KR20240143214A - A microorganism having Threonine/homoserine exporter variant and a method for producing L-amino acid using the same - Google Patents

Abstract

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

Description

{A microorganism having Threonine/homoserine exporter variant and a method for producing L-amino acid using the same}

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

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

In order to produce L-amino acids such as L-homoserine and other useful substances, various studies are being conducted to develop high-efficiency production microorganisms and fermentation process technologies. For example, target substance-specific approaches such as increasing the expression of genes encoding enzymes involved in L-homoserine biosynthesis or removing genes unnecessary for biosynthesis are mainly used.

However, research is still needed to effectively increase the production of L-amino acids, including L-homoserine, in microorganisms.

One embodiment of the present application provides a mutant threonine/homoserine exporter protein, wherein the amino acid corresponding to position 119 in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid.

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

Another aspect of the present application provides a polynucleotide encoding a variant threonine/homoserine exporter protein of the present application.

Another aspect of the present application provides a microorganism comprising at least one selected from the group consisting of a variant threonine/homoserine exporter protein of the present application; a polynucleotide encoding the same; or a vector comprising the same.

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

In another specific example, 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.

A microorganism according to any one of the preceding specific examples, wherein the microorganism may be a microorganism of the genus Corynebacterium.

As a microorganism according to any one of the preceding specific examples, the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum.

Another aspect of the present application provides a method for producing L-amino acid, comprising the step of culturing a microorganism comprising at least one selected from the group consisting of a variant threonine/homoserine exporter protein of the present application; a polynucleotide encoding the same; or a vector comprising the same; in a medium.

Another aspect of the present application provides a composition for producing L-amino acid, comprising a microorganism comprising at least one selected from the group consisting of a variant threonine/homoserine efflux protein of the present application; a polynucleotide encoding the same; or a vector comprising the same; a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof.

Hereinafter, this will be described in detail. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of this application is not limited by the specific description described below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated into this specification as a reference in their entirety so that the level of the technical field to which this application belongs and the content of this application are more clearly explained.

definition

As used in the specification and the appended claims of this application, the singular articles "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless the context clearly dictates otherwise, the singular terms include the plural and the plural terms include the singular. In the specification and the appended claims of this application, unless the context clearly dictates otherwise, the use of "or" is intended to include "and/or."

In this application, the term "about" may be presented before a specific numerical value. The term "about" as used in this application includes not only the exact number described after the term, but also a range that is or is nearly that number. Whether the number is or is nearly the specific number referred to may be determined based on the context in which it is presented. As an example, the term "about" may refer to a range of -10% to +10% of a numerical value. As another example, the term "about" may refer to a range of -5% to +5% of a given numerical value. However, the present invention is not limited thereto.

In this application, the terms "first, second, third," "i), ii), iii)..." or "(a), (b), (c), (d)..." are used to distinguish similar configurations, and these terms do not imply that they are performed consecutively or in order. For example, when the terms are used in connection with steps of a method, use, or analysis, these steps may be performed simultaneously, or may be performed at intervals of seconds, minutes, hours, days, or months.

In this application, the term "consisting essentially of" means that a non-specified component may be present, provided that the characteristics of the subject matter claimed in this application are not substantially affected by the presence of the non-specified component.

In this application, the term "consisting of" means that the proportion of a specific component(s) totals 100%. The components or features listed below the term "consisting of" may be essential or mandatory. In some embodiments, other than the components or features listed below "consisting of," other optional or non-essential components may be excluded.

In this application, the term "comprising" means the presence of the features, steps or components described below the term, and does not exclude the presence or addition of one or more features, steps or components. The components or features described below "comprising" in this application may be essential or mandatory, but in some embodiments, other optional or non-essential components or features may be further included.

Polypeptide

In this application, the term "protein" or "polypeptide" means a polymer or oligomer of consecutive amino acid residues. In this application, "polypeptide," "protein," and "peptide" may be used interchangeably with "amino acid sequence."

In some cases, an amino acid sequence that exhibits activity may be referred to as an "enzyme." In this application, amino acid sequences are described in N-terminal → C-terminal orientation unless otherwise indicated.

In this application, the term "mature polypeptide" means a polypeptide in a form without a signal sequence or a propeptide sequence. The mature protein/polypeptide/peptide can be a functional form of the protein/polypeptide/peptide. The mature polypeptide can be a final form that has undergone translational or post-translational modifications. Examples of post-translational modifications include, but are not limited to, N- or C-terminal modifications, glycosylation, phosphorylation, and leader sequence removal.

In the present application, with respect to an amino acid sequence, even if it is described as a polypeptide "comprising" an amino acid sequence set forth in a specific sequence number, a polypeptide "consisting of" an amino acid sequence set forth in a specific sequence number, or a polypeptide or protein "having" an amino acid sequence set forth in a specific sequence number, it is obvious that a protein having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted or added is also included within the scope of the present application if it has the same or corresponding activity as a protein consisting of the amino acid sequence of the corresponding sequence number. For example, if it has the same or corresponding activity as the above-mentioned mutant protein, it does not exclude addition of a sequence that does not change the function of the protein before or after the above-mentioned amino acid sequence, a mutation that may occur naturally, a silent mutation or conservative substitution thereof, and it is obvious that even if it has such sequence addition or mutation, it falls within the scope of the present application.

For example, the polypeptide of the present invention may have sequence additions, naturally occurring mutations, silent mutations or conservative substitutions at the N-terminus, C-terminus and/or internally of the amino acid sequence that do not alter the function of the polypeptide. For example, the polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus of the protein that is involved in the transfer of the protein co-translationally or post-translationally. The polypeptide may also be conjugated to other sequences or linkers so that the polypeptide can be identified, purified or synthesized.

In this application, the term "conservative substitution" means replacing one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids having nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; Amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, amino acids with charged side chains include arginine, lysine, histidine, glutamic acid, and aspartic acid, and amino acids with uncharged side chains include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. As another example, valine, leucine, and isoleucine can be classified as branched amino acids. As another example, the 20 amino acids can be classified into five groups based on size, starting from a group of amino acids with relatively small volume: glycine, alanine, and serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, and glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, and tyrosine. However, this is not necessarily limited to these. Typically, conservative substitutions may have little or no effect on the activity of the polypeptide.

polynucleotide

In this application, the term "gene" means a polynucleotide that encodes a polypeptide and a polynucleotide comprising regions preceding and following the coding region. In some embodiments, a gene may have a sequence (intron) inserted between each coding region (exon).

The term "polynucleotide, nucleic acid or nucleic acid molecule" in this application means a polymer of nucleotides in which nucleotide units (monomers) are covalently bonded to form a long chain, and a strand of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA) of a certain length or longer.

Homology, identity

As used herein, the terms "homology" or "identity" refer to the degree to which two given amino acid sequences or base sequences are related, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be utilized together with a default gap penalty established by the program being used. In practice, homologous or identical sequences are generally capable of hybridizing under moderate or high stringency conditions along at least about 50%, 60%, 70%, 80% or 90% of the entire or full-length sequence. It will be appreciated that hybridization also includes polynucleotides containing common codons or codons that are considered codon degeneracy in the polynucleotide.

Whether any two polynucleotide or polypeptide sequences are homologous, similar or identical can be determined using well-known computer algorithms such as the "FASTA" program using default parameters as in, for example, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, 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) can be determined using the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [Ed.,] Academic Press, San Diego,1994, and [CARILLO et al .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST, or ClustalW from the National Center for Biotechnology Information Database.

Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482, or using, for example, the GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443. In brief, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). The default parameters for the GAP program are (1) unitary matrices (containing values of 1 for identity and 0 for non-identity), the PAM Matrix (as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation (1978)), Gribskov et al(1986) Nucl. Acids Res. 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or gap opening penalty 10, gap extension penalty 0.5); and (3) no penalty for terminal gaps.

Additionally, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by comparing the sequences by Southern hybridization experiments under defined stringent conditions, and the defined appropriate hybridization conditions are within the scope 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.

In this application, the term "stringent conditions" means 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, the conditions under which polynucleotides having high homology or identity hybridize with each other, or polynucleotides 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 hybridize, and polynucleotides having lower homology or identity do not hybridize, or the conditions of washing once, specifically twice to three times, at a salt concentration and temperature corresponding to 60°C, 1ХSSC, 0.1% SDS, specifically 60°C, 0.1ХSSC, 0.1% SDS, and more specifically 68°C, 0.1ХSSC, 0.1% SDS, which are washing conditions of conventional southern hybridization, can be listed.

