Mutant threonine / homoserine excretion protein and method for producing L-amino acids using the same

Abstract

The present application relates to a mutant threonine / homoserine excretion protein and a method for producing L-amino acids using the same.

Description

[Technical Field]

[0001] The present application relates to a mutant threonine / homoserine excretion protein and a method for producing L-amino acids using the same. [Background technology]

[0002] L-homoserine is an intermediate in the methionine and threonine biosynthetic pathways (WO2008 / 013432) and is used as a precursor for the production of methionine and threonine. L-homoserine is synthesized by reduction of L-aspartate-4-semialdehyde with homoserine dehydrogenase (hom).

[0003] Various studies have been conducted to develop highly efficient production microorganisms and fermentation process technologies for the production of L-amino acids such as L-homoserine and other useful substances. Target-specific approaches, such as increasing the expression of genes encoding enzymes involved in L-homoserine biosynthesis or deleting genes unnecessary for biosynthesis, have been mainly used.

[0004] However, research is still needed to effectively increase the ability of microorganisms to produce L-amino acids, including L-homoserine. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2008 / 013432 [Patent Document 2] US Patent US 7662943 B2 [Patent Document 3] US Patent US 10584338 B2 [Patent Document 4] US Patent US 10273491 B2 [Patent Document 5] Korean Registration License No. 10-2183209 [License 6] Korean Patent No. 10-2019-0003019 [License 7] Korean publication number 10-2020-0136813 [Non-licensed literature]

[0006] [Non-licensed Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444 [Non-licensed Document 2] Rice et al., 2000, Trends Genet. 16:276-277 [Non-licensed Document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453 [Non-licensed Document 4] Devereux,J.,et al,Nucleic Acids Research 12:387 (1984) [Non-licensed Document 5] Atschul,[S.] [F.,] [ET AL,J MOLEC BIOL 215]:403 (1990) [Non-licensed Document 6] Guide to Huge Computers,Martin J. Bishop,[ED.,] Academic Press,San Diego, 1994 [Non-licensed Document 7] [CARILLO et al.](1988) SIAM J Applied Math 48:1073 [Non-licensed Document 8] Smith and Waterman,Adv. Appl. Math (1981) 2:482 [Non-licensed Document 9] Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation (1978)

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Non-licensed Document 15

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Non-licensed Document 18

[0007] The present inventors have completed the present application by confirming that a microorganism expressing the mutant threonine / homoserine excretion protein of the present application produces L-amino acids in high yields.

[0008] One aspect of the present application provides a mutant threonine / homoserine efflux protein in which the amino acid corresponding to position 123 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid.

[0009] In one embodiment, the other amino acid may be an amino acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, tryptophan, and tyrosine.

[0010] In another embodiment, the other amino acid may be an amino acid selected from the group consisting of alanine, cysteine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, serine, threonine, tryptophan, and tyrosine.

[0011] Another aspect of the present application provides a polynucleotide encoding a mutant threonine / homoserine excretion protein of the present application.

[0012] Another aspect of the present application provides a microorganism comprising any one or more selected from the mutant threonine / homoserine excretion protein of the present application; a polynucleotide encoding the same; or a vector comprising the same.

[0013] In one specific example, the microorganism may have increased L-amino acid production ability compared to a microorganism containing a wild-type threonine / homoserine export protein having the amino acid sequence of SEQ ID NO: 1 or a polynucleotide encoding the same.

[0014] In another embodiment, the L-amino acid may be any one or more selected from the group consisting of L-homoserine, O-acetyl-L-homoserine, O-succinyl-L-homoserine, L-methionine, and L-threonine.

[0015] As a microorganism according to any one of the above-mentioned embodiments, the microorganism may be a Corynebacterium microorganism.

[0016] In any one of the above-mentioned embodiments, the Corynebacterium microorganism may be Corynebacterium glutamicum.

[0017] Another aspect of the present application provides a method for producing an L-amino acid, comprising culturing in a medium a microorganism containing any one or more selected from the mutant threonine / homoserine excretion protein of the present application; a polynucleotide encoding the same; or a vector containing the same.

[0018] Another aspect of the present application provides a composition for producing L-amino acids, comprising a microorganism containing any one or more selected from the mutant threonine / homoserine excretion protein of the present application; a polynucleotide encoding the same; or a vector containing the same; a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more of these. [Effects of the Invention]

[0019] Microorganisms expressing the mutant threonine / homoserine excretion proteins of the present application can produce L-amino acids in high yields and can be usefully utilized for the production of L-amino acids. DETAILED DESCRIPTION OF THE INVENTION

[0020] This will be explained in more detail as follows: Meanwhile, each description and embodiment disclosed in this application also applies 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.

[0021] definition As used in this specification and the appended claims, the singular articles "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless the context dictates otherwise, singular terms include plurals and plural terms include the singular. In this specification and the appended claims, the use of "or" is intended to include "and / or" unless specifically stated otherwise.

[0022] In this application, the term "about" is used before a specific numerical value. As used in this application, the term "about" includes not only the exact number listed after the term, but also approximately that number or a range close to that number. Whether a number is close to or approximately the specific number mentioned can be determined by considering the context in which the number is presented. As an example, the term "about" refers to a range of 10% to +10% of the numerical value. As another example, the term "about" refers to a range of -5% to +5% of the given numerical value. However, the present invention is not limited to this.

[0023] In this application, terms such as "first, second, third," "i), ii), iii)...," or "(a), (b), (c), (d)..." are used to distinguish between similar features and do not imply sequential or orderly performance. For example, when the terms are used in connection with steps of a method, use, or analysis, the steps may be spaced apart, may occur simultaneously, or may be separated by a few seconds, minutes, hours, days, or months.

[0024] In this application, the term "consisting essentially of" means that unspecified components may be present if the characteristics of the subject matter claimed in this application are not substantially affected by the presence of the unspecified components.

[0025] In this application, the term "consisting of" means that the percentages of the specified component(s) total 100%. The components or features following the term "consisting of" may be essential or mandatory. In some embodiments, other optional or non-essential components may be excluded, other than the components or features following "consisting of."

[0026] In this application, the term "comprising" means the presence of the feature, step or component described below that term, and does not exclude the presence or addition of one or more features, steps or components. In this application, the component or feature described below as "comprising" may be essential or mandatory, but some embodiments may further include other optional or non-essential components or features.

[0027] Polypeptides As used herein, the term "protein" or "polypeptide" refers to a polymer or oligomer of consecutive amino acid residues. In this application, "polypeptide," "protein," and "peptide" are used interchangeably with "amino acid sequence."

[0028] In some cases, amino acid sequences that exhibit activity can be referred to as “enzymes.” In this application, amino acid sequences are written in the N-terminal to C-terminal orientation unless otherwise indicated.

[0029] As used herein, the term "mature polypeptide" refers to a form of a polypeptide that lacks a signal sequence or propeptide sequence. A mature protein / polypeptide / peptide may be a functional form of a protein / polypeptide / peptide. A mature polypeptide may be a final form that has undergone post-translational or post-translational modifications. Examples of post-translational modifications include, but are not limited to, N- or C-terminal modifications, glycosylation, phosphorylation, removal of a leader sequence, etc.

[0030] In the present application, in relation to an amino acid sequence, a polypeptide "comprising" an amino acid sequence set forth in a particular SEQ ID NO, a polypeptide "consisting of" an amino acid sequence set forth in a particular SEQ ID NO, or a polypeptide or protein "having" an amino acid sequence set forth in a particular SEQ ID NO, is clearly within the scope of the present application, provided that it has the same or corresponding activity as a protein consisting of the amino acid sequence of the SEQ ID NO. For example, 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 are not excluded, and it is clearly within the scope of the present application even if such additions or mutations are present, provided that they have the same or corresponding activity as the mutant protein.

[0031] For example, the polypeptide may have an addition of a sequence, a naturally occurring mutation, a silent mutation, or a conservative substitution at the N-terminus, C-terminus, and / or internally of the amino acid sequence that does not alter the function of the polypeptide of the present application. For example, the polypeptide may be conjugated to an N-terminal signal (or leader) sequence of a protein involved in co-translational or post-translational protein transfer. The polypeptide may also be conjugated to other sequences or linkers that allow the polypeptide to be identified, purified, or synthesized.

[0032] As used herein, the term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made based on 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; amino acids with nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) include 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 by size and divided into five groups, starting with the amino acid group with the smallest volume: glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, and tyrosine. However, conservative substitutions are not necessarily limited to these. Typically, conservative substitutions have little or no effect on the activity of a polypeptide.

[0033] Polynucleotides In this application, the term "gene" refers to a polynucleotide that encodes a polypeptide and includes regions before and after the coding region. In some embodiments, a gene may have sequences (introns) inserted between each coding region (exon).

[0034] In this application, the term "polynucleotide, nucleic acid, or nucleic acid molecule" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and a DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA) chain of a certain length or greater.

[0035] homology, identity In this application, the term "homology" or "identity" means the degree of relatedness between two given amino acid or nucleotide sequences, and can be expressed as a percentage. The terms homology and identity can often be used interchangeably.

[0036] Homology or identity of conserved polynucleotide or polypeptide sequences can be determined by standard sequence algorithms, optionally with default gap penalties established by the program used. Substantially homologous or identical sequences are generally hybridizable under moderately or highly stringent conditions over at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or over the entire length of the sequence. Hybridization obviously also includes polynucleotides containing common codons in polynucleotides or codons that take codon degeneracy into account.

[0037] 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, e.g., as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later), as implemented in the GCG program package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.] Academic Press, San (See, for example, BLAST or ClustalW from the National Center for Biotechnology Information.) Homology, similarity, or identity can be determined using, for example, BLAST or ClustalW from the National Center for Biotechnology Information.

