletion of all or part of the gene encoding a polypeptide; 2) Modification of the gene expression regulatory region (or gene expression regulatory sequence) so that the expression of the polypeptide-coding gene is weakened; 3) Modification of the amino acid sequence constituting the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence) so as to remove or weaken the activity of the polypeptide; 4) Modification of the polynucleotide sequence encoding the polypeptide so as to remove or weaken the polypeptide's activity (e.g., deletion / substitution / addition of one or more nucleic acid bases on the nucleic acid sequence of the polypeptide gene so as to encode a polypeptide that has been modified so as to remove or weaken the polypeptide's activity); 5) Modification of the start codon or 5'-UTR region sequence of a polypeptide-coding gene; 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence to the leading end of the Shine-Dalgarno sequence of the polypeptide-encoding gene in order to form a secondary structure that prevents ribosome attachment; 8) Addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the polynucleotide sequence encoding the polypeptide (reverse transcription engineering, RTE); or 9) A combination of two or more selected from items 1) to 8) above is also acceptable, but is not particularly limited thereto.

[0118] for example, The deletion of part or all of the gene encoding the polypeptide described in 1) above may be the removal of the entire polynucleotide encoding the endogenous target polypeptide within the chromosome, replacement with a polynucleotide in which a portion of the nucleotide sequence is deleted, or replacement with a marker gene.

[0119] Methods for deleting some or all of such polynucleotides include, but are not limited to, methods for deleting polynucleotides by homologous recombination via a chromosome insertion vector within a microorganism, or methods for inducing mutations using light such as ultraviolet light or chemical substances, and selecting strains lacking the target gene from the resulting mutants. The methods for deleting some or all of the gene may include methods using DNA recombination technology. For example, the deletion of some or all of the gene may be achieved by injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination. The injected nucleotide sequence or vector may, but is not limited to, contain a dominant selection marker.

[0120] Furthermore, the modification of the regulatory expression sequence described in 2) above may involve the occurrence of a mutation on the regulatory expression region (or regulatory expression sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacement with a sequence having weaker activity. The regulatory expression region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and decoding.

[0121] Furthermore, the modifications of the amino acid sequence or polynucleotide sequence described in 3) and 4) above may be, but are not limited to, deletion, insertion, non-conservative or conservative substitution or combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, thereby causing sequence mutations, or replacement with an amino acid sequence or polynucleotide sequence modified to have weaker activity or an amino acid sequence or polynucleotide sequence modified to have no activity, in order to weaken the activity of the polypeptide. For example, gene expression may be inhibited or weakened by introducing mutations within the polynucleotide sequence to form a termination codon, but are not limited to this.

[0122] Furthermore, the sequence modification of the start codon or 5'-UTR region of the gene encoding the polypeptide (5) may, but is not limited to, being replaced with another start codon that has a lower polypeptide expression rate compared to the endogenous start codon.

[0123] The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide (6) can be done by referring to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0124] 7) In order to form a secondary structure that prevents ribosome attachment, the addition of a sequence complementary to the Shine-Dalgarno sequence to the leading end of the Shine-Dalgarno sequence of the polypeptide-encoding gene may make mRNA translation impossible or reduce its rate.

[0125] The addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the polynucleotide sequence encoding the polypeptide (reverse transcription engineering, RTE) may be performed by creating an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide, thereby weakening its activity.

[0126] Another aspect of this application provides a method for producing L-glutamic acid, comprising the step of culturing the microorganism of this application in a culture medium.

[0127] Specifically, the method for producing L-glutamic acid according to this application may include, but is not limited to, a step of culturing a microorganism containing the mutant, polynucleotide, or vector of this application in a culture medium.

[0128] In this application, the term "culture" means growing the microorganisms of this application under appropriately controlled environmental conditions. The culture process of this application can be carried out according to suitable culture media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culture may be batch, continuous, and / or fed-batch.

[0129] In this application, the term "culture medium" refers to a substance mixed primarily with nutrients necessary for culturing the microorganisms of this application, supplying nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganisms of this application can be any culture medium used for culturing ordinary microorganisms without special restrictions. However, the microorganisms of this application can be cultured under aerobic conditions in a normal culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, while adjusting the temperature, pH, etc. For example, a culture medium for Corynebacterium strains can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0130] In this application, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc.; glycerol, propanediol, etc. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and other appropriate amounts of carbon sources can be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0131] The nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; or organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their decomposition products, defatted soy cake or its decomposition products. These nitrogen sources may be used individually or in combination of two or more, and are not limited thereto.