The above hybridization requires that the two nucleotides have complementary sequences, although mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present application may also include isolated nucleic acid fragments that are complementary in their entirety, as well as substantially similar base sequences.

For example, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. In addition, the Tm value may be, but is not limited to, 60°C, 63°C, or 65°C and can be appropriately adjusted by a person skilled in the art depending on the purpose.

The appropriate stringency to hybridize the above polynucleotides depends on the length and degree of complementarity of the polynucleotides, variables which are well known in the art (e.g., J. Sambrook et al., supra).

Nucleic acid structures, vectors, transformation

The term "nucleic acid construct" in this application means a single or double-stranded nucleic acid molecule that contains one or more regulatory sequences and is artificially synthesized, engineered to contain a specific sequence in a way that does not exist in nature, or isolated from nature.

The term "vector" as used in this application means a DNA construct comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating the termination of transcription and translation. The vector, after being transformed into a suitable host cell, may replicate or function independently of the host genome, and may be integrated into the genome itself.

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

For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome through a vector for intracellular chromosome insertion. The insertion of the polynucleotide into the chromosome can be accomplished by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker for confirming whether the chromosome has been inserted can be additionally included. The selection marker is for selecting cells transformed with the vector, that is, confirming whether the target nucleic acid molecule has been inserted, and markers that confer a selectable phenotype, such as drug resistance, nutrient requirement, resistance to cytotoxic agents, or expression of a surface polypeptide, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, so that the transformed cells can be selected.

In this application, the term "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide can include both the position of being inserted into the chromosome of the host cell or being located outside the chromosome, as long as it can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding the target polypeptide. The polynucleotide can be introduced in any form as long as it can be introduced into the host cell and expressed. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for self-expression. The expression cassette can typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal that are operably linked to the polynucleotide. The expression cassette can be in the form of an expression vector capable of self-replication. Additionally, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

As used herein, the term "operably linked" means a configuration in which a regulatory sequence is positioned at an appropriate location such that the regulatory sequence directs the expression of a coding sequence. Accordingly, "operably linked" includes a regulatory region of a functional domain having a known or desired activity, such as a promoter, a terminator, a signal sequence, or an enhancer region, attached or linked to a target (gene or polypeptide) so that the expression, secretion, or function of the target can be controlled according to said known or desired activity. For example, it means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a polypeptide of the present application is functionally linked to said polynucleotide sequence.

The term "expression" as used herein 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.

As used herein, the term “expression vector” means a linear or circular nucleic acid molecule comprising a coding sequence and regulatory sequences operably linked to it for expression.

In the present application, the term "regulatory sequence" refers to a polynucleotide sequence necessary for the expression of a coding sequence. Each regulatory sequence may be a native sequence (having the same origin) or a foreign sequence (derived from another gene) to the coding sequence. 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, a transcription and translation termination sequence.

The term "recombinant" in relation to a cell, polynucleotide, polypeptide, or vector, as used herein, means that the cell, polynucleotide, polypeptide or vector has been modified by the introduction of a heterologous nucleic acid or polypeptide or by alteration of a native polynucleotide or polypeptide, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell may express a gene that is not found in the native (non-recombinant) form of the cell, or may express a native gene that is expressed or not expressed at all, or otherwise abnormally expressed.

microorganism

In this application, the term "microorganism (or strain)" includes both wild-type microorganisms and microorganisms that have undergone genetic modification naturally or artificially, and may be microorganisms that have a specific mechanism weakened or increased due to causes such as insertion of an external gene or increased or inactivated activity of an endogenous gene, and may be microorganisms that include genetic modification for the production of a desired polypeptide, protein or product. In this application, "microorganism" and "strain" may be used interchangeably without limitation with the same meaning.

For example, the microorganism of the present application may be a microorganism comprising a variant threonine/homoserine export protein or a polynucleotide encoding the same; or a microorganism genetically modified to include a variant threonine/homoserine export protein or a polynucleotide encoding the same (e.g., a recombinant strain).

The strain of the present application may be a strain that naturally has L-amino acid production ability, or a microorganism in which L-amino acid production ability is imparted to a strain that does not have L-amino acid production ability. As an example, it may be a microorganism in which the mutant threonine/homoserine excretion protein of the present application or a polynucleotide encoding the same is introduced to increase L-amino acid production ability, but is not limited thereto.

The strain of the present application may be a microorganism having increased L-amino acid productivity compared to a parent strain or a wild-type Mycobacterium strain that does not include the variant of the present application. The microorganism may have improved L-amino acid productivity by introduction of the variant threonine/homoserine efflux protein of the present application having increased threonine/homoserine efflux activity compared to a wild-type threonine/homoserine efflux protein, or a polynucleotide encoding the same. Specifically, the strain of the present application may have increased L-amino acid productivity compared to a Corynebacterium microorganism including a wild-type threonine/homoserine efflux protein having the amino acid sequence of SEQ ID NO: 1, or a polynucleotide encoding the same.

In the present application, the term "microorganism having L-amino acid production ability" refers to a prokaryotic or eukaryotic microbial strain capable of producing L-amino acid within the organism, and may include both a microorganism in which L-amino acid production ability is imparted to a parent strain without L-amino acid production ability, or a microorganism inherently having L-amino acid production ability. The L-amino acid production ability may be imparted or enhanced by species improvement.

In the present application, the term "unmodified microorganism" does not exclude a strain containing a mutation that may occur naturally in a microorganism, and may mean a wild-type strain or a natural strain itself, or a strain before its phenotype is changed due to a genetic mutation caused by natural or artificial factors. The "unmodified microorganism" may be used interchangeably with "pre-modified strain", "pre-modified microorganism", "unmutated strain", "unmodified strain", "unmutated microorganism", "pre-mutated parent strain", "wild-type microorganism", "reference microorganism" or "reference microorganism". In addition, the term "threonine/homoserine efflux protein unmodified microorganism" in the present application may mean a strain into which the mutant threonine/homoserine efflux protein described herein is not introduced or before it is introduced. The unmodified microorganism of the present application for the threonine/homoserine efflux protein does not exclude strains containing modifications of other proteins or other genes other than modifications of the threonine/homoserine efflux protein or the polynucleotide encoding it. In addition, the unmodified microorganism in the present application may be a microorganism containing an amino acid sequence consisting of SEQ ID NO: 1 or a polynucleotide consisting of SEQ ID NO: 2, but is not limited thereto.

Increased polypeptide activity

As used herein, the term "increase" of polypeptide activity means that the activity of the polypeptide is increased compared to its intrinsic activity. The increase may be used interchangeably with terms such as activation, up-regulation, overexpression, and enhancement.

The increase may include exhibiting an activity that was not originally present, or exhibiting an activity that is enhanced compared to the inherent activity or activity prior to modification.

For example, the above "showing an activity that was not originally present" may be, but is not limited to, "introduction of a protein." The introduction of the protein means that a gene that the microorganism did not originally have is expressed in the microorganism, thereby causing the activity of a specific protein to be exhibited, or that the activity of the protein is increased or improved compared to the intrinsic activity or activity before modification. For example, a polynucleotide encoding a specific protein may be introduced into the chromosome of the microorganism, or a vector including a polynucleotide encoding a specific protein may be introduced into the microorganism, thereby causing its activity to be exhibited.

The above “intrinsic activity” refers to the activity of a specific polypeptide that the parent strain or unmodified microorganism originally had before the change in trait when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification.”

An increase in the activity of a polypeptide compared to its intrinsic activity means that the activity and/or concentration (expression amount) of a specific polypeptide is improved compared to the activity and/or concentration (expression amount) that the parent strain or unmodified microorganism originally had before the transformation.

By way of example only, the increase may be, but is not limited to, an increase in the activity or concentration of the corresponding protein, typically 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 more, relative to the activity or concentration in the wild-type protein or the initial microbial strain.

An increase in the activity of the above polypeptide can be achieved by introducing an exogenous polypeptide or by increasing the activity of the endogenous polypeptide. Whether the activity of the above polypeptide is increased can be confirmed by an increase in the level of activity of the polypeptide, the amount of expression, or the amount of a product secreted from the polypeptide.