[0038] 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) unitary matrices (containing values ​​of 1 for identity and 0 for non-identity), the PAM Matrix (see the disclosure by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation (1978)), 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); (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.

[0039] Furthermore, whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be confirmed by comparing the sequences in a Southern hybridization experiment under defined stringent conditions. Suitable defined hybridization conditions are within the skill of the art and can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto.

[0040] In the present application, the term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Sambrook et al., supra, 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, 1×SSC, and 0.1% SDS, specifically 60°C, 0.1×SSC, and 0.1% SDS, more specifically 68°C, 0.1×SSC, and 0.1% SDS.

[0041] Such hybridization requires that the two nucleotides have complementary sequences, even though mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to one another. For example, with respect to DNA, adenosine 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 the entire sequence, as well as those with substantially similar base sequences.

[0042] For example, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the above-mentioned hybridization conditions, including a hybridization step at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0043] The appropriate stringency for hybridizing such polynucleotides depends on the length and degree of complementation of the polynucleotides, variables well known in the art (eg, J. Sambrook et al., supra).

[0044] Nucleic acid constructs, vectors, and transformation As used herein, the term "nucleic acid construct" refers to a single- or double-stranded nucleic acid molecule that contains one or more regulatory sequences and that has been artificially synthesized, engineered to contain a specific sequence in a manner that does not occur in nature, or isolated from nature.

[0045] The term "vector" as used in this application refers to a DNA construct comprising a polynucleotide sequence encoding a polypeptide of interest operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the polypeptide in a suitable host. The expression control region may comprise 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 host cell, a vector can replicate or function independently of the host genome, or it can be integrated into the genome itself.

[0046] The vectors used in this application are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pDZ, pDC, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Examples of vectors that can be used include pDZ, pDC, pACYC177, pACYC184, pCL, pCL1920, pSHK130, pDCM2, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC.

[0047] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome of a cell using a vector for chromosomal insertion. The insertion of the polynucleotide into a chromosome can be performed by any method known in the art, including, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of the 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 the insertion of the target nucleic acid molecule. A marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide, is 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.

[0048] In this application, the term "transformation" refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, thereby enabling the expression of the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may include any polynucleotide, whether it is located intrachromosomally or extrachromosomally, as long as it is expressible in the host cell. The polynucleotide may also include DNA and RNA encoding the target polypeptide. The polynucleotide may be introduced in any form that can be introduced and expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. The expression cassette typically includes 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 an autonomously replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form, operably linked to sequences necessary for expression in the host cell, but is not limited thereto.

[0049] As used herein, the term "operably linked" refers to a configuration in which a regulatory sequence is positioned appropriately so that it directs the expression of a coding sequence. Thus, "operably linked" includes a regulatory region, such as a promoter, terminator, signal sequence, or enhancer region, that is a functional domain having a known or desired activity, attached or linked to a target (gene or polypeptide) so as to regulate the expression, secretion, or function of the target in accordance with the known or desired activity. For example, it refers to the operably linking of a polynucleotide sequence encoding a polypeptide of the present application with a promoter sequence that initiates and mediates transcription of the polynucleotide.

[0050] In this application, the term "expression" includes any step involved in the production of a polypeptide, such as, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0051] In this application, the term "expression vector" means a linear or circular nucleic acid molecule that contains a coding sequence and regulatory sequences operably linked thereto for the expression thereof.

[0052] In this application, the term "regulatory sequence" refers to a polynucleotide sequence required for the expression of a coding sequence. Each regulatory sequence may be native to the coding sequence (having the same origin) or foreign (derived from another gene). Examples of the regulatory sequence include a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination. The minimum unit of the regulatory sequence may include a promoter, and a transcription and translation termination sequence.

[0053] As used herein, the term "recombinant" in reference to a cell, polynucleotide, polypeptide, or vector means that the cell, polynucleotide, polypeptide, or vector has been altered by the introduction of a heterologous nucleic acid or polypeptide or the alteration of a naturally occurring polynucleotide or polypeptide, or that the cell is derived from a cell so altered. Thus, for example, a recombinant cell may express genes that are not found within the native (non-recombinant) form of the cell, or may express naturally occurring genes that are not expressed, not expressed at all, or that are abnormally expressed.

[0054] microorganisms In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially, and may be a microorganism in which a specific mechanism has been weakened or enhanced by inserting an exogenous gene or by increasing 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 limitation.

[0055] For example, the microorganism of the present application may be a microorganism containing a mutant threonine / homoserine export protein or a polynucleotide encoding the same; or a microorganism that has been genetically modified (e.g., a recombinant strain) to contain a mutant threonine / homoserine export protein or a polynucleotide encoding the same.

[0056] The strain of the present application may be a microorganism in which L-amino acid production ability has been imparted to a strain that naturally has the ability to produce L-amino acids or a strain that does not have the ability to produce L-amino acids, for example, but is not limited to, a microorganism in which L-amino acid production ability has been increased by introducing a mutant threonine / homoserine export protein of the present application or a polynucleotide encoding the same.

[0057] The strain of the present application may be a microorganism having increased L-amino acid production ability compared to a parent strain or a wild-type Corynebacterium strain not containing the mutant of the present application. The microorganism may have improved L-amino acid production ability due to the introduction of a mutant threonine / homoserine export protein of the present application, which has increased threonine / homoserine export activity compared to the wild-type threonine / homoserine export protein, or a polynucleotide encoding the same. Specifically, the strain of the present application may have increased L-amino acid production ability compared to a Corynebacterium microorganism containing a wild-type threonine / homoserine export protein having the amino acid sequence of SEQ ID NO: 1 or a polynucleotide encoding the same.

[0058] In this application, the term "microorganism capable of producing an L-amino acid" refers to a prokaryotic or eukaryotic microbial strain capable of producing an L-amino acid in vivo, and includes both microorganisms in which the ability to produce an L-amino acid has been imparted to a parent strain that does not have the ability to produce an L-amino acid, and microorganisms that inherently have the ability to produce an L-amino acid. The ability to produce an L-amino acid can be imparted or enhanced by species improvement.

[0059] In the present application, the term "unaltered 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 characteristics are altered by genetic mutations due to natural or artificial factors. The term "unaltered microorganism" may be used interchangeably with "unaltered strain," "unaltered microorganism," "unaltered strain," "unaltered microorganism," "unaltered parent strain," "wild-type microorganism," "reference microorganism," or "reference microorganism." Furthermore, in the present application, the term "unaltered microorganism with a threonine / homoserine export protein" refers to a strain into which a mutant threonine / homoserine export protein described herein has not been introduced or has not yet been introduced. The term "unaltered microorganism with a threonine / homoserine export protein" in the present application does not exclude strains containing modifications of other proteins or genes in addition to modifications of the threonine / homoserine export protein or the polynucleotide encoding it. Furthermore, in the present application, the unaltered microorganism may be, but is not limited to, a microorganism containing the amino acid sequence of SEQ ID NO: 1 or the polynucleotide of SEQ ID NO: 2.

[0060] Increased polypeptide activity In the present application, the term "increase" of polypeptide activity means that the activity of the polypeptide is increased compared to the endogenous activity. The increase may be interchangeably used with terms such as activation, up-regulation, overexpression, and enhancement.

[0061] The increase can include both the display of an activity that the protein did not originally have, and the display of an activity that is improved compared to the intrinsic activity or the activity before the modification.

[0062] For example, the "exhibition of an activity not originally possessed" may be, but is not limited to, "introduction of a protein." The introduction of a protein means that a gene not originally possessed by a microorganism is expressed in the microorganism, thereby exhibiting the activity of a specific protein, or that the activity of the protein is increased or improved compared to the endogenous activity or activity before modification. For example, the introduction may mean that a polynucleotide encoding a specific protein is introduced into a chromosome in a microorganism, or that a vector containing a polynucleotide encoding a specific protein is introduced into a microorganism, resulting in the activity of the protein being exhibited.

[0063] The term "intrinsic activity" refers to the activity of a specific polypeptide that a parent strain or an unaltered microorganism originally possessed before the trait change, when the trait has been changed due to genetic mutation caused by natural or artificial factors. This term may be used interchangeably with "activity before transformation."

[0064] An increase in polypeptide activity compared to the endogenous activity means that the activity and / or concentration (expression level) of a particular polypeptide is improved compared to the activity and / or concentration (expression level) that the parent strain or untransformed microorganism originally possessed before transformation.

[0065] By way of example, the increase may indicate that the corresponding protein activity was absent or that its activity or concentration is increased, generally by about 1%, about 10%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%, up to about 1000% or about 2000% or more, relative to the activity or concentration of the wild-type protein or the initial microbial strain, but is not limited thereto.

[0066] The increase in the activity of the polypeptide can be achieved by introducing an exogenous polypeptide or by increasing the activity of an endogenous polypeptide. The increase in 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.

[0067] The activity of the polypeptide can be increased 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 increased compared to that of the microorganism before transformation. Specifically, the activity may be increased by 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, pp. 1-16; Sambrook et al., Molecular Cloning 2012, etc.).

[0068] Specifically, the increase in activity of the polypeptide of the present application is 1) an increase in the intracellular copy number of the polynucleotide encoding the polypeptide; 2) Modification of the expression regulatory region of the gene on the chromosome encoding the polypeptide (e.g., mutation within the expression regulatory region, replacement with a sequence having stronger activity, or insertion of a sequence having stronger activity); 3) a modification of the nucleotide sequence encoding the initiation codon or 5'UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so as to increase the polypeptide activity; 5) modifying a polynucleotide sequence encoding the polypeptide so as to increase the polypeptide's activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide that has been modified so as to increase 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 altering 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.