[0132] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or their corresponding sodium-containing salts. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate, and may also include amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the culture medium in batches or continuously, but are not limited thereto.

[0133] Furthermore, during the cultivation of the microorganisms of this application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture medium in an appropriate manner to adjust the pH of the culture medium. In addition, during cultivation, antifoaming agents such as fatty acid polyglycol esters can be used to suppress the formation of bubbles. Furthermore, in order to maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas can be injected into the culture medium, or in order to maintain an anaerobic and microaerobic state, no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limited to these methods.

[0134] In the culture described in this application, the culture temperature can be maintained at 20-45°C, specifically 25-40°C, and the culture time can be continued until the desired amount of the target substance is obtained, and can be approximately 10-160 hours, but is not limited to this.

[0135] The L-glutamic acid produced by the culture described in this application is either secreted into the culture medium or remains within the cells.

[0136] In one specific example, the method for producing L-glutamic acid of the present application may further include, for example, a step of preparing the microorganism of the present application, a step of preparing a culture medium for culturing the strain, or a combination thereof (in any order), before the culturing step.

[0137] In one specific example, the method for producing L-glutamic acid according to this application may further include a step of recovering L-glutamic acid from the cultured microorganism, the culture of the microorganism, the fermented product of the microorganism, or the culture medium. The recovery step may further include a step after the culture step.

[0138] The aforementioned recovery may also involve collecting L-glutamic acid using appropriate methods known in the art, such as batch, continuous, or fed-batch culture methods, as described in this application. For example, L-glutamic acid can be recovered from the culture medium or microorganism using appropriate methods known in the art, such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting out), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof.

[0139] Furthermore, the L-glutamic acid production method of this application may further include a purification step. The purification can be carried out using appropriate methods known in the art. For example, if the L-glutamic acid production method of this application includes both a recovery step and a purification step, the recovery step and the purification step can be carried out sequentially or discontinuously, regardless of order, or simultaneously or integrated into a single step, but are not limited thereto.

[0140] In the method of this application, the mutant polypeptide, polynucleotide, and L-glutamic acid, etc., are as described in the other aspects described above.

[0141] Another aspect of this application provides a composition for L-glutamic acid production comprising: the mutant polypeptide of this application; a polynucleotide encoding the mutant polypeptide; a vector containing the polynucleotide; or a microorganism comprising the mutant polypeptide of this application, the polynucleotide encoding the mutant polypeptide, or a vector containing the polynucleotide; a culture of the microorganism; or a combination of two or more of these.

[0142] The composition of this application may further contain any suitable excipients commonly used in compositions for L-glutamic acid production, such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0143] As one specific example, each component present in the composition of this application may be included in a microbiologically effective amount or in an amount that can be adequately present in a production composition.

[0144] In the composition of this application, the mutant polypeptide, polynucleotide, and L-glutamic acid, etc., are as described in the other aspects described above.

[0145] Another aspect of this application provides the use of a microbial organism comprising a mutant polypeptide, a polynucleotide encoding the mutant polypeptide, or a vector containing the polynucleotide for L-glutamic acid production.

[0146] The variant polypeptides, polynucleotides, vectors, microorganisms, and L-glutamic acid of this application are as described in the other aspects mentioned above.

[0147] The present application will be described in more detail below through experimental examples. However, the following embodiments are merely preferred embodiments for illustrative purposes of the present application and are not intended to limit the scope of the rights of the present application. On the other hand, technical matters not described herein can be easily understood and performed by a person of ordinary skill who is skilled in the art of the present application or a similar art.

[0148] Example 1. Selection of mutant strains with increased glutamate production capacity through artificial mutation. Example 1-1. Induction of artificial mutagenesis through UV irradiation To select mutant strains with improved glutamic acid production capacity, the target product of fermentation, wild-type Corynebacterium glutamicum (ATCC13869) was first inoculated onto a nutrient medium containing agar and cultured at 30°C for 16 hours. The resulting hundreds of colonies were then irradiated with UV light at room temperature to induce random mutations in the genome of the strains.

[0149] Examples 1-2. Experiments on the fermentation titer of mutagenic strains and strain selection. In the above-mentioned Example 1-1, experiments were conducted on the fermentation titer of mutant strains that had undergone random mutation.

[0150] Each colony was subcultured in nutrient medium and then cultured in fermentation medium for 5 hours. Subsequently, 25% Tween40 was added to each medium at a concentration of 0.4%, and each colony was cultured for a further 32 hours.