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

Specifically, the increase in activity of the polypeptide of the present application is

1) Increase in the intracellular copy number of a polynucleotide encoding a polypeptide;

2) Modification of the expression control region of a gene on a chromosome encoding a polypeptide (e.g., occurrence of a mutation in the expression control region, replacement with a sequence having stronger activity, or insertion of a sequence having stronger activity);

3) Modification of the base sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide;

4) Modification of the amino acid sequence of the polypeptide so as to increase the polypeptide activity;

5) Modification of a polynucleotide sequence encoding said polypeptide such that the activity of said polypeptide is increased (e.g., modification of a polynucleotide sequence of said polypeptide gene such that the polynucleotide sequence of said polypeptide is modified such that the activity of said polypeptide is increased);

6) Introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same;

7) Codon optimization of polynucleotides encoding polypeptides;

8) Analyzing the tertiary structure of the polypeptide and selecting the exposed portion to modify or chemically modify; or

9) It may be a combination of two or more of the above 1) to 8), but is not particularly limited thereto.

for example,

The increase in the intracellular copy number of the polynucleotide encoding the polypeptide described above 1) may be caused by the introduction of a vector into the host cell that is capable of replicating and functioning independently of the host, to which the polynucleotide encoding the polypeptide is operably linked. Alternatively, the polynucleotide encoding the polypeptide may be introduced into the chromosome of the host cell in one copy or two or more copies. The introduction into the chromosome may be performed by the introduction into the host cell of a vector capable of inserting the polynucleotide into the chromosome of the host cell, but is not limited thereto. The vector is as described above.

The above 2) replacement of the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence having strong activity may be, for example, a mutation in the sequence such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof to further increase the activity of the expression control region, or replacement with a sequence having stronger activity. The expression control region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. As an example, the original promoter may be replaced with a strong promoter, but is not limited thereto.

Examples of known strong promoters include, but are not limited to, the cj1 to cj7 promoters (US Patent No. US 7662943 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 (US Patent No. US 10584338 B2), the O2 promoter (US Patent No. US 10273491 B2), the tkt promoter, and the yccA promoter.

The above 3) modification of the base sequence of the initiation codon or 5'-UTR region of the gene encoding the polypeptide may be, for example, a substitution with another initiation codon having a higher polypeptide expression rate than the endogenous initiation codon, but is not limited thereto.

The modification of the amino acid sequence or polynucleotide sequence of the above 4) and 5) may be, but is not limited to, a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to increase the activity of the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence improved to have stronger activity, or an amino acid sequence or polynucleotide sequence improved to have increased activity. The replacement may be specifically performed by inserting the polynucleotide into a chromosome by homologous recombination, but is not limited thereto. The vector used at this time may additionally include a selection marker to confirm whether or not the chromosome has been inserted. The selection marker is as described above.

The introduction of the foreign polynucleotide exhibiting the activity of the above 6) polypeptide may be the introduction into the host cell of a foreign polynucleotide encoding a polypeptide exhibiting the same/similar activity as the above polypeptide. The foreign polynucleotide is not limited in its origin or sequence as long as it exhibits the same/similar activity as the above polypeptide. The method used for the above introduction can be performed by a person skilled in the art appropriately selecting a known transformation method, and the introduced polynucleotide is expressed in the host cell, thereby producing the polypeptide and increasing its activity.

The above 7) codon optimization of a polynucleotide encoding a polypeptide may be codon optimization of an endogenous polynucleotide to increase transcription or translation within a host cell, or codon optimization of a foreign polynucleotide to achieve optimized transcription or translation within a host cell.

The above 8) analyzing the tertiary structure of a polypeptide and selecting an exposed portion to modify or chemically modify may be done by, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing the sequence information of known proteins, determining a template protein candidate based on the degree of sequence similarity, confirming the structure based on this, and selecting an exposed portion to modify or chemically modify and modifying or modifying it.

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 the wild-type or unmodified microbial strain, or an increase in the amount of a product produced from the polypeptide.

In the microorganism of the present application, modification of part or all of the polynucleotide may be induced by, but is not limited to, (a) homologous recombination using a vector for chromosome insertion into the microorganism or genome editing using engineered nuclease (e.g., CRISPR-Cas9) and/or (b) treatment with light such as ultraviolet rays and radiation and/or chemicals. The method for modifying part or all of the gene may include a method using DNA recombination technology. For example, a nucleotide sequence or vector including a nucleotide sequence homologous to a target gene may be injected into the microorganism to cause homologous recombination, thereby causing deletion of part or all of the gene. The injected nucleotide sequence or vector may include, but is not limited to, a dominant selection marker.

culture

In this application, the term "cultivation" means growing the strain of this application under appropriately controlled environmental conditions. The culturing process of this application can be performed according to appropriate media and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art according to the selected strain. Specifically, the culturing may be batch, continuous, and/or fed-batch, but is not limited thereto.

In this application, the term "medium" means a material containing nutrients as its main component, which are necessary for culturing the microorganism of the present application, and supplies nutrients and growth factors, including water, which is essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the strain of the present application may be any medium used for culturing general microorganisms without particular limitation, but the microorganism of the present application may be cultured in a general medium containing an appropriate carbon source, nitrogen source, phosphorus, inorganic compounds, amino acids, and/or vitamins, under aerobic conditions while controlling temperature, pH, etc. For example, a culture medium for a strain of the genus Corynebacterium can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington D.C., USA, 1981)].

In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane residue, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugar) may be used, and other appropriate amounts of carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

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

The above-mentioned personnel may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. The inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and/or appropriate precursors may be included. These components or precursors may be added to the medium in batch or continuous manner. However, the present invention is not limited thereto.

In addition, during the cultivation of the microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. may be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or in order to maintain the anaerobic and microaerobic state, no gas may be injected, or nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.

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.

In the present application, the term "culture" means a culture solution, a concentrated culture solution, a dried product of a culture solution, a culture filtrate, a concentrated culture filtrate, or a dried product of a culture filtrate obtained by culturing a specific microorganism in a culture medium, wherein the culture solution means that it contains a specific microorganism, and the culture filtrate means that it does not substantially contain a specific microorganism (here, substantially means excluding a specific microorganism separated by filtration or the like, but does not mean that the filtrate completely excludes microorganisms). The culture is not limited in its formulation, and may be, for example, a liquid, an emulsion, or a solid.

In this application, the term "fermentation" means a process in which microorganisms decompose organic matter using their own enzymes, but it is not a putrefaction reaction. Fermentation and putrefaction reactions proceed through similar processes, but if a useful substance is created as a result of the decomposition, it is called fermentation, and if an unpleasant-smelling or harmful substance is created, it is called putrefaction.

The method for obtaining a fermented product from the strain in the present application is not particularly limited, and can be obtained according to a method commonly used in the relevant technical field or a similar field.

In the present application, the term "fermentation" includes all kinds of materials including a fermentation product generated from the strain, such as not only the fermented material itself, but also a culture medium of the strain in which the strain and the culture coexist, a fermentation product obtained by filtering the strain from the culture medium, a fermentation product obtained by sterilizing the strain from the culture medium and filtering it, an extract obtained by extracting the fermentation product or the culture medium containing it, a diluted solution obtained by diluting the fermentation product or the extract thereof, a concentrate obtained by drying the fermentation product or the extract thereof, a dried product obtained by capturing and crushing the cells of the strain, and the like.

Specific description of this application

Hereinafter, specific examples of the present application will be described in more detail as follows.

One embodiment of the present application provides a mutant threonine/homoserine exporter protein, wherein the amino acid corresponding to position 119 in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid.

The term "variant threonine/homoserine export protein" in the present application means any polypeptide having threonine and homoserine export activity or a variant comprising a substitution of an amino acid corresponding to position 119 from the N-terminus of SEQ ID NO: 1 in a threonine/homoserine export protein with another amino acid. The "variant threonine/homoserine export protein" may also be referred to as "threonine/homoserine export protein variant", "variant RhtA", "RhtA variant", etc.