[0069] for example, The increase in intracellular copy number of the polynucleotide encoding the polypeptide (1) may be achieved by introducing into the host cell a vector operably linked to the polynucleotide encoding the polypeptide, which can replicate and function independently of the host. Alternatively, one or more copies of the polynucleotide encoding the polypeptide may be introduced into a chromosome in the host cell. The introduction into the chromosome can be achieved by, but is not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into the chromosome of the host cell. The vector is as described above.

[0070] The 2) 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 be, for example, by introducing a mutation in the sequence through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further increase the activity of the expression regulatory region, or by replacing the sequence 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.

[0071] 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 rhtB 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.

[0072] The modification of the nucleotide sequence of the start codon or 5'UTR region of the gene encoding the polypeptide in 3) may be, for example, but is not limited to, substituting another start codon that results in a higher polypeptide expression rate than the endogenous start codon.

[0073] 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, or an amino acid sequence or polynucleotide sequence modified to have stronger activity or to have increased activity, so as to increase the activity of the polypeptide. Specifically, the modification can be performed by inserting the polynucleotide into a chromosome by homologous recombination, but is not limited to this. 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.

[0074] The introduction of an exogenous polynucleotide that exhibits the activity of a polypeptide (6) may be the introduction of an exogenous polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide into a host cell. The exogenous polynucleotide is not limited in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be any known transformation method appropriately selected by those skilled in the art. The introduced polynucleotide is expressed in a host cell to produce a polypeptide, and its activity can be increased.

[0075] The codon optimization of the polynucleotide encoding the polypeptide (7) may be an optimization of the codons of an endogenous polynucleotide to increase transcription and translation in a host cell, or an optimization of the codons of an exogenous polynucleotide to optimize transcription and translation in a host cell.

[0076] 8) Analyzing the tertiary structure of a polypeptide and selecting and modifying or chemically modifying exposed sites may involve, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and modifying exposed sites to be modified or chemically modified.

[0077] Such an increase in polypeptide activity may be, but is not limited to, an increase in the activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in a wild-type or untransformed microbial strain, or an increase in the amount of product produced from the polypeptide.

[0078] Modification of a portion or all of a polynucleotide in the microorganism of the present application can 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 all of the gene include methods using DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene can be injected into the microorganism to cause homologous recombination, thereby deleting a portion or all of the gene. The injected nucleotide sequence or vector can include, but is not limited to, a dominant selectable marker.

[0079] culture In the present application, the term "culturing" refers to growing the strain 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 microorganism. Specifically, the culturing may be, but is not limited to, a batch, continuous, and / or fed-batch culture.

[0080] In the present application, the term "culture medium" refers to a mixture of nutrients, primarily those required for culturing the microorganisms of the present application, and provides nutrients and growth factors, including water, essential for survival and growth. Specifically, the culture medium and other culture conditions used to culture the strains 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, with temperature, pH, and other parameters adjusted. 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)].

[0081] In the present application, the carbon source may 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; and amino acids such as glutamic acid, methionine, and lysine. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid may also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used. A variety of other carbon sources may also be used in appropriate amounts without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0082] Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its degradation products, and defatted soybean cake or its degradation products. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0083] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or the corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and other compounds, including amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the culture medium in a batch or continuous manner. However, the present invention is not limited to these compounds.

[0084] During the cultivation of the microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the medium in an appropriate manner to adjust the pH of the medium. 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.

[0085] 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 can be performed for about 10 to 160 hours, but is not limited thereto.

[0086] In this application, the term "culture" refers to a culture solution, concentrated culture solution, dried culture solution, culture filtrate, concentrated culture filtrate, or dried culture filtrate obtained by culturing a specific microorganism in a culture medium, and the culture solution refers to one containing the specific microorganism, while the culture filtrate refers to one that is substantially free of the specific microorganism (here, "substantially" means that the specific microorganism separated by filtration or the like has been removed, and does not mean that the microorganism has been completely removed in the filtrate). The form of the culture is not limited, and may be, for example, a liquid, emulsion, or solid.

[0087] In this application, the term "fermentation" refers to the process in which microorganisms use their own enzymes to decompose organic matter, but is not a putrefaction reaction. Fermentation and putrefaction proceed through similar processes, but if useful substances are produced as a result of decomposition, it is called fermentation, and if foul odors or harmful substances are produced, it is called putrefaction.

[0088] In the present application, the method for obtaining a fermented product from the strain is not particularly limited, and the product can be obtained by a method commonly used in the technical field or a similar field.

[0089] In the present application, the term "fermented product" refers not only to the fermented substance itself but also to any kind of substance containing the fermented product generated from the strain, such as a culture medium of the strain in which the strain and the culture coexist, a fermented product obtained by filtering the strain from the culture medium, a fermented product obtained by sterilizing the strain from the culture medium and filtering it, an extract obtained by extracting the fermented product or a culture medium containing the fermented product, a diluted solution or concentrate obtained by diluting the fermented product or an extract thereof, a dried product obtained by drying the fermented product or an extract thereof, and a lysate obtained by collecting and disrupting the cells of the strain.

[0090] Specific Description of the Application A specific example of the present application will be described in more detail below.

[0091] One aspect of the present application provides a mutant threonine / homoserine efflux protein in which the amino acid corresponding to position 123 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid.

[0092] In the present application, the term "mutant threonine / homoserine export protein" refers to any polypeptide having threonine and homoserine export activity or a mutant threonine / homoserine export protein comprising a substitution of the amino acid corresponding to the 123rd position from the N-terminus of SEQ ID NO: 1 with another amino acid. The "mutant threonine / homoserine export protein" may also be referred to as a "threonine / homoserine export protein mutant," "mutant RhtA," "RhtA mutant," etc.

[0093] The threonine / homoserine export protein to be mutagenized in the present application may also be referred to as "RhtA protein" or "RhtA" and may be a protein having threonine and homoserine export activity. Specifically, the protein may comprise the amino acid sequence of SEQ ID NO: 1 and have threonine and homoserine export activity, but is not limited thereto. Proteins that have the same or corresponding activity as a protein containing the amino acid sequence of SEQ ID NO: 1, excluding meaningless additions of sequences before or after the amino acid sequence of SEQ ID NO: 1, naturally occurring mutations, or silent mutations, are included in the present application. For example, the protein to be mutagenized in the present application may be a protein consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. Furthermore, it is obvious that proteins having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted or added are also included within the scope of proteins that are subject to mutation in this application, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the protein.

[0094] The amino acid sequence before modification of the threonine / homoserine excretion protein to be mutated in the present application, i.e., the amino acid before modification corresponding to the 123rd amino acid of SEQ ID NO: 1 in the parent sequence, may be valine (V).

[0095] The mutant threonine / homoserine excretion protein of the present application may be one in which the amino acid at the position corresponding to the 123rd amino acid in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid different from the amino acid before substitution.

[0096] For example, the mutant threonine / homoserine excretion protein may be one in which the amino acid corresponding to position 123 in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid other than valine, the amino acid before substitution.

[0097] In another example, the mutant threonine / homoserine export protein may be one in which the amino acid corresponding to position 123 in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, tryptophan, and tyrosine. Specifically, the mutant threonine / homoserine export protein may be one in which the amino acid corresponding to position 123 in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of alanine, cysteine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, serine, threonine, tryptophan, and tyrosine.

[0098] As yet another example, the mutant threonine / homoserine efflux protein of the present application may have, comprise, consist of, or essentially consist of the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31 or SEQ ID NO:33 or SEQ ID NO:35 or SEQ ID NO:37 or SEQ ID NO:39.

[0099] The mutant threonine / homoserine excretion protein of the present application may include an amino acid sequence in which the amino acid corresponding to the 123rd position in the amino acid sequence of SEQ ID NO: 1 is an amino acid other than valine, such as an amino acid selected from the group consisting of alanine, cysteine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, serine, threonine, tryptophan, and tyrosine, and which has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, or 99.3% homology or identity to the amino acid sequence set forth in SEQ ID NO: 1. Furthermore, it is obvious that mutant threonine / homoserine export proteins 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 mutant threonine / homoserine export protein of the present application.

[0100] For example, the amino acid sequence may contain additions or deletions of sequences at the N-terminus, C-terminus and / or internally that do not change the function of the mutant threonine / homoserine excretion protein of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0101] The term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0102] As used herein, the term "mutant protein" or "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 amino acid sequence of the variant prior to the mutation 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 variant. That is, the performance of the variant may be increased, unchanged, or decreased compared to the polypeptide prior to the mutation. 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 removed. Other variants may include variants in which portions have been removed from the N- and / or C-termini of the mature protein. The term "mutant protein" may be used interchangeably with terms such as mutation, variant, mutant polypeptide, mutated protein, mutation, and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited to these terms as long as they are used to mean mutated. For the purposes of this application, the variant may be a polypeptide in which the amino acid corresponding to position 123 in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, tryptophan, and tyrosine, and specifically, a polypeptide in which the amino acid is substituted with an amino acid selected from the group consisting of alanine, cysteine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, serine, threonine, tryptophan, and tyrosine.For example, it may have or comprise an amino acid sequence that is 60% or more, 65% 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 homologous or identical to SEQ ID NO: 1, or it may comprise, consist of, or consist essentially of an amino acid sequence that is 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 homologous or identical to said sequence but less than 100%. As a more specific example, the variant may be, but is not limited to, a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:9 or SEQ ID NO:11 or SEQ ID NO:13 or SEQ ID NO:15 or SEQ ID NO:17 or SEQ ID NO:19 or SEQ ID NO:21 or SEQ ID NO:23 or SEQ ID NO:25 or SEQ ID NO:27 or SEQ ID NO:29 or SEQ ID NO:31 or SEQ ID NO:33 or SEQ ID NO:35 or SEQ ID NO:37 or SEQ ID NO:39.