[0151] <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

[0152] <Fermentation medium> Glucose 6%, Calcium carbonate 5%, Ammonium sulfate 2.25%, Monopotassium phosphate 0.1%, Magnesium sulfate 0.04%, Iron sulfate 10 mg / L, Biotin 0.3 mg / L, Thiamine hydrochloride 0.2 mg / L

[0153] Each colony was cultured under the aforementioned conditions, and mutant strains producing L-glutamic acid equivalent to or greater than that of wild-type Corynebacterium glutamicum (ATCC13869) were selected. Subsequently, the L-glutamic acid concentration of the selected mutant strains was measured using HPLC. The measured L-glutamic acid concentrations are shown in Table 1 below.

[0154] [Table 1]

[0155] Based on Table 1 above, "ATCC13869-t11" and "ATCC13869-t15" were selected as mutant strains that increased glutamic acid production compared to the wild-type strain.

[0156] Example 2. Confirmation of mutations through gene sequencing To confirm the genetic mutations of the aforementioned mutant strains, the genes of the ATCC13869-t11 and ATCC13869-t15 strains selected in Examples 1-2 were compared with those of the wild-type strain.

[0157] As a result, it was confirmed that the ATCC13869-t11 and ATCC13869-t15 strains contain the same mutation (substitution of nucleotide 691 of the polynucleotide sequence shown in SEQ ID NO: 2) at a specific position in the gene terC (SEQ ID NO: 2) that codes for the tellurium resistance membrane protein transporter.

[0158] Therefore, in Examples 3 and 4 below, we decided to investigate whether the mutation affects the amount of glutamic acid produced by microorganisms of the genus Corynebacterium.

[0159] Example 3. Production of a mutated strain and confirmation of glutamic acid production. Example 3-1. Production of a strain into which a mutation has been introduced. We decided to create a mutant strain into which the mutation confirmed through Example 2 had been introduced. Specifically, in order to introduce the aforementioned mutation (substitution of nucleotide 691 with T in the polynucleotide sequence shown in SEQ ID NO: 2) into wild-type Corynebacterium glutamicum (ATCC13869 and ATCC13032), we prepared a gene substitution vector to replace the 231st proline with serine in the tellurium-resistant membrane protein transporter shown in SEQ ID NO: 1, which contains the aforementioned strain. The gene fragment for vector preparation was obtained by PCR using ATCC13869 genomic DNA as a template. Based on information on the Corynebacterium glutamicum (ATCC13869) gene and surrounding nucleotide sequences registered in the National Institutes of Health GenBank (NIH GenBank), primers containing polynucleotides of SEQ ID NOs: 5-8 were prepared.

[0160] PCR was performed by denaturing at 95°C for 5 minutes, denaturing at 95°C for 20 seconds, annealing at 55°C for 20 seconds, polymerization at 72°C for 30 seconds, and repeating this 30 times, followed by polymerization at 72°C for 5 minutes. More specifically, 500 bp polynucleotides amplified using primers SEQ ID NOs. 5 and 6, and 500 bp polynucleotides amplified using primers SEQ ID NOs. 8 were obtained. A gene substitution vector was constructed by ligating the two gene fragments obtained above into a pDZ vector (Registered Patent No. 10-0924065 of the Republic of Korea and International Publication No. 2008-033001) obtained by cleaving with restriction enzymes BamHI and SalI using an infusion enzyme, and this was named "pDZ-terC(P231S)". The primer sequence information used for the above vector construction is shown in Table 2 below.

[0161] [Table 2]

[0162] Next, the gene substitution vector was used to transform wild-type strains via homologous recombination on the chromosomes (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector was inserted onto the chromosomes by homologous sequence recombination were selected using a culture medium containing 25 mg / l of kanamycin. Subsequently, gene sequence analysis was performed on the Corynebacterium glutamicum transformed strains after secondary recombination was completed. The results confirmed that the target mutation had been introduced into the strains, and the strains in which the mutation was introduced were named "ATCC13869::terC(P231S)" and "ATCC13032::terC(P231S)".

[0163] Example 3-2. Confirmation of glutamic acid production. The mutant strains ATCC13869::terC(P231S) and ATCC13032::terC(P231S) produced through Example 3-1, and their respective wild-type Corynebacterium glutamicum (ATCC13869 and ATCC13032), were cultured in the same manner as in Example 1-2.

[0164] After the culture was complete, the concentration of L-glutamic acid in each culture medium was measured. The measured concentrations of L-glutamic acid are shown in Table 3 below.

[0165] [Table 3]

[0166] As shown in Table 3 above, the L-glutamic acid concentration produced by the mutated Corynebacterium glutamicum ATCC13869::terC(P231S) strain was found to be approximately 2.2 g / L (approximately 31%) higher than that produced by the wild-type Corynebacterium glutamicum ATCC13869.