The threonine/homoserine export protein to which the mutation is introduced in the present application may also be referred to as "RhtA protein" or "RhtA", and may be a protein having an export activity of threonine and homoserine. Specifically, the protein may be one having an amino acid sequence of SEQ ID NO: 1 and having an export activity of threonine and homoserine, but is not limited thereto. This does not exclude meaningless sequence additions before and after the amino acid sequence of SEQ ID NO: 1, mutations that may occur naturally, or silent mutations thereof, and if it has an activity identical or corresponding to that of the protein including the amino acid sequence of SEQ ID NO: 1, it may correspond to the protein to which the mutation is introduced in the present application. For example, the protein that is the target of the mutation introduction of the present application may be a protein composed of an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that has 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. In addition, if it is an amino acid sequence that has such homology or identity and exhibits an effect corresponding to the protein, it is obvious that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted, or added is also included within the scope of the protein that is the target of the mutation of the present application.

In the present application, the amino acid sequence before modification of the threonine/homoserine export protein to be mutated, i.e., the amino acid before modification corresponding to position 119 of sequence number 1 in the sequence that can be the parent sequence, may be proline (P).

The variant threonine/homoserine export protein of the present application may have an amino acid at position 119 in the amino acid sequence of SEQ ID NO: 1 substituted with an amino acid different from the amino acid before substitution.

For example, the mutant threonine/homoserine export protein may be one in which the amino acid corresponding to position 119 in the amino acid sequence of sequence number 1 is substituted with an amino acid other than proline, which is the amino acid before substitution.

As another example, the mutant threonine/homoserine export protein may be one in which the amino acid corresponding to position 119 in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of threonine, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, valine, tryptophan, and tyrosine.

As another example, the variant threonine/homoserine export protein of the present application can have, comprise, consist of or consist essentially of an 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.

The variant threonine/homoserine export protein of the present application may include an amino acid sequence in which the amino acid corresponding to the 119th position based on the amino acid sequence of SEQ ID NO: 1 is an amino acid other than proline, for example, an amino acid selected from the group consisting of threonine, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, valine, tryptophan, and tyrosine, and 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 with the amino acid sequence described in SEQ ID NO: 1. In addition, it is obvious that a variant threonine/homoserine export protein having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted or added is also included within the scope of the present application, provided that the amino acid sequence has such homology or identity and exhibits an effect corresponding to the variant threonine/homoserine export protein of the present application.

For example, in the case where the amino acid sequence has sequence additions or deletions, naturally occurring mutations, silent mutations or conservative substitutions that do not alter the function of the variant threonine/homoserine exporter protein of the present application at the N-terminus, C-terminus and/or internally.

The above "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and/or chemical properties. Such amino acid substitutions may generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of the protein or polypeptide.

As used herein, the term "variant protein" or "variant" refers to a polypeptide in which one or more amino acids are conservatively substituted and/or modified, thereby differing from the amino acid sequence of the variant before the mutation, but retaining functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. In addition, some variants may include variants in which one or more portions, such as the N-terminal leader sequence or the transmembrane domain, are deleted. Other variants may include variants in which portions are deleted from the N- and/or C-terminus of the mature protein. The above term "mutant protein" may be used interchangeably with terms such as mutant, modification, mutant polypeptide, mutant protein, mutation, and variant (in English expressions, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited thereto if the term is used in the meaning of mutation. For the purpose of the present application, the mutant may be a polypeptide in which the amino acid corresponding to position 119 in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid selected from the group consisting of threonine, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, valine, tryptophan, and tyrosine. For example, the variant may be, but is not limited to, a polypeptide comprising an 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.

Additionally, the variant may include deletions or additions of amino acids that have minimal impact 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 that is involved in co-translationally or post-translationally translocation of the protein. Additionally, the variant may be conjugated to other sequences or linkers so that it can be identified, purified, or synthesized.

In addition, the polynucleotide encoding the variant threonine/homoserine efflux protein of the present application may include a base sequence encoding the variant threonine/homoserine efflux protein in which the amino acid corresponding to position 119 in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid. For example, the polynucleotide of the present application may include a base sequence encoding an 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. As a more specific example of the present application, a polynucleotide encoding a variant threonine/homoserine exporter protein of the present application can have or comprise a 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. Additionally, the polynucleotide encoding the variant threonine/homoserine exporter protein of the present application may consist of, or consist essentially of, a 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.

The polynucleotide of the present application can have various modifications in the coding region within a range that does not change the amino acid sequence of the variant threonine/homoserine export protein of the present application, taking into account the degeneracy of the codon or the preferred codon in an organism that is intended to express the variant threonine/homoserine export protein of the present application. Specifically, the polynucleotide encoding the variant threonine/homoserine export protein of the present application has or includes a base sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity with 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 has at least 70%, It may consist of or essentially consist of a base sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the base sequence, but is not limited thereto. At this time, in the sequence having the above homology or identity, the codon encoding the amino acid corresponding to the 119th position of the sequence number 1 may be one of the codons encoding an amino acid selected from the group consisting of amino acids other than proline, for example, threonine, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, valine, tryptophan and tyrosine.

In this application, the term "corresponding to" refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the residue listed in the polypeptide. Identifying an amino acid at a corresponding position can be determining a particular amino acid in a sequence that references a particular sequence. As used herein, a "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue of the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described in the present application can identify the position of an amino acid, or the position at which a modification, such as a substitution, insertion, or deletion, occurs, by comparing it to a query sequence (also referred to as a “reference sequence”).

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

In the present application, the term "threonine/homoserine exporter (RhtA)" means a protein having the activity of exporting threonine and homoserine. Specifically, the "threonine/homoserine exporter" of the present application can be used interchangeably with "RhtA". The threonine/homoserine exporter is known in the art, and specifically, may be one of NCBI Accession No. WP_264865234.1, WP_161647838.1, HAP0341854.1, but is not limited thereto. The amino acid and polynucleotide sequences of the threonine/homoserine exporter can be obtained from a known database, and examples thereof include, but are not limited to, NCBI's GenBank.

For example, the threonine/homoserine efflux protein may comprise the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 60% homology or identity thereto, but is not limited thereto, as long as it has threonine/homoserine efflux protein activity. Specifically, the polypeptide having the threonine/homoserine efflux protein activity may have, comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity thereto. For example, the threonine/homoserine efflux protein may refer to, but is not limited to, a protein endogenously present in a microorganism of the genus Corynebacterium or Corynebacterium glutamicum, and specifically, may refer to, but is not limited to, a threonine/homoserine efflux protein consisting of an amino acid sequence of sequence number 1 endogenously present in a microorganism of the genus Corynebacterium or Corynebacterium glutamicum.

In addition, the threonine/homoserine efflux protein having the amino acid sequence of the above sequence number 1 may be encoded by a polynucleotide having or including a base 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 with the sequence of the above sequence number 2, or consisting of or consisting essentially of the above base sequence, but is not limited thereto. In addition, the base sequence of the above sequence number 2 can be obtained from a known database, such as NCBI's GenBank, but is not limited thereto.

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.

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 threonine/homoserine efflux protein of the present application due to codon degeneracy or in consideration of the codon preferred in an organism that is intended to express the threonine/homoserine efflux protein of the present application. Therefore, it is obvious that the polynucleotide that can be translated into a polypeptide consisting of the amino acid sequence of the threonine/homoserine efflux protein of the present application or a polypeptide having homology or identity therewith due to codon degeneracy may also be included. For example, the polynucleotide encoding the threonine/homoserine efflux protein of the present application may be SEQ ID NO: 2 or a degenerated sequence thereof.

As another example, the polynucleotide of the present application may have or include a base sequence that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous or identical to SEQ ID NO: 2, or may consist of or consist essentially of a base sequence that is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous or identical to SEQ ID NO: 2, but is not limited thereto.

In addition, the polynucleotide of the present application may include, without limitation, a probe that can be prepared from a known genetic 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, so long as it is a sequence encoding a threonine/homoserine exporter protein of the present application.

Another aspect of the present application provides a vector comprising the polynucleotide of the present application.

The above vector may be an expression vector for expressing the above polynucleotide in a host cell, but is not limited thereto.

The above vector is as described above.

Another aspect of the present application provides a microorganism comprising at least one selected from the group consisting of a variant threonine/homoserine exporter protein of the present application; a polynucleotide encoding the same; or a vector comprising the same.

The microorganism of the present application may have the ability to produce L-amino acid.

In the present application, “L-amino acid” includes all L-homoserine, derivatives of L-homoserine, and L-amino acids biosynthesized using L-homoserine as a precursor.