[0103] In the mutant threonine / homoserine export protein of the present application, the amino acid corresponding to position 193 of SEQ ID NO: 1 may be isoleucine, alanine, cysteine, glycine, histidine, lysine, leucine, threonine, valine, or tryptophan. For example, in the mutant threonine / homoserine export protein of the present application, the amino acid corresponding to position 193 of SEQ ID NO: 1 may be isoleucine or valine, but is not limited thereto.

[0104] The variants may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be conjugated to a signal (or leader) sequence involved in co- or post-translational protein translocation. The variants may also be conjugated to other sequences or linkers to allow identification, purification, or synthesis.

[0105] Furthermore, a polynucleotide encoding a mutant threonine / homoserine export protein of the present application may comprise a nucleotide sequence encoding a mutant threonine / homoserine export protein in which the amino acid corresponding to position 123 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid. As an example, a polynucleotide of the present application may comprise a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 9 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 17 or SEQ ID NO: 19 or SEQ ID NO: 21 or SEQ ID NO: 23 or SEQ ID NO: 25 or SEQ ID NO: 27 or SEQ ID NO: 29 or SEQ ID NO: 31 or SEQ ID NO: 33 or SEQ ID NO: 35 or SEQ ID NO: 37 or SEQ ID NO: 39. For example, the nucleic acid sequence may have or contain a base sequence that is 60% or more, 65% 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 homologous or identical to SEQ ID NO: 2, or may contain, consist of, or essentially consist of a base sequence that is 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 homologous or identical to said sequence but less than 100%. As a more specific example of the present application, a polynucleotide encoding a mutant threonine / homoserine export protein of the present application may have or include the sequence of SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID NO:18 or SEQ ID NO:20 or SEQ ID NO:22 or SEQ ID NO:24 or SEQ ID NO:26 or SEQ ID NO:28 or SEQ ID NO:30 or SEQ ID NO:32 or SEQ ID NO:34 or SEQ ID NO:36 or SEQ ID NO:38 or SEQ ID NO:40. Furthermore, a polynucleotide encoding a mutant threonine / homoserine export protein of the present application may consist of or essentially consist of the sequence of SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID NO:18 or SEQ ID NO:20 or SEQ ID NO:22 or SEQ ID NO:24 or SEQ ID NO:26 or SEQ ID NO:28 or SEQ ID NO:30 or SEQ ID NO:32 or SEQ ID NO:34 or SEQ ID NO:36 or SEQ ID NO:38 or SEQ ID NO:40.

[0106] The polynucleotide of the present application may have various modifications in the coding region within a range that does not change the amino acid sequence of the mutant threonine / homoserine excretion protein of the present application, taking into account codon degeneracy or codons preferred in the organism in which the mutant threonine / homoserine excretion protein of the present application is to be expressed. Specifically, the polynucleotide encoding the mutant threonine / homoserine export protein of the present application may have or comprise a nucleotide sequence that is 60%, 65%, 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 homologous or identical to the sequence of SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID NO:18 or SEQ ID NO:20 or SEQ ID NO:22 or SEQ ID NO:24 or SEQ ID NO:26 or SEQ ID NO:28 or SEQ ID NO:30 or SEQ ID NO:32 or SEQ ID NO:34 or SEQ ID NO:36 or SEQ ID NO:38 or SEQ ID NO:40, or may consist of or essentially consist of a nucleotide sequence that is 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 homologous or identical to the sequence set forth above, but is not limited thereto. In this case, in the homologous or identical sequence, the codon encoding the amino acid corresponding to position 123 of SEQ ID NO: 1 may be one of the codons encoding an amino acid other than proline, for example, an amino acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, tryptophan, and tyrosine, and specifically, may be one of the codons encoding an amino acid selected from the group consisting of alanine, cysteine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, serine, threonine, tryptophan, and tyrosine.

[0107] As used herein, the term "corresponding to" refers to the amino acid residue at the recited position in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the recited residue in a polypeptide. Identifying the amino acid at the corresponding position can also determine the specific amino acid of the sequence to which a particular sequence refers. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0108] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue in the amino acid sequence can be numbered by reference to the numeric position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described in this application can identify the position of an amino acid relative to a query sequence (also referred to as a "reference sequence"), or the position at which a variation such as a substitution, insertion, or deletion occurs.

[0109] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16:276-277), etc. can be used, but are not limited to these, and sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the art can be used as appropriate.

[0110] As used herein, the term "threonine / homoserine exporter (RhtA)" refers to a protein having the activity of exporting threonine and homoserine. Specifically, the term "threonine / homoserine exporter" in the present application may be used interchangeably with "RhtA." The threonine / homoserine exporter is known in the art and may be, but is not limited to, one of NCBI Accession Nos. WP_264865234.1, WP_161647838.1, and WP_179150183.1. The amino acid and polynucleotide sequences of the threonine / homoserine exporter can be obtained from publicly known databases, including, but not limited to, NCBI GenBank.

[0111] For example, the threonine / homoserine export protein may comprise 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 threonine / homoserine export protein activity. Specifically, the polypeptide having threonine / homoserine export protein activity may have, comprise, consist of, or essentially consist of SEQ ID NO: 1 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. For example, the threonine / homoserine excretion protein refers to a protein endogenously present in Corynebacterium microorganisms or Corynebacterium glutamicum, but is not limited thereto. Specifically, the threonine / homoserine excretion protein may be endogenously present in Corynebacterium microorganisms or Corynebacterium glutamicum and have the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.

[0112] Furthermore, the threonine / homoserine export protein having the amino acid sequence of SEQ ID NO: 1 may be encoded by a polynucleotide having, comprising, consisting of, or 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, or 99% or more homology or identity to the sequence of SEQ ID NO: 2, but is not limited thereto. The nucleotide sequence of SEQ ID NO: 2 can be obtained from publicly known databases, such as, but not limited to, GenBank of NCBI.

[0113] In the present application, the gene comprising the base sequence of SEQ ID NO: 2 may be used interchangeably with a polynucleotide comprising the base sequence of SEQ ID NO: 2, or a gene or polynucleotide having the base sequence of SEQ ID NO: 2.

[0114] The polynucleotide of the present application may have various modifications in its coding region, taking into account codon degeneracy or preferred codons in the organism in which the threonine / homoserine export protein of the present application is to be expressed, without changing the amino acid sequence of the threonine / homoserine export protein of the present application. Therefore, it is clear that the polynucleotide also includes polynucleotides that, due to codon degeneracy, can be translated into a polypeptide consisting of the amino acid sequence of the threonine / homoserine export protein of the present application or a polypeptide having homology or identity thereto. For example, a polynucleotide encoding the threonine / homoserine export protein of the present application may be SEQ ID NO: 2 or a degenerated sequence thereof.

[0115] As another example, the polynucleotide encoding the threonine / homoserine excretion protein of the present application may have or contain a base 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, or 99% or more homologous or identical to SEQ ID NO: 2, or may consist of or essentially consist of a base 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, or 99% or more homologous or identical to SEQ ID NO: 2, but is not limited thereto.

[0116] Furthermore, the polynucleotide of the present application may include, without limitation, a probe prepared from a known gene sequence, for example, a sequence that hybridizes under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present application and encodes the threonine / homoserine excretion protein of the present application.

[0117] Another aspect of the present application is to provide a vector comprising a polynucleotide of the present application.

[0118] The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0119] The vector is as described above.

[0120] Another aspect of the present application is to provide a microorganism comprising any one or more selected from the mutant threonine / homoserine excretion protein of the present application; a polynucleotide encoding the same; or a vector comprising the same.

[0121] The microorganism of the present application may be capable of producing an L-amino acid.

[0122] In the present application, the term "L-amino acid" includes all of L-homoserine, derivatives of L-homoserine, and L-amino acids biosynthesized using L-homoserine as a precursor.

[0123] In the present application, the "derivative of L-homoserine" may be, for example, O-acetyl-L-homoserine, O-succinyl-L-homoserine, etc., but is not necessarily limited thereto. Any derivative obtained during fermentation and in which a substituent is linked to the terminal oxygen of L-homoserine in the art is included within the scope of the present application.

[0124] In the present application, "L-amino acids biosynthesized using L-homoserine as a precursor" may be, for example, methionine, threonine, isoleucine, etc., which have L-homoserine as a precursor, but are not necessarily limited thereto, and all L-amino acids biosynthesized using L-homoserine as a precursor may be included in the scope of the present application.

[0125] For example, the "L-amino acid" may be any one or more selected from the group consisting of L-homoserine, O-acetyl-L-homoserine, O-succinyl-L-homoserine, L-methionine, and L-threonine.

[0126] For the purposes of this application, the microorganism of this application may be a microorganism containing a mutant threonine / homoserine export protein or a polynucleotide encoding the same; or a microorganism that has been genetically modified (e.g., a recombinant strain) to contain a mutant threonine / homoserine export protein or a polynucleotide encoding the same.

[0127] The strain of the present application may be a microorganism in which L-amino acid production ability has been imparted to a strain that naturally has the ability to produce L-amino acids or a strain that does not have the ability to produce L-amino acids, for example, but is not limited to, a microorganism in which L-amino acid production ability has been increased by introducing a mutant threonine / homoserine export protein of the present application or a polynucleotide encoding the same.

[0128] The strain of the present application may be a microorganism having increased L-amino acid production ability compared to a parent strain or a wild-type Corynebacterium strain not containing the mutant of the present application. The microorganism may have improved L-amino acid production ability due to the introduction of a mutant threonine / homoserine export protein of the present application, which has increased threonine / homoserine export activity compared to the wild-type threonine / homoserine export protein, or a polynucleotide encoding the same. Specifically, the strain of the present application may have increased L-amino acid production ability compared to a Corynebacterium microorganism containing a wild-type threonine / homoserine export protein having the amino acid sequence of SEQ ID NO: 1 or a polynucleotide encoding the same.