[0167] Furthermore, it was confirmed that the L-glutamic acid concentration produced by the mutated Corynebacterium glutamicum ATCC13032::terC(P231S) strain was approximately 1.2 g / L (approximately 34%) higher than that produced by the wild-type Corynebacterium glutamicum ATCC13032.

[0168] In other words, we confirmed that the variant of this application increases the L-glutamate production capacity of microorganisms.

[0169] Example 4. Confirmation of glutamate production in the KFCC11074 strain into which the mutation was introduced. Example 4-1. Production of a strain into which a mutation has been introduced. In order to confirm whether the mutation would have the same effect on strains with increased glutamate production capacity, in addition to the wild-type strain, we decided to introduce the mutation into the KFCC11074 strain (Korean Patent Publication No. 10-0292299), which is known as a glutamate-producing strain.

[0170] Specifically, in order to replace the 231st proline position indicated by Sequence ID No. 1, which contains the aforementioned strain, with serine, the pDZ-terC(P231S) vector prepared through Example 3-1 was used to transform the KFCC11074 strain by homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector was inserted on the chromosome by homologous sequence recombination were selected using a medium containing 25 mg / l of kanamycin. Subsequently, gene sequence analysis was performed on the Corynebacterium glutamicum transformed strains after secondary recombination was completed, and it was confirmed that the target mutation had been introduced into the strains. The strains in which the mutation was introduced were named "KFCC11074_terC(P231S)".

[0171] Example 4-2. Confirmation of glutamic acid production. Corynebacterium glutamicum KFCC11074 without the introduced mutation and the KFCC11074_terC(P231S) strain in which the mutation was introduced through Example 4-1 were each cultured in the same manner as in Example 1-2.

[0172] After the culture was complete, the concentration of L-glutamic acid in each culture medium was measured. The measured L-glutamic acid concentrations are shown in Table 4 below.

[0173] [Table 4]

[0174] As shown in Table 4 above, the concentration of L-glutamic acid produced by the mutated Corynebacterium glutamicum KFCC11074_terC(P231S) strain was found to be approximately 2.7 g / L (approximately 48%) higher than that produced by the unmutated Corynebacterium glutamicum KFCC11074.

[0175] In other words, we confirmed that the mutation described in this application increases the L-glutamate production capacity of microorganisms, even in strains with increased glutamate production capacity.

[0176] In summary, the mutants of this application can enhance the activity of tellurium-resistant membrane protein transporters in wild-type or glutamate-producing strains through mutated gene activity, thereby increasing the production capacity of glutamate, the target product of fermentation. Therefore, they can be usefully used in a variety of industrial fields seeking to produce glutamate in high yield.

[0177] From the above description, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

Claims

1. A tellurium-resistant membrane protein transporter mutant polypeptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, in which the amino acid corresponding to position 231 of SEQ ID NO: 1 is substituted with serine.

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

3.

3. A polynucleotide encoding a mutant polypeptide according to claim 1 or 2.

4. A microorganism comprising a mutant polypeptide according to claim 1 or 2, a polynucleotide encoding the mutant polypeptide, or a vector containing the polynucleotide.

5. The microorganism according to claim 4, wherein the microorganism has increased L-glutamic acid production capacity compared to a microorganism containing the polypeptide of Sequence ID No. 1 or a polynucleotide encoding it.

6. The microorganism according to claim 4, wherein the microorganism is a microorganism of the genus Corynebacterium.

7. The microorganism according to claim 6, wherein the Corynebacterium genus microorganism is Corynebacterium glutamicum.

8. A method for producing L-glutamic acid, comprising the step of culturing the microorganism described in claim 4 in a culture medium.

9. The method for producing L-glutamic acid according to claim 8, further comprising the step of recovering L-glutamic acid from the cultured microorganism, the culture of the microorganism, the fermented product of the microorganism, or the culture medium.

10. A composition for L-glutamic acid production comprising: a mutant polypeptide according to claim 1 or 2; a polynucleotide encoding the mutant polypeptide; a vector containing the polynucleotide; or a microorganism containing the mutant polypeptide, the polynucleotide encoding the mutant polypeptide, or the vector containing the polynucleotide; a culture of the microorganism; or a combination of two or more of these.

11. Use of a microorganism comprising the mutant polypeptide described in claim 1 or 2, a polynucleotide encoding the mutant polypeptide, or a vector containing the polynucleotide for L-glutamic acid production.

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