In the present application, the “derivative of L-homoserine” may include, for example, O-acetyl-L-homoserine, O-succinyl-L-homoserine, etc., but is not necessarily limited thereto, and any derivative obtained through a fermentation process and having a substituent linked to the terminal oxygen of L-homoserine in the relevant technical field may be included in the scope of the present application.

In the present application, “L-amino acid biosynthesized using L-homoserine as a precursor” may include, for example, methionine, threonine, isoleucine, etc., which use L-homoserine as a precursor, but is not necessarily limited thereto, and any L-amino acid biosynthesized using L-homoserine as a precursor may be included in the scope of the present application.

For example, 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.

For the purposes of the present application, the microorganism of the present application may be a microorganism comprising a variant threonine/homoserine export protein or a polynucleotide encoding same; or a microorganism (e.g., a recombinant strain) genetically modified to include a variant threonine/homoserine export protein or a polynucleotide encoding same.

The strain of the present application may be a strain that naturally has L-amino acid production ability, or a microorganism in which L-amino acid production ability is imparted to a strain that does not have L-amino acid production ability. As an example, it may be a microorganism in which the mutant threonine/homoserine excretion protein of the present application or a polynucleotide encoding the same is introduced to increase L-amino acid production ability, but is not limited thereto.

The strain of the present application may be a microorganism having increased L-amino acid productivity compared to a parent strain or a wild-type Mycobacterium strain that does not include the variant of the present application. The microorganism may have improved L-amino acid productivity by introduction of the variant threonine/homoserine efflux protein of the present application having increased threonine/homoserine efflux activity compared to a wild-type threonine/homoserine efflux protein, or a polynucleotide encoding the same. Specifically, the strain of the present application may have increased L-amino acid productivity compared to a Corynebacterium microorganism including a wild-type threonine/homoserine efflux protein having the amino acid sequence of SEQ ID NO: 1, or a polynucleotide encoding the same.

For example, a microorganism having L-amino acid production ability is a prokaryotic or eukaryotic microorganism strain that can produce L-amino acids within the organism, and may include all microorganisms that inherently have L-amino acid production ability or are genetically modified to include a variant threonine/homoserine efflux protein of the present application or a polynucleotide encoding the same in a parent strain that does not have L-amino acid production ability, thereby imparting L-amino acid production ability due to increased threonine/homoserine efflux protein activity. The L-amino acid production ability can be imparted or enhanced by species improvement.

For example, the recombinant microorganism having L-amino acid production ability of the present application may include any microorganism capable of producing L-amino acids by being transformed via a vector to express a mutant threonine/homoserine efflux protein of the present application having increased threonine/homoserine efflux protein activity.

For example, the microorganism producing the L-amino acid may be a microorganism which endogenously comprises a protein consisting of an amino acid sequence of SEQ ID NO: 1, or a protein consisting of an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% homology or identity with SEQ ID NO: 1.

For example, the microorganism producing the L-amino acid may be a microorganism that inherently includes a polynucleotide sequence capable of encoding a protein comprising an amino acid sequence having at least 80% homology with the sequence number 1, a base sequence of the sequence number 2, or a base sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or identity with the base sequence of the sequence number 2.

The microorganism of the present application may include all microorganisms in which the activity of the threonine/homoserine efflux protein is increased compared to the intrinsic activity by expressing the variant threonine/homoserine efflux protein of the present application having increased threonine/homoserine efflux protein activity by various known methods.

As a specific example, a microorganism having an increased activity of a threonine/homoserine efflux protein of the present application compared to its intrinsic activity may be, but is not limited to, a microorganism having a modified amino acid sequence of a threonine/homoserine efflux protein or a polynucleotide sequence encoding the same. The modification of the amino acid sequence or the polynucleotide sequence may include deletion, substitution, insertion, etc. The modification in the sequence is as described above. As another specific example, a microorganism having an increased activity of a threonine/homoserine efflux protein of the present application compared to its intrinsic activity may be, but is not limited to, a microorganism having a chromosomal gene expression control region (or expression control sequence) encoding a threonine/homoserine efflux protein replaced with a sequence having a strong activity. The expression control region may be, for example, a promoter. The replacement with a sequence having a strong activity is as described above. In another specific example, in a microorganism in which the activity of the threonine/homoserine efflux protein of the present application is increased compared to the intrinsic activity, the increased activity of the threonine/homoserine efflux protein may be achieved by a combination of the above specific examples.

As an example of the present application, the microorganism of the present application may have the ability to produce L-amino acids.

The above increased threonine/homoserine efflux protein activity may be defined as, but is not limited to, increased L-amino acid productivity of the microorganism of the present application compared to the productivity of a natural wild-type microorganism or an unmodified microorganism (e.g., a microorganism expressing a polypeptide having wild-type threonine/homoserine efflux protein activity (e.g., a polypeptide of SEQ ID NO: 1) or a strain in which the threonine/homoserine efflux protein activity of the present application is not increased or is increased compared to the intrinsic activity).

For example, the threonine/homoserine efflux protein activity can be measured by, but is not limited to, measuring L-amino acid productivity or yield. For example, the threonine/homoserine efflux protein activity can be measured by, but is not limited to, measuring L-amino acid productivity as described in Example 2-4.

For example, the microorganism with increased L-amino acid productivity of the present application may be a microorganism with increased L-amino acid productivity compared to a non-modified microorganism, but is not limited thereto. For example, the non-modified microorganism, which is a target strain for comparing whether or not the L-amino acid productivity is increased, may be 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 efflux protein is replaced with a gapA promoter, but is not limited thereto.

For example, the L-amino acid productivity of the microorganism with increased L-amino acid productivity is about 1% or more, specifically, about 1% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 55.6% or more, about 60% or more, about 66.7% or more, about 70% or more, about 77.8% or more, about 80% or more, about 88.9% or more, about 90% or more, about 100% or more, about 110% or more, about 111.1% or more, about 120% or more, about 122.2% or more, about 130% or more, about 133.3% or more, about 140% or more, about 150% or more, about 160% or more, about 170% or more, It may be increased by about 180% or more, about 190% or more, about 200% or more, about 210% or more, about 211.1% or more, about 220% or more, about 230% or more, about 233.3% or more, about 240% or more, about 244.4% or more, about 250% or more, about 260% or more, about 270% or more, about 277.8% or more, about 280% or more, or about 288.9% or more (the upper limit is not specifically limited, and 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). It is not limited thereto, as long as it has a positive increase in value compared to the productivity of the parent strain or non-mutated microorganism before mutation. In another example, the recombinant strain with increased L-amino acid productivity has an L-amino acid productivity of about 1.01 times or more, about 1.1 times or more, about 1.2 times or more, about 1.3 times or more, about 1.4 times or more, about 1.5 times or more, about 1.556 times or more, about 1.6 times or more, about 1.667 times or more, about 1.7 times or more, about 1.778 times or more, about 1.8 times or more, about 1.889 times or more, about 1.9 times or more, about 2 times or more, about 2.1 times or more, about 2.111 times or more, about 2.2 times or more, about 2.222 times or more, about 2.3 times or more, about 2.333 times or more, about 2.4 times or more, about 2.5 times or more, or about 2.6 times, compared to the parent strain before mutation or the unmodified microorganism. The upper limit may be increased by, but is not limited to, about 2.7 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 3.111 times or more, about 3.2 times or more, about 3.3 times or more, about 3.333 times or more, about 3.4 times or more, about 3.444 times or more, about 3.5 times or more, about 3.6 times or more, about 3.7 times or more, about 3.778 times or more, about 3.8 times or more, or about 3.889 times or more (there is no particular limitation on the upper limit, and 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).

In 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 ammoniagenes , It may be Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens , and specifically, but not limited to, Corynebacterium glutamicum.

Meanwhile, it was already known that microorganisms of the genus Corynebacterium can produce L-homoserine, derivatives of L-homoserine, or L-amino acids biosynthesized using L-homoserine as a precursor as L-amino acids; however, their productivity is significantly low, and the genes and mechanisms involved in the production mechanism have not been fully elucidated. Therefore, the Corynebacterium microorganism having the ability to produce L-homoserine, a derivative of L-homoserine, or an L-amino acid biosynthesized using L-homoserine as a precursor of the present application may include a natural wild-type microorganism itself, a Corynebacterium microorganism having improved L-homoserine, a derivative of L-homoserine, or an L-amino acid biosynthesized using L-homoserine as a precursor production ability by increasing or decreasing the activity of a gene related to the production mechanism of L-homoserine, a derivative of L-homoserine, or an L-amino acid biosynthesized using L-homoserine as a precursor, or a Corynebacterium microorganism having improved L-homoserine, a derivative of L-homoserine, or an L-amino acid biosynthesized using L-homoserine as a precursor production ability by introducing or increasing the activity of an external gene.