[0129] For example, a microorganism capable of producing an L-amino acid is a prokaryotic or eukaryotic microbial strain capable of producing an L-amino acid within the organism, and may include any microorganism that has an endogenous ability to produce an L-amino acid or a parent strain that does not have the ability to produce an L-amino acid, but has been genetically modified to contain a mutant threonine / homoserine export protein of the present application or a polynucleotide encoding the same, thereby conferring the ability to produce an L-amino acid due to increased threonine / homoserine export protein activity. The ability to produce an L-amino acid can be imparted or enhanced by species improvement.

[0130] As an example, the recombinant microorganism capable of producing an L-amino acid of the present application may include any microorganism that can produce an L-amino acid by being transformed with a vector and expressing a mutant threonine / homoserine export protein of the present application having increased threonine / homoserine export protein activity.

[0131] For example, the microorganism that produces the L-amino acid may be a microorganism that endogenously contains a protein consisting of the amino acid sequence of SEQ ID NO: 1, or a protein consisting of an amino acid sequence that has at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity to SEQ ID NO: 1.

[0132] For example, the microorganism that produces the L-amino acid may be a microorganism that endogenously contains a polynucleotide sequence that can encode a protein comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, the nucleotide sequence of SEQ ID NO: 2, or a nucleotide 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, or 99% or more homologous or identical to the nucleotide sequence of SEQ ID NO: 2.

[0133] The microorganisms of the present application may include all microorganisms in which the activity of a threonine / homoserine export protein is increased compared to its endogenous activity by expressing a mutant threonine / homoserine export protein of the present application having increased threonine / homoserine export protein activity by various known methods.

[0134] As one specific example, a microorganism in which the activity of the threonine / homoserine export protein of the present application is increased compared to its endogenous activity may be, but is not limited to, a microorganism in which the amino acid sequence of the threonine / homoserine export protein or the polynucleotide sequence encoding the same has been modified. The modification of the amino acid sequence or polynucleotide sequence can include deletion, substitution, insertion, and the like. The sequence modification is as described above. As another specific example, a microorganism in which the activity of the threonine / homoserine export protein of the present application is increased compared to its endogenous activity may be, but is not limited to, a microorganism in which the expression regulatory region (or expression regulatory sequence) of a gene on the chromosome encoding the threonine / homoserine export protein has been replaced with a sequence with stronger activity. The expression regulatory region may, for example, be a promoter. The replacement with a sequence with stronger activity is as described above. In yet another specific example, in a microorganism in which the activity of the threonine / homoserine export protein of the present application is increased compared to its endogenous activity, the increased activity of the threonine / homoserine export protein may be achieved by combining the above specific examples.

[0135] As an example of the present application, the microorganism of the present application may be one capable of producing an L-amino acid.

[0136] The increased threonine / homoserine excretion protein activity may be defined as, but is not limited to, increased L-amino acid production ability of the microorganism of the present application compared to the production ability of a natural wild-type microorganism or a non-transformed microorganism (e.g., a microorganism expressing a polypeptide having wild-type threonine / homoserine excretion protein activity (e.g., the polypeptide of SEQ ID NO: 1) or a strain in which the threonine / homoserine excretion protein activity of the present application has not been increased compared to its endogenous activity or has not yet been increased).

[0137] For example, the threonine / homoserine export protein activity can be measured by, but is not limited to, measuring the L-amino acid production ability or yield, for example, the threonine / homoserine export protein activity can be measured by, but is not limited to, the L-amino acid production ability described in Examples 2-4.

[0138] For example, the microorganism of the present application having increased L-amino acid production ability may be, but is not limited to, a microorganism having increased L-amino acid production ability compared to a non-modified microorganism. For example, the non-modified microorganism, which is the subject strain for comparing the increased L-amino acid production ability, may be, but is not limited to, a Corynebacterium glutamicum strain (Cgl-HS-2) having a hom mutation (G378E, R398Q) and a lysC mutation (L377K) and a Corynebacterium glutamicum strain (Cgl-HS-3) in which the promoter of the threonine / homoserine excretion protein has been replaced with the gapA promoter.

[0139] For example, the L-amino acid producing ability of the microorganism having increased L-amino acid producing ability is about 1% or more, specifically about 1% or more, about 10% or more, about 11.1% or more, about 20% or more, about 22.2% or more, about 30% or more, about 33.3% or more, about 40% or more, about 44.4% or more, about 50% or more, about 55.6% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 110% or more, about 120% or more, about 130% or more, about 140% or more, about 150% or more, about 160% or more, about 170% or more, about 177.8% or more, about 180% or more, The increase may be about 190% or more, about 200% or more, about 210% or more, about 220% or more, about 222.2% or more, about 230% or more, about 233.3% or more, about 240% or more, about 250% or more, or about 255.6% or more (there is no particular upper limit, and the increase may be, for example, about 300% or less, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, or about 15% or less), but is not limited thereto as long as there is an increase in the productivity compared to the parent strain or untransformed microorganism before mutation. In other examples, the recombinant strain having increased L-amino acid producing ability has an L-amino acid producing ability that is about 1.01-fold or more, about 1.1-fold or more, about 1.111-fold or more, about 1.2-fold or more, about 1.222-fold or more, about 1.3-fold or more, about 1.333-fold or more, about 1.4-fold or more, about 1.444-fold or more, about 1.5-fold or more, about 1.556-fold or more, about 1.6-fold or more, about 1.7-fold or more, about 1.8-fold or more, about 1.9-fold or more, about 2-fold or more, about 2.1-fold or more, about 2.2-fold or more, about 2.3-fold or more, about 2.4-fold or more, compared to the parent strain or unmodified microorganism before mutation. The increase may be, but is not limited to, about 2.5 times or more, about 2.6 times or more, about 2.7 times or more, about 2.778 times or more, about 2.8 times or more, about 2.9 times or more, about 3 times or more, about 3.1 times or more, about 3.2 times or more, about 3.222 times or more, about 3.3 times or more, about 3.333 times or more, about 3.4 times or more, about 3.5 times or more, or about 3.556 times or more (there is no particular restriction on the upper limit, and it may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, about 1.5 times or less, or about 1.2 times or less).

[0140] In yet another example of the present application, the microorganism of the present application is Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium The bacterial strain may be Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, and specifically may be Corynebacterium glutamicum, but is not limited thereto.

[0141] On the other hand, it has already been known that Corynebacterium microorganisms can produce L-amino acids such as L-homoserine, L-homoserine derivatives, or L-amino acids biosynthesized from L-homoserine as a precursor. However, their production capacity is significantly low, and the genes and mechanistic principles affecting the production mechanism have not yet been fully elucidated. Therefore, the Corynebacterium microorganisms of the present application having the ability to produce L-homoserine, L-homoserine derivatives, or L-amino acids biosynthesized using L-homoserine as a precursor may include all of the following: natural wild-type microorganisms themselves; Corynebacterium microorganisms that have improved ability to produce L-homoserine, L-homoserine derivatives, or L-amino acids biosynthesized using L-homoserine as a precursor by increasing or decreasing the activity of a gene related to the mechanism of L-amino acid production biosynthesized using L-homoserine as a precursor; and Corynebacterium microorganisms that have improved ability to produce L-homoserine, L-homoserine derivatives, or L-amino acids biosynthesized using L-homoserine as a precursor by introducing or increasing the activity of an exogenous gene.

[0142] The microorganism capable of producing an L-amino acid of the present application may be a microorganism in which the activity of homoserine dehydrogenase or lysine-sensitive aspartokinase 3 is further increased or decreased in the biosynthetic pathway of L-homoserine, an L-homoserine derivative, or an L-amino acid biosynthesized using L-homoserine as a precursor, or a microorganism in which the ability to produce L-homoserine, an L-homoserine derivative, or an L-amino acid biosynthesized using L-homoserine as a precursor is improved, but is not limited thereto.

[0143] Examples of the homoserine dehydrogenase or lysine-sensitive aspartokinase 3 mutations applicable to the present application are disclosed in Korean Patent Registration No. 10-2183209 or Korean Patent Publication No. 10-2019-0003019, respectively, the entire specifications of which are incorporated herein by reference.

[0144] Another aspect of the present application provides a method for producing an L-amino acid, comprising culturing in a medium a microorganism containing any one or more selected from the mutant threonine / homoserine excretion protein of the present application; a polynucleotide encoding the same; or a vector containing the same.

[0145] In the method of the present application, the microorganism can be cultured under any culture conditions and by any culture method known in the art, and such a culture process can be easily adjusted by a person skilled in the art depending on the selected strain.

[0146] The L-amino acids produced by the culture of the present invention are either secreted into the medium or remain intracellularly.

[0147] The L-amino acid may be at least one selected from the group consisting of L-homoserine, O-acetyl-L-homoserine, O-succinyl-L-homoserine, L-methionine, and L-threonine, as described above.

[0148] In one embodiment, the method for producing an L-amino acid of the present application may further include a step of preparing a 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.

[0149] The method for producing an L-amino acid of the present application may further include a step of recovering a target substance, specifically an L-amino acid, from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium. The recovery step may be further included after the culturing step.

[0150] The recovery may involve collecting the target L-amino acid using an appropriate 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. The target substance, specifically, the L-amino acid, can be recovered from the medium or the microorganism using an appropriate method known in the art.

[0151] The method for producing an L-amino acid of the present application may further include a purification step. The purification can be performed using an appropriate method known in the art. In one example, when the method for producing an L-amino 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.

[0152] In the method of the present application, the mutant threonine / homoserine excretion protein and the L-amino acid are as described above in other aspects.

[0153] Another aspect of the present application provides a composition for producing L-amino acids, comprising a microorganism containing any one or more selected from the mutant threonine / homoserine excretion protein of the present application; a polynucleotide encoding the same; or a vector containing the same; a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more of these.

[0154] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing L-amino acids, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonicity agents.

[0155] In one embodiment, each component present in the compositions of the present application may be included in a microbiologically effective amount, or in an amount that may be suitably present in a production composition.

[0156] In the composition of the present application, the mutant threonine / homoserine excretion protein and the L-amino acid are as described above in other aspects.

[0157] Another aspect of the present application provides use of the mutant threonine / homoserine excretion protein of the present application in the production of L-amino acids.

[0158] Another aspect of the present application provides use of a microorganism containing any one or more selected from the mutant threonine / homoserine excretion protein of the present application; a polynucleotide encoding the same; or a vector containing the same; for producing L-amino acids.

[0159] As used herein, the mutant threonine / homoserine excretion proteins and L-amino acids are as defined above in other aspects. [Example]

[0160] The present application will be described in more detail below with reference to 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 implemented by those of ordinary skill in the technical field of the present application or a similar technical field.

[0161] Example 1. Threonine / homoserine efflux protein (RhtA) mutation library screening Example 1-1. Construction of RhtA Mutation Library and Screening Strain To prepare a template for error-prone PCR, a wild-type rhtA gene fragment encoding RhtA was obtained by PCR using the genomic DNA of Escherichia coli W3110 as a template and the primer pair of SEQ ID NOs: 41 and 42. Furthermore, a promoter fragment of the rhtB gene was obtained by PCR using the genomic DNA of E. coli W3110 as a template and the primer pair of SEQ ID NOs: 43 and 44.

[0162] The PCR reaction uses the polymerase Solg TM The reaction was carried out using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes.

[0163] The primer sequences used here are as shown in Table 1 below.

[0164] [Table 1]

[0165] The resulting gene fragment was cloned into pCL1920 vector (Nucleic Acids Research, 18, (1990) 4631) digested with SmaI restriction enzyme using the Gibson assembly method (D.G. Gibson et al., NATURE METHOD, VOL. 6 NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain the recombinant plasmid pCL1920-PrhtB_rhtA(wt). Cloning was performed by mixing the Gibson assembly reagent with each gene fragment in the calculated molar amounts and storing at 50°C for 1 hour.

[0166] Error-prone PCR was performed to induce random mutagenesis in the wild-type rhtA gene, which encodes the threonine / homoserine excretion protein. The diversify PCR random mutagenesis kit (Takara) was used for the error-prone PCR. To select the mutation rate, error-prone PCR was performed under two conditions, as shown below, depending on the amount of MnSO added. pCL1920-PrhtB_rhtA(wt) was used as the DNA template for mutation introduction, and PCR was performed using the primer pair of SEQ ID NOs: 41 and 42. PCR was performed under the following conditions: denaturation at 95°C for 30 seconds, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 68°C for 30 seconds (25 cycles), followed by polymerization at 68°C for 60 seconds.

[0167] The composition of the composition for performing error-prone PCR is shown in Table 2 below.

[0168] [Table 2]

[0169] The error-prone PCR product was treated with DpnI to remove the template plasmid, and the resulting DNA was cloned into pCL1920 vector cleaved with SmaI restriction enzyme using the Gibson assembly method to obtain the recombinant mutant plasmid library pCL1920-PrhtB_rhtA(mt).

[0170] Example 1-2. Construction of strains for productivity screening of RhtA mutant libraries To prepare the W3110(△rhtABC) strain for library screening, we first performed PCR using E. coli genomic DNA as a template and the primer pair of SEQ ID NOs: 45 and 46 and the primer pair of SEQ ID NOs: 47 and 48 to delete the rhtBC gene, thereby amplifying homologous region fragments upstream and downstream of rhtBC, respectively.

[0171] The rhtBC upstream and downstream homology fragments obtained in this process were cloned into pSKH130 vector cleaved with SmaI restriction enzyme using the Gibson assembly method to obtain the recombinant plasmid pSHK130△rhtBC. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated molar amounts and storing them at 50°C for 1 hour.

[0172] Next, to delete the rhtA gene, PCR was performed using the genomic DNA of the E. coli W3110 strain as a template and the primer pair of SEQ ID NOs: 49 and 50 and the primer pair of SEQ ID NOs: 51 and 52 to amplify homologous region fragments upstream and downstream of rhtA, respectively.

[0173] The rhtA upstream and downstream homologous region fragments obtained in the above process were cloned into pSKH130 vector cleaved with SmaI restriction enzyme using the Gibson assembly method to obtain the recombinant plasmid pSHK130△rhtA.

[0174] The PCR reaction uses the polymerase Solg TM The reaction was carried out using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, and polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes.

[0175] The constructed pSHK130△rhtA and pSHK130△rhtBC vectors were transformed into the E. coli W3110 strain by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), and then a second crossover process was carried out to obtain the W3110(△rhtABC) strain, in which the rhtABC genes were deleted on the chromosome.

[0176] PCR and genome sequencing were performed using the primer pair of sequence numbers 53 and 54, and the primer pair of sequence numbers 55 and 56, which can respectively amplify the external sites of the upstream and downstream regions of the homologous recombination where the gene was inserted, to confirm the genetic manipulation.

[0177] The primer sequences used here are as shown in Table 3 below.

[0178] [Table 3]

[0179] Example 1-3. Productivity screening of RhtA mutant library The mutant libraries pCL1920-PrhtB_rhtA(mt) and pCL1920-PrhtB_rhtA(wt), obtained in Example 1-1, were transformed into the W3110(ΔrhtABC) strain and cultured on LB plates containing 50 μg / L spectinomycin. 50 colonies were selected from the W3110(ΔrhtABC) strain transformed with the mutant library and sequenced to determine the mutation rate and whether mutations occurred at various positions.

[0180] As a result of sequencing, the mutation rate under case #1 conditions in Example 1-1 was 1.2 kb -1 , 2.0 kb under case #2 conditions -1Based on this, it was determined that both cases #1 and #2 had mutation rates suitable for securing a mutant library, and effective mutations were selected using the library constructed under the above conditions.

[0181] The W3110(△rhtABC) strains transformed with pCL1920-PrhtB_rhtA(mt) and pCL1920-PrhtB_rhtA(wt) were inoculated into 96-Deep Well Plate-Dome (Bioneer) containing 400 μL of seed medium, and cultured in a plate shaking incubator (TAITEC) at 32°C and 12,000 rpm for approximately 12 hours.

[0182] <Seed medium (pH 7.2)> Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4·7H2O 0.5g, biotin 100μg, thiamine HCl 1000μg, calcium pantothenate 2000μg, nicotinamide 2000μg

[0183] For the primary screening of approximately 1,000 cultured colonies, each colony was serially diluted in seed medium containing 200 g / L of L-homoserine and subjected to MIC (minimum inhibitory concentration) testing. 29 colonies were found to have significantly higher MIC values ​​than the control strain, W3110(△rhtABC) transformed with pCL1920-PrhtB_rhtA(wt).

[0184] Each of the obtained colonies was inoculated into a 96-deep well plate-dome containing 400 μL of seed medium, and cultured in a plate shaking incubator at 32° C. and 12,000 rpm for approximately 12 hours.

[0185] For secondary screening of the 29 cultured colonies, the final cultured colonies were matched to the initial optical density (OD) values ​​and serially diluted in seed medium containing 200 g / L L-homoserine for MIC testing. Seven colonies were selected that showed significantly higher MIC values ​​than the control strain, W3110(△rhtABC) (K12 / pCL1920-Pn_rhtA) transformed with pCL1920-PrhtB_rhtA(wt) (Table 4).

[0186] [Table 4]

[0187] The pCL1920-Pn_rhtA mutant plasmid was extracted from the seven selected colonies and then sequenced to confirm the mutation. As a result, it was confirmed that a mutation had occurred in the coding sequence (CDS).

[0188] The mutant plasmids derived from the seven selected colonies were named pCL1920-PrhtB_rhtA(m1), pCL1920-PrhtB_rhtA(m2), pCL1920-PrhtB_rhtA(m3), pCL1920-PrhtB_rhtA(m4), pCL1920-PrhtB_rhtA(m5), pCL1920-PrhtB_rhtA(m6), and pCL1920-PrhtB_rhtA(m7), respectively. Specifically, pCL1920-PrhtB_rhtA(m1) was confirmed to contain the rhtA(T260A) mutation, pCL1920-PrhtB_rhtA(m2) was confirmed to contain the rhtA(S183A) mutation, pCL1920-PrhtB_rhtA(m3) was confirmed to contain the rhtA(V123A / I193V) mutation, pCL1920-PrhtB_rhtA(m4) was confirmed to contain the rhtA(F66Y) mutation, pCL1920-PrhtB_rhtA(m5) was confirmed to contain the rhtA(V15A / A229V) mutation, pCL1920-PrhtB_rhtA(m6) was confirmed to contain the rhtA(L210S) mutation, and pCL1920-PrhtB_rhtA(m7) was confirmed to contain the rhtA(P119T) mutation.

[0189] Example 1-4. Construction of RhtA mutant plasmid for introduction into Corynebacterium sp. strains To introduce the RhtA mutations selected in Examples 1-3 into Corynebacterium sp. strains and compare and evaluate RhtA activity, rhtA mutant plasmids were constructed as follows.