The microorganism having L-amino acid production ability of the present application may be a microorganism having additionally increased or decreased homoserine dehydrogenase or lysine-sensitive aspartokinase 3 activity in the biosynthetic pathway of L-homoserine, a derivative of L-homoserine, or an L-amino acid biosynthesized using L-homoserine as a precursor, or may be a microorganism having enhanced production ability of L-amino acid biosynthesized using L-homoserine, a derivative of L-homoserine, or L-homoserine as a precursor, but is not limited thereto.

Examples of the homoserine dehydrogenase or lysine-sensitive aspartokinase 3 mutants 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 disclosures of the above patents may be incorporated as reference material of the present application.

Another aspect of the present application provides a method for producing L-amino acid, comprising the step of culturing a microorganism comprising at least one selected from the group consisting of a variant threonine/homoserine exporter protein of the present application; a polynucleotide encoding the same; or a vector comprising the same; in a medium.

In the method of the present application, any culture conditions and culture methods known in the art can be used for culturing microorganisms. Those skilled in the art can easily adjust and use this culture process according to the selected strain.

L-amino acids produced by the culture of the present invention may be secreted into the medium or remain within the cells.

The above 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. This is as described above.

In one specific embodiment, the method for producing L-amino acid of the present application may further comprise 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, prior to the culturing step.

The method for producing 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, a culture of the microorganism, a fermented product of the microorganism, or the culture medium. The recovering step may be additionally included after the culturing step.

The above recovery may be to collect the target L-amino acid using a suitable method known in the art according to the culture method of the microorganism of the present application, for example, a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC or a combination of these methods may be used, and the target substance, specifically, an L-amino acid, can be recovered from a medium or microorganism using a suitable method known in the art.

In addition, the method for producing L-amino acid of the present application may additionally include a purification step. The purification may be performed using a suitable method known in the art. In one example, when the method for producing L-amino acid of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously regardless of the order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

In the method of the present application, the mutant threonine/homoserine efflux protein and L-amino acid, etc. are as described in the other embodiments above.

Another aspect of the present application provides a composition for producing L-amino acid, comprising a microorganism comprising at least one selected from the group consisting of a variant threonine/homoserine efflux protein of the present application; a polynucleotide encoding the same; or a vector comprising the same; a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof.

The composition of the present application may further comprise 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, buffering agents, stabilizers, or isotonic agents.

In one specific example, each component present in the composition of the present application may be included in a microbiologically effective amount, or an amount that can be suitably present in the production composition.

In the composition of the present application, the mutant threonine/homoserine efflux protein and L-amino acid, etc. are as described in the other embodiments above.

Another aspect of the present application provides a use of the variant threonine/homoserine efflux protein of the present application for producing L-amino acids.

Another aspect of the present application provides a use of a microorganism for producing L-amino acids, the microorganism comprising at least one selected from the group consisting of a variant threonine/homoserine exporter protein of the present application; a polynucleotide encoding the same; or a vector comprising the same.

For the purposes of the present application, the mutant threonine/homoserine efflux protein and L-amino acids, etc. are as described in the other embodiments above.

A microorganism expressing the mutant threonine/homoserine efflux protein of the present invention can produce L-amino acids in high yield, and thus can be usefully utilized in the production of L-amino acids.

Hereinafter, the present application will be described in more detail by way of examples. However, the following examples are merely preferred embodiments for illustrating the present application and therefore are not intended to limit the scope of the rights of the present application. Meanwhile, technical matters not described in this specification can be sufficiently understood and easily implemented by a person skilled in the technical field of the present application or a similar technical field.

Example 1. Production of L-homoserine producing strain

Example 1-1. Production of homoserine dehydrogenase (hom) mutant strain

In order to increase L-homoserine production, a mutation (G378E, R398Q) trait (Korean Patent No. 10-2183209) for relieving feedback inhibition of hom on L-homoserine was introduced into a Corynebacterium strain. For this purpose, a hom mutant plasmid was constructed using the following method.

First, a fragment of the promoter upstream region of the hom gene was amplified by PCR 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.

And in order to apply the hom mutation (G378E, R398Q), the upper fragment of the gene coding for CDS including the 1st to 378th amino acid from the N-terminus in the amino acid sequence of the hom protein, the hom G378E/R398Q gene fragment, and the lower fragment of the hom R398Q gene where homologous recombination on the chromosome occurs were obtained. 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 NO: 67 and SEQ ID NO: 68, the primer pairs of SEQ ID NO: 69 and SEQ ID NO: 70, and the primer pairs of SEQ ID NO: 71 and SEQ ID NO: 72 to obtain the upper fragment of the coding sequence including the 1st to 378th amino acid from the N-terminus in the amino acid sequence of the hom protein, the G378E/R398Q gene fragment, and the lower fragment of the R398Q gene, respectively.

PCR reactions were performed using Solg ^TM Pfu-X DNA polymerase as the polymerase, denaturing at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 60 seconds, and then polymerization at 72°C for 5 minutes.

The gene fragments obtained through the above process were cloned into the pDCM2 vector (Korean Publication No. 10-2020-0136813) digested 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 a calculated molar number and then storing at 50°C for 1 hour. The constructed recombinant plasmid was named pDCM2-hom (G378E, R398Q).

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

The genetic manipulation was confirmed by PCR and genome sequencing using primer pairs of SEQ ID NOs: 45 and 46, which can amplify external regions of the upstream and downstream regions of the homologous recombination region into which the gene was inserted, respectively.

The primer sequences used here were as shown in Table 1 below.

Sequence number Sequence name Sequence (5'->3') 65 up F TCGAGCTCGGTACCCTGTCTCCGTATGCAGTGAGC 66 up R GGATGTTTCTTTGGAGCTTCGCTCAATCAT 67 hom F TATAGGAGAACAATCATGACCTCAGCATCTGCCCC 68 G378E R GCCAAAACCTCCACGCGATCTT 69 G378E F AAGATCGCGTGGAGGTTTTGGC 70 R398Q R GCGCTCTTCCTGTTGGATTGTACGC 71 R398Q F GCGTACAATCCAACAGGAAGAGCGC 72 hom R CTCTAGAGGATCCCCGACTGCGGAATGTTGTTGTG 45 hom conf F TGGGTAGGTCGAGTTGTTAA 46 hom conf R CAGCGCAGTCGCACGAATAT

The transformed strain obtained above was named Cgl-HS-1.

Example 1-2. Production of lysine-sensitive aspartokinase 3 (LysC) mutant strain

We aimed to introduce a mutation (L377K) trait (Korean Patent Publication No. 10-2019-0003019) to enhance the expression of the lysC gene and to relieve feedback inhibition of L-lysine and L-homoserine in a Corynebacterium strain.

First, the upstream region of the lysC gene promoter and the upstream and downstream regions of the lysC L377K gene where homologous recombination on the chromosome occurs were obtained. 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 NO: 47 and SEQ ID NO: 48, the primer pairs of SEQ ID NO: 49 and SEQ ID NO: 50, and the primer pairs of SEQ ID NO: 51 and SEQ ID NO: 52 to obtain gene fragments of 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.

PCR reactions were performed using Solg ^TM Pfu-X DNA polymerase as the polymerase, denaturing at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, polymerization at 72°C for 60 seconds, and then polymerization at 72°C for 5 minutes.

The gene fragments obtained through the above process were cloned into the pDCM2 vector digested 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 a calculated molar amount and then storing it at 50°C for 1 hour. The constructed recombinant plasmid was named pDCM2-_lysC L377K.

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

The genetic manipulation was confirmed by PCR using primers of SEQ ID NO: 53 and SEQ ID NO: 54 that can amplify external regions of the upstream and downstream regions of the homologous recombination site where the gene was inserted, respectively, and by genome sequencing.

The primer sequences used here were as shown in Table 2 below.