[0190] First, for homologous recombination, PCR was performed using the genomic DNA of wild-type Corynebacterium glutamicum ATCC13032 strain as a template and the primer pair of SEQ ID NOs: 57 and 58 and the primer pair of SEQ ID NOs: 63 and 64 to amplify the upstream and downstream regions of Ncgl0998, respectively. To utilize the gapA promoter as the promoter for the mutant rhtA gene, PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and the primer pair of SEQ ID NOs: 59 and 60 to amplify the gapA promoter. In addition, the mutant plasmids pCL1920-PrhtB_rhtA(m1), pCL1920-PrhtB_rhtA(m2), pCL1920-PrhtB_rhtA(m3), pCL1920-PrhtB_rhtA(m4), pCL1920-PrhtB_rhtA(m5), pCL1920-PrhtB_rhtA(m6), pCL1920 PCR was performed using the primer pair of SEQ ID NO: 61 and SEQ ID NO: 62 with -PrhtB_rhtA(m7) as a template to amplify the mutant rhtA fragments from the mutant plasmids selected in Examples 1-3, thereby obtaining fragments of rhtA, rhtA(m1), rhtA(m2), rhtA(m3), rhtA(m4), rhtA(m5), rhtA(m6), and rhtA(m7).

[0191] The PCR reaction uses the polymerase Solg TM The reaction was carried out using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes.

[0192] The primer sequences used here are as shown in Table 5 below.

[0193] [Table 5]

[0194] The gene fragments obtained in this process were cloned into pDCM2 vector (Korean Publication No. 10-2020-0136813) digested with SmaI restriction enzyme using the Gibson assembly method to obtain eight recombinant plasmids containing wild-type rhtA or mutant rhtA. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated molar amounts and storing at 50°C for 1 hour. The eight recombinant plasmids constructed were named pDCM2-PgapA_rhtA, pDCM2-PgapA_rhtA(m1), pDCM2-PgapA_rhtA(m2), pDCM2-PgapA_rhtA(m3), pDCM2-PgapA_rhtA(m4), pDCM2-PgapA_rhtA(m5), pDCM2-PgapA_rhtA(m6), and pDCM2-PgapA_rhtA(m7), respectively.

[0195] Example 2. Construction of an L-homoserine-producing strain Example 2-1. Construction of homoserine dehydrogenase (hom) mutant strain We attempted to increase L-homoserine production by introducing mutations (G378E, R398Q) (Korean Patent Registration No. 10-2183209) into a Corynebacterium strain to remove feedback inhibition of hom on L-homoserine. To this end, a hom mutant plasmid was constructed as follows.

[0196] First, PCR was performed using the genomic DNA of the wild-type Corynebacterium glutamicum ATCC13032 strain as a template and the primer pair of SEQ ID NO: 65 and SEQ ID NO: 66 to amplify a fragment of the promoter upstream region of the hom gene.

[0197] To apply the hom mutations (G378E, R398Q), we obtained an upstream fragment of the CDS gene encoding the hom protein containing amino acids 1 to 378 from the N-terminus, the hom G378E / R398Q gene fragment, and a downstream fragment of the hom R398Q gene, which undergoes homologous recombination on the chromosome. Specifically, PCR was performed using the genomic DNA of wild-type Corynebacterium glutamicum ATCC13032 strain as a template and the primer pairs of SEQ ID NOs: 67 and 68, 69 and 70, and 71 and 72 to obtain the upstream fragment of the coding sequence containing amino acids 1 to 378 from the N-terminus of the hom protein, the G378E / R398Q gene fragment, and the downstream fragment of the R398Q gene, respectively.

[0198] The PCR reaction uses the polymerase Solg TM The reaction was carried out using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes.

[0199] The gene fragments obtained in this process were cloned into pDCM2 vector cleaved with SmaI restriction enzyme using the Gibson assembly method to obtain a recombinant plasmid. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated molar amounts and storing at 50°C for 1 hour. The resulting recombinant plasmid was named pDCM2-hom(G378E, R398Q).

[0200] The constructed pDCM2-hom(G378E, R398Q) vector was transformed into the wild-type Corynebacterium glutamicum ATCC13032 strain by electroporation, and then a second crossover process was carried out to obtain a strain in which the wild-type hom gene was replaced with the mutant hom(G378E, R398Q) gene on the chromosome.

[0201] The genetic manipulation was confirmed by PCR and genome sequencing using the primer pair of sequence numbers 73 and 74, which can respectively amplify the external sites of the upstream and downstream regions of the homologous recombination where the gene was inserted.

[0202] The primer sequences used here are as shown in Table 6 below.

[0203] [Table 6]

[0204] The transformed strain obtained above was designated Cgl-HS-1.

[0205] Example 2-2. Construction of lysine-sensitive aspartokinase 3 (LysC) mutant strain We decided to introduce a mutation (L377K) into a Corynebacterium strain to enhance expression of the lysC gene and to release feedback inhibition of L-lysine and L-homoserine (Korean Patent Publication No. 10-2019-0003019).

[0206] First, we obtained the upstream region of the lysC gene promoter and the upstream and downstream regions of the lysC L377K gene, where homologous recombination occurs on the chromosome. Specifically, PCR was performed using the genomic DNA of the wild-type Corynebacterium glutamicum ATCC13032 strain as a template and the primer pairs of SEQ ID NOs: 75 and 76, 77 and 78, and 79 and 80 to obtain gene fragments for the upstream region of the lysC gene promoter, the downstream region of the lysC L377K gene, and the upstream region of the lysC L377K gene, respectively.

[0207] The PCR reaction uses the polymerase Solg TMThe reaction was carried out using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 60 seconds, repeated 30 times, followed by polymerization at 72°C for 5 minutes.

[0208] The gene fragments obtained in this process were cloned into pDCM2 vector cleaved with SmaI restriction enzyme using the Gibson assembly method to obtain a recombinant plasmid. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated molar amounts and storing at 50°C for 1 hour. The resulting recombinant plasmid was named pDCM2-_lysC L377K.

[0209] The constructed pDCM2-_lysC L377K vector was transformed into the Cgl-HS-1 strain of Example 2-1 by electroporation, and then a secondary crossover process was carried out to obtain a strain in which the wild-type lysC gene was replaced with the mutant lysC (L377K) gene on the chromosome.

[0210] The genetic manipulation was confirmed through PCR and genome sequencing using primers of SEQ ID NO: 81 and SEQ ID NO: 82, which can amplify the upstream and downstream regions of the homologous recombination where the gene was inserted, respectively.

[0211] The primer sequences used here are as shown in Table 7 below.

[0212] [Table 7]

[0213] The transformed strain obtained above was designated Cgl-HS-2.

[0214] Example 2-3. Construction of Corynebacterium strains with RhtA mutations The pDCM2-PgapA_rhtA, pDCM2-PgapA_rhtA(m1), pDCM2-PgapA_rhtA(m2), pDCM2-PgapA_rhtA(m3), pDCM2-PgapA_rhtA(m4), pDCM2-PgapA_rhtA(m5), pDCM2-PgapA_rhtA(m6), and pDCM2-PgapA_rhtA(m7) vectors constructed in Example 1-4 were transformed into the Cgl-HS-2 strain of Example 2-2 by electroporation, and then a secondary crossover process was carried out to obtain strains having seven wild-type rhtA or mutant rhtA genes inserted on the chromosome. The genetic manipulation was confirmed through PCR and genome sequencing using the primer pair of SEQ ID NO: 83 and SEQ ID NO: 84, which can respectively amplify the upstream and downstream regions of the homologous recombination where the gene was inserted.

[0215] The primer sequences used here are as shown in Table 8 below.

[0216] [Table 8]

[0217] The transformed strains obtained above were designated Cgl-HS-3, Cgl-HS-3(m1), Cgl-HS-3(m2), Cgl-HS-3(m3), Cgl-HS-3(m4), Cgl-HS-3(m5), Cgl-HS-3(m6), and Cgl-HS-3(m7), respectively.

[0218] Example 2-4. L-homoserine production ability of Corynebacterium strains with RhtA mutations To compare the L-homoserine production of Cgl-HS-2 (Example 2-2) and Cgl-HS-3, Cgl-HS-3(m1), Cgl-HS-3(m2), Cgl-HS-3(m3), Cgl-HS-3(m4), Cgl-HS-3(m5), Cgl-HS-3(m6), and Cgl-HS-3(m7) (Example 2-3), they were cultured as follows. Specifically, each strain was inoculated into a 250 ml corner-baffled flask containing 25 ml of production medium and cultured at 30°C for 24 hours with shaking at 200 rpm. After the culture was completed, the L-homoserine production was measured by HPLC, and the results are shown in Table 9.

[0219] <Production medium (pH 7.0)> Glucose 45g, (NH4)2SO4 20g, MgSO4·7H2O 1.2g, KH2PO4 1.1g, biotin 900μg, thiamine hydrochloride 4500μg, calcium pantothenate 4500μg, CaCO3 30g (based on 1 liter of distilled water)

[0220] [Table 9]

[0221] As shown in Table 9, the Cgl-HS-2 strain of Example 2-2 produced 0.3 g / L of L-homoserine, and the Cgl-HS-3 strain, which was transformed with the PgapA_rhtA wild-type trait and used the Cgl-HS-2 strain as a parent strain, produced 0.9 g / L of L-homoserine. Furthermore, the Cgl-HS-3(m1), Cgl-HS-3(m2), Cgl-HS-3(m3), Cgl-HS-3(m4), Cgl-HS-3(m5), Cgl-HS-3(m6), and Cgl-HS-3(m7), which were transformed with the PgapA_rhtA mutant trait, produced 1.9 g / L, 2.5 g / L, 3.2 g / L, 1.7 g / L, 1.1 g / L, 1.0 g / L, and 3.0 g / L of L-homoserine, respectively.