Sequence number Sequence name Sequence (5'->3') 47 lysC promoter Up 1 TCGAGCTCGGTACCCGACAGGACAAGCACTGGTTG 48 lysC promoter Up 2 AGTAGCGCTGGGATGTTTTCTCTTTGTGCACCTTTC 49 lysC Down 1 GAACATCGAAAAGATTTCCACCTCTGAGAT 50 lysC Down 2 CTCTAGAGGATCCCCGTTCACCTCAGAGACGATTA 51 lysC 1 CGAAAGGAAACACTCATGGCCCTGGTCGTACAGAA 52 lysC 2 GGTGGAAATCTTTTTCGATGTTCACGTTGAC 53 confirm lysC 1 ACATTCCACCCATTACTGCA 54 confirm lysC 2 TCTTCATCGGTTTCGAAGGT

The transformed strain obtained above was named Cgl-HS-2.

Example 2. Production of strain introducing RhtA mutation

Example 2-1. Production of RhtA mutant

To produce a template to be used in error-prone PCR, a wild-type rhtA gene fragment encoding RhtA was obtained by performing PCR using the genomic DNA of Escherichia coli W3110 strain as a template and the primer pair of SEQ ID NO: 41 and SEQ ID NO: 42. In addition, a promoter fragment of the rhtB gene was obtained by performing PCR using the genomic DNA of E. coli W3110 strain as a template and the primer pair of SEQ ID NO: 43 and SEQ ID NO: 44.

PCR reactions were performed using Solg ^TM Pfu-X DNA polymerase as the polymerase, denaturing at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, polymerization at 72°C for 60 seconds, and then polymerization at 72°C for 5 minutes.

The primer sequences used here were as shown in Table 3 below.

Sequence number Sequence name Sequence (5'->3') 41 rhtA F AGACACACCGGGAGTTCATCATGCCTGGTTTCATTACGT 42 rhtA R AATTCGAGTCGGTACCCTTAATTAATGTCTAATTCT 43 PrhtB F GTCGACTCTAGAGGATCCCCCGGGTAATCTGCCTCTTA 44 PrhtB R ACGTAATGAACCAGGCATGATGAACTCCCGGTGTGTCT

The gene fragments obtained above were cloned into the pCL1920 vector (Nucleic Acids Rersearch, 18, (1990) 4631) digested with SmaI restriction enzyme using the Gibson assembly method (D. G. Gibson et al., NATURE MEHSODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix), obtaining the recombinant plasmid pCL1920-PrhtB_rhtA (wt). Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in a calculated molar amount and then storing at 50°C for 1 hour. Error-prone PCR was performed to induce random mutagenesis in the wild-type rhtA gene encoding threonine/homoserine efflux protein, and a diversify PCR random mutagenesis kit (Takara) was used for error-prone PCR. In order to select the mutation rate conditions, error-prone PCR was performed under two conditions depending on the amount of MnSO ₄ added, as follows. PCR was performed using pCL1920-PrhtB_rhtA (wt) as the DNA template to introduce mutations and the primer pair of SEQ ID NO: 41 and SEQ ID NO: 42 as primers. PCR was performed under the following conditions: denaturation at 95°C for 30 sec, followed by 25 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, and polymerization at 68°C for 30 sec, and then polymerization at 68°C for 60 sec.

The composition of the composition for performing error-inducing PCR was as shown in Table 4 below.

case # case #1(MnSO ₄ 1 μl) case #2 (MnSO ₄ 2 μl) 10X Titanium taq Buffer 5 5 MnSO ₄ (8mM) 1 2 dGTP (2mM) 1 1 50 X dNTP Mix 1 1 Titanium Taq Polymerase 1 1 Forward primer(5pmol) 2 2 Reverse primer(5pmol) 2 2 Template DNA 1 1 _dH2O 36 35 Total 50 50

The error-causing PCR product obtained above was treated with DpnI to remove the template plasmid, and the DNA was cloned into the pCL1920 vector digested with SmaI restriction enzyme using the Gibson assembly method to obtain a recombinant mutant plasmid library pCL1920-PrhtB_rhtA(mt). As a result of performing sequencing to confirm the mutation, a mutation was found in the coding sequence (CDS), and a plasmid containing a mutation (P119T) from proline to threonine at the 119th amino acid in the RhtA amino acid sequence was confirmed, and this was named pCL1920-PrhtB_rhtA(m7).

Example 2-2. Production of RhtA mutant plasmid for introduction of Corynebacterium strains

To introduce the RhtA mutation obtained in Example 2-1 into a Corynebacterium strain, a rhtA mutation plasmid was constructed using the following method.

First, for homologous recombination, 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: 57 and 58 and the primer pairs of SEQ ID NOs: 63 and 64 to amplify the upstream and downstream regions of Ncgl0998, respectively. In order to utilize the gapA promoter as a promoter of the mutant rhtA gene, PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and the primer pairs of SEQ ID NOs: 59 and 60 to amplify the gapA promoter. In addition, PCR was performed using the primer pairs of SEQ ID NO: 61 and SEQ ID NO: 62, using the wild-type plasmid pCL1920-PrhtB_rhtA and the mutant plasmid pCL1920-PrhtB_rhtA(m7) obtained in the above Example 2-1 as templates, to amplify fragments of the wild-type and mutant rhtA from the wild-type and mutant plasmids of the above Example 2-1, respectively, thereby obtaining fragments of rhtA and rhtA(m7).

PCR reactions were performed using Solg ^TM Pfu-X DNA polymerase as the polymerase, with the following conditions: denaturation at 95°C for 5 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 60 seconds, and then polymerization at 72°C for 5 minutes.

The primer sequences used here were as shown in Table 5 below.

Sequence number Sequence name Sequence (5'->3') 57 Ncgl0998 up F CGAGCTCGGTACCCTAATAATTCTGCTTTCCCGACG 58 Ncgl0998 up R CCACAAATTAAGGGACGGGTCGATGTCGAGG 59 PgapA F CTCGACATCGACCGGTCCCTTAATTTGTGGATTCAC 60 PgapA R CGTAATGAACCAGGCATGTTGTGTCTCCTCTAAA 61 rhtA_m F TTAGAGGAGACACAACATGCCTGGTTCATTACGTA 62 rhtA_m R TCGGGCTCTTCCGGTTCCCTTAATTAATGTCTAAT 63 Ncgl0998 dn F AGACATTAATTAAGGGAACCGGAAGAGCCCGATTG 64 Ncgl0998 dn R TAGAGGATCCCGAAAAGGAAGAAGTCTGAGAAGAAGG

The gene fragments obtained through the above process were cloned into the pDCM2 vector digested with SmaI restriction enzyme using the Gibson assembly method to obtain recombinant plasmids containing wild-type rhtA or mutant rhtA. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in a calculated molar number and then storing at 50°C for 1 hour. The two types of constructed recombinant plasmids were named pDCM2-PgapA_rhtA and pDCM2-PgapA_rhtA(m7), respectively.

Example 2-3. Production of Corynebacterium strains introducing RhtA mutations

The pDCM2-PgapA_rhtA and pDCM2-PgapA_rhtA(m7) vectors constructed in the above Example 2-2 were each transformed into the Cgl-HS-2 strain of the above Example 1-2 by electroporation, and then, through a second crossing over process, each strain having a wild-type rhtA or mutant rhtA gene inserted on the chromosome was obtained. The genetic manipulation was confirmed through PCR using the primer pair of SEQ ID NOs: 55 and 56 capable of amplifying the external regions of the upstream and downstream regions of the homologous recombination region where the gene was inserted, and genome sequencing.

The primer sequences used here were as shown in Table 6 below.

Sequence number Sequence name Sequence (5'->3') 55 outside F CACGTGCAGATTCGGTCTCG 56 outside R TTCGGTTCTTCAACGTCTGG

The transformed strains obtained above were named Cgl-HS-3 and Cgl-HS-3(m7), respectively.

Example 2-4. L-homoserine production ability of Corynebacterium strains containing RhtA mutations

In order to compare the L-homoserine production of the Cgl-HS-2 strains of Example 1-2 and the Cgl-HS-3, Cgl-HS-3(m7) strains of Example 2-3, they were cultured using the following method. Specifically, each strain was inoculated into a 250 mL corner-baffle flask containing 25 mL of production medium, and cultured with shaking at 200 rpm at 30°C for 24 hours. After the culture was completed, the production of L-homoserine was measured by HPLC, and the results of how it changed compared to the CgI-HS-3 strain are shown in Table 7 below.