[0222] The L-homoserine fermentation yield of Cgl-HS-3, which was transformed with the wild-type PgapA_rhtA gene, was increased by 1.2% compared to Cgl-HS-2. Meanwhile, the L-homoserine fermentation yield of Cgl-HS-3(m1), Cgl-HS-3(m2), Cgl-HS-3(m3), Cgl-HS-3(m4), Cgl-HS-3(m5), Cgl-HS-3(m6), and Cgl-HS-3(m7), which were transformed with the PgapA_rhtA mutant gene, was increased by 3%, 4.4%, 5.8%, 2.8%, 1.6%, 1.4%, and 5.4%, respectively, compared to Cgl-HS-2. In particular, the yield increase rates of Cgl-HS-3(m3) and Cgl-HS-3(m7) over Cgl-HS-2 were approximately 4.8-fold and 4.5-fold, respectively, compared to the yield increase rate of Cgl-HS-3 over Cgl-HS-2.

[0223] Example 3. rhtA Saturated Mutagenesis Since the Cgl-HS-3(m3) strain containing the rhtA(V123A) mutation was found to have superior L-homoserine production ability compared to a strain expressing wild-type rhtA in Examples 1-3 and Table 9, we decided to mutate valine, the 123rd amino acid in the amino acid sequence of RhtA, with another amino acid to examine the effectiveness of improving L-homoserine excretion at position 123. To mutate valine, the 123rd amino acid in the amino acid sequence of RhtA, with one of 19 amino acids other than valine, site-directed mutagenesis was performed as follows.

[0224] Specifically, PCR was performed using pDCM2-PgapA_rhtA from Example 1-4 as a template, with the PCR composition prepared according to Table 10 below. PCR was performed under the following conditions: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 1 minute, and polymerization at 68°C for 10 minutes, repeated 30 times. The mutagenic primer set listed in Table 11 below was used in PCR.

[0225] [Table 10]

[0226] [Table 11] JPEG2026507874000012.jpg225149 JPEG2026507874000013.jpg204149

[0227] The resulting gene fragments were treated with 1 μl of DpnI restriction enzyme at 37°C for 1 hour. Each DpnI-treated gene fragment was cloned into the pDCM2 vector using the Gibson assembly method to obtain recombinant mutant plasmids. 3 μl of recombinant plasmid DNA was then transferred to DH5a competent cells. pDCM2-PgapA_rhtA mutant plasmids were obtained by transforming them into rhtA(V123A), rhtA(V123C), rhtA(V123D), rhtA(V123E), rhtA(V123F), rhtA(V123G), rhtA(V123H), rhtA(V123I), rhtA(V123K), rhtA(V123L), rhtA(V123M), rhtA(V123N), rhtA(V123P), rhtA(V123Q), rhtA(V123R), rhtA(V123S), rhtA(V123T), rhtA(V123W), and rhtA(V123Y) mutations.

[0228] pDCM2-PgapA_rhtA V123A, pDCM2-PgapA_rhtA V123C, pDCM2-PgapA_rhtA V123D, pDCM2-PgapA_rhtA V123E, pDCM2-PgapA_rhtA produced above V123F, pDCM2-PgapA_rhtA V123G, pDCM2-PgapA_rhtA V123H, pDCM2-PgapA_rhtA V123I, pDCM2-PgapA_rhtA V123K, pDCM2-PgapA_rhtA V123L, pDCM2-PgapA_rhtA V123M, pDCM2-PgapA_rhtA V123N, pDCM2-PgapA_rhtA The Cgl-HS-3 strain was transformed by electroporation with the pDCM2-PgapA_rhtA V123P, pDCM2-PgapA_rhtA V123Q, pDCM2-PgapA_rhtA V123R, pDCM2-PgapA_rhtA V123S, pDCM2-PgapA_rhtA V123T, pDCM2-PgapA_rhtA V123W, and pDCM2-PgapA_rhtA V123Y vectors as described in Example 2-3, and then subjected to a secondary crossover process to obtain strains with 19 mutant rhtA genes inserted into the chromosome. The genetic manipulation was confirmed by PCR and genome sequencing using primer pairs of SEQ ID NOs: 83 and 84, which can amplify the upstream and downstream regions of the homologous recombination region where the genes were inserted, respectively.

[0229] The transformed strains obtained above were designated as Cgl-HS-3(V123A), Cgl-HS-3(V123C), Cgl-HS-3(V123D), Cgl-HS-3(V123E), Cgl-HS-3(V123F), Cgl-HS-3(V123G), Cgl-HS-3(V123H), Cgl-HS-3(V123I), Cgl-HS-3(V123K), and Cgl-HS-3(V123H). gl-HS-3(V123L), Cgl-HS-3(V123M), Cgl-HS-3(V123N), Cgl-HS-3(V123P), Cgl-HS-3(V123Q), Cgl -HS-3(V123R), Cgl-HS-3(V123S), Cgl-HS-3(V123T), Cgl-HS-3(V123W), and Cgl-HS-3(V123Y).

[0230] Example 4. L-homoserine production ability of Corynebacterium strains with RhtA mutations To compare the L-homoserine production amounts of Cgl-HS-2 in Example 2-2, Cgl-HS-3 in Example 2-3, and the strain constructed in Example 3, they were cultured in the same manner as in Example 2-4. After the culture was completed, the L-homoserine production amount was measured by HPLC, and the results are shown in Table 12 below.

[0231] [Table 12]

[0232] As shown in Table 12, the Cgl-HS-3 strain into which the rhtA(V123A), rhtA(V123C), rhtA(V123D), rhtA(V123F), rhtA(V123G), rhtA(V123H), rhtA(V123I), rhtA(V123L), rhtA(V123M), rhtA(V123N), rhtA(V123Q), rhtA(V123S), rhtA(V123T), rhtA(V123W), and rhtA(V123Y) mutations were introduced showed increased L-homoserine fermentation yield compared to the Cgl-HS-3 strain into which the PgapA_rhtA wild-type trait was introduced. As a result, it was confirmed that strains expressing RhtA in which the 123rd amino acid in the amino acid sequence of RhtA, valine, is mutated with alanine, cysteine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, serine, threonine, tryptophan, or tyrosine, produce more L-homoserine than strains expressing wild-type RhtA.

[0233] Example 5. Threonine-producing ability of RhtA-mutated Corynebacterium strains To compare the threonine production among the Cgl-HS-2 strain of Example 2-2, the Cgl-HS-3 strain of Example 2-3, and the Cgl-HS-3 strain of Example 4, which contains a mutant in which valine, the 123rd amino acid in the amino acid sequence of RhtA, is mutated with another amino acid, they were cultured according to the method of Example 2-4, and Cgl-HS-3 (V123A) was used as a representative Cgl-HS-3 strain containing the mutant, which is listed in Table 12. After the cultivation was completed, the threonine production was measured by HPLC, and the results are shown in Table 13 below.

[0234] [Table 13]

[0235] As shown in Table 13, the Cgl-HS-3 strain into which the V123A mutation was introduced had a higher threonine fermentation yield than the Cgl-HS-3 strain into which the wild-type PgapA_rhtA trait was introduced. This confirmed that the strain expressing RhtA in which the 123rd amino acid, valine, is substituted with another amino acid is superior to the strain expressing wild-type RhtA in threonine production.

[0236] From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including within the meaning and scope of the claims below, and any modifications or variations derived from the equivalent concepts thereof, rather than the above detailed description.

Claims

1. A mutant threonine / homoserine excretion protein in which the amino acid corresponding to position 123 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid.

2. The mutant threonine / homoserine export protein of claim 1, wherein the other amino acid is an amino acid selected from the group consisting of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, tryptophan, and tyrosine.

3. The mutant threonine / homoserine export protein of claim 2, wherein the other amino acid is an amino acid selected from the group consisting of alanine, cysteine, aspartic acid, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, serine, threonine, tryptophan, and tyrosine.

4. The mutant threonine / homoserine excretion protein of claim 1, wherein the protein has a sequence identity of 80% or more but less than 100% with SEQ ID NO:

1.

5. A polynucleotide encoding the mutant threonine / homoserine excretion protein according to any one of claims 1 to 3.

6. The polynucleotide of claim 5, wherein the polynucleotide has a sequence identity of 80% or more but less than 100% with SEQ ID NO:

2.

7. A microorganism comprising one or more of the following: a mutant threonine / homoserine excretion protein in which the amino acid corresponding to position 123 in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid; a polynucleotide encoding the same; or a vector containing the same.

8. The microorganism according to claim 7, wherein the microorganism has an increased ability to produce L-amino acids compared to a microorganism containing a wild-type threonine / homoserine excretion protein having the amino acid sequence of SEQ ID NO: 1 or a polynucleotide encoding the same.

9. 8. The microorganism according to claim 7, wherein the L-amino acid is at least one selected from the group consisting of L-homoserine, O-acetyl-L-homoserine, O-succinyl-L-homoserine, L-methionine, and L-threonine.

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

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

12. A method for producing an L-amino acid, comprising the step of culturing in a medium a microorganism containing one or more selected from the following: a mutant threonine / homoserine excretion protein in which the amino acid corresponding to position 123 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid; a polynucleotide encoding the same; or a vector containing the same.

13. 13. The method of claim 12, wherein the L-amino acid is at least one selected from the group consisting of L-homoserine, O-acetyl-L-homoserine, O-succinyl-L-homoserine, L-methionine, and L-threonine.

14. Use of a mutant threonine / homoserine excretion protein in which the amino acid corresponding to position 123 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid for L-amino acid production.

15. Use of a microorganism containing one or more of the following for producing L-amino acids: a mutant threonine / homoserine excretion protein in which the amino acid corresponding to position 123 in the amino acid sequence of SEQ ID NO: 1 has been replaced with another amino acid; a polynucleotide encoding the same; or a vector containing the same.

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