<Production medium (pH 7.0)>

Glucose 45 g, _{( NH4} ) _2SO4 20 g, MgSO4 _· 7H2O _1.2 g, _KH2PO4 1.1 g, biotin ₉₀₀ μg, thiamine hydrochloride 4500 μg, calcium-pantothenic acid 4500 μg, CaCO3 ₃₀ g (based on 1 liter of distilled water)

Strain name L-homoserine production (g/L) L-homoserine yield
(*100 g/g, %) Cgl-HS-2 0.3 0.6 Cgl-HS-3 0.9 1.8 Cgl-HS-3(m7) 3.0 6.0

As shown in Table 7 above, the Cgl-HS-2 strain of Example 1-2 produced 0.3 g/L of L-homoserine, and the Cgl-HS-3 strain, into which the PgapA_rhtA wild-type trait was introduced using the strain as the parent strain, produced 0.9 g/L of L-homoserine. And Cgl-HS-3(m7) into which the PgapA_rhtA mutant trait was introduced produced 3.2 g/L and 3.0 g/L of L-homoserine, respectively.

The fermentation yield of L-homoserine was significantly increased in Cgl-HS-3 strain with the PgapA_rhtA wild-type trait introduced compared to Cgl-HS-2, with a 1.2% difference, whereas in Cgl-HS-3(m7) strain with the PgapA_rhtA mutant trait introduced compared to Cgl-HS-2, the difference was 5.4%. In addition, the yield increase rate of Cgl-HS-Cgl-HS-3(m7) compared to Cgl-HS-2 was approximately 4.5 times higher than that of Cgl-HS-3 compared to Cgl-HS-2.

Example 3. Saturated mutagenesis of rhtA

In the above Example 2-4, it was confirmed that the Cgl-HS-3(m7) strain containing the rhtA(P119T) mutation had superior L-homoserine production ability compared to the strain expressing wild-type rhtA. Therefore, the effectiveness of improving L-homoserine excretion at position 119 by mutating proline, the 119th amino acid in the amino acid sequence of rhtA, to another amino acid was verified. Site-directed mutagenesis was performed by the following method to mutate proline, the 119th amino acid in the amino acid sequence of rhtA, to 19 other amino acids other than proline.

Specifically, using pDCM2-PgapA_rhtA of Example 2-2 as a template, a PCR composition was prepared according to Table 8 below, and PCR was performed. PCR was performed under the conditions of denaturing at 95°C for 5 minutes, followed by 30 cycles of denaturing at 95°C for 30 seconds, annealing at 55°C for 1 minute, and polymerization at 68°C for 10 minutes. When performing PCR, the mutagenic primer set in Table 9 below was used.

furtherance Content (μl) 10X pfu-X Buffer 5 10mM dNTP Mix 1 pfu-X Polymerase 1 Mutagenic forward primer (5pmol) 2 Mutagenic reverse primer (5pmol) 2 pDCM2-PgapA_rhtA (template DNA, 200 ng/μl) 1 _dH2O 38 Total 50

rhtA mutant plasmid Sequence number Sequence name Sequence (5'-> 3') pDCM2-PgapA_rhtA P119A 73 pDCM2-PgapA_rhtA P119A F CTGTTCTCTTCTCGTCGCGCTGTAGATTTCGTCTGGGTTG 74 pDCM2-PgapA_rhtA P119A R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119C 75 pDCM2-PgapA_rhtA P119C F CTGTTCTCTTCTCGTCGCTGCGTAGATTTCGTCTGGGTTG 76 pDCM2-PgapA_rhtA P119C R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119D 77 pDCM2-PgapA_rhtA P119D F CTGTTCTCTTCTCGTCGCGACGTAGATTTCGTCTGGGTTG 78 pDCM2-PgapA_rhtA P119D R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA
P119E 79 pDCM2-PgapA_rhtA
P119E F CTGTTCTCTTCTCGTCGCGAAGTAGATTTCGTCTGGGTTG 80 pDCM2-PgapA_rhtA
P119E R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119F 81 pDCM2-PgapA_rhtA P119F F CTGTTCTCTTCTCGTCGCTTCGTAGATTTCGTCTGGGTTG 82 pDCM2-PgapA_rhtA P119F R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119G 83 pDCM2-PgapA_rhtA P119G F CTGTTCTCTTCTCGTCGCGGCGTAGATTTCGTCTGGGTTG 84 pDCM2-PgapA_rhtA P119G R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119H 85 pDCM2-PgapA_rhtA P119H F CTGTTCTCTTCTCGTCGCCACGTAGATTTCGTCTGGGTTG 86 pDCM2-PgapA_rhtA P119H R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119I 87 pDCM2-PgapA_rhtA P119I F CTGTTCTCTTCTCGTCGCATCGTAGATTTCGTCTGGGTTG 88 pDCM2-PgapA_rhtA P119I R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119K 89 pDCM2-PgapA_rhtA P119K F CTGTTCTCTTCTCGTCGCAAGGTAGATTTCGTCTGGGTTG 90 pDCM2-PgapA_rhtA P119K R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119L 91 pDCM2-PgapA_rhtA P119L F CTGTTCTCTTCTCGTCGCTCGTAGATTTCGTCTGGGTTG 92 pDCM2-PgapA_rhtA P119L R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119M 93 pDCM2-PgapA_rhtA P119M F CTGTTCTCTTCTCGTCGCATGGTAGATTTCGTCTGGGTTG 94 pDCM2-PgapA_rhtA P119M R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119N 95 pDCM2-PgapA_rhtA P119N F CTGTTCTCTTCTCGTCGCAACGTAGATTTCGTCTGGGTTG 96 pDCM2-PgapA_rhtA P119N R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119Q 97 pDCM2-PgapA_rhtA P119Q F CTGTTCTCTTCTCGTCGCCAGGTAGATTTCGTCTGGGTTG 98 pDCM2-PgapA_rhtA P119Q R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119R 99 pDCM2-PgapA_rhtA P119R F CTGTTCTCTTCTCGTCGCCCGGTAGATTTCGTCTGGGTTG 100 pDCM2-PgapA_rhtA P119R R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119S 101 pDCM2-PgapA_rhtA P119S F CTGTTCTCTTCTCGTCGCTCCGTAGATTTCGTCTGGGTTG 102 pDCM2-PgapA_rhtA P119S R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119V 103 pDCM2-PgapA_rhtA P119V F CTGTTCTCTTCTCGTCGCGTAGTAGATTTCGTCTGGGTTG 104 pDCM2-PgapA_rhtA P119V R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119W 105 pDCM2-PgapA_rhtA P119W F CTGTTCTCTTCTCGTCGCTGGGTAGATTTCGTCTGGGTTG 106 pDCM2-PgapA_rhtA P119W R GCGACGAGAAGAGAACAGCG pDCM2-PgapA_rhtA P119Y 107 pDCM2-PgapA_rhtA P119Y F CTGTTCTCTTCTCGTCGCTACGTAGATTTCGTCTGGGTTG 108 pDCM2-PgapA_rhtA P119Y R GCGACGAGAAGAGAACAGCG

After adding 1 ㎕ of DpnI restriction enzyme to the gene fragments obtained above, treatment was performed at 37°C for 1 hour, and each gene fragment treated with DpnI was cloned into the pDCM2 vector using the Gibson assembly method to obtain a recombinant mutant plasmid. 3 ㎕ of the recombinant plasmid DNA was transformed into DH5a competent cells to obtain the pDCM2-PgapA_rhtA mutant plasmid, and sequence analysis was performed to determine that the mutant plasmids were rhtA(P119A), rhtA(P119C), rhtA(P119D), rhtA(P119E), rhtA(P119F), rhtA(P119G), rhtA(P119H), rhtA(P119I), rhtA(P119K), rhtA(P119L), rhtA(P119M), rhtA(P119N), rhtA(P119Q), rhtA(P119R), rhtA(P119S), rhtA(P119V), It was confirmed to contain rhtA(P119W) and rhtA(P119Y) mutations.

pDCM2-PgapA_rhtA P119A, pDCM2-PgapA_rhtA P119C, pDCM2-PgapA_rhtA P119D, pDCM2-PgapA_rhtA P119E, pDCM2-PgapA_rhtA P119F, pDCM2-PgapA_rhtA P119G , pDCM2-PgapA_rhtA P119H, pDCM2-PgapA_rhtA P119I, pDCM2-PgapA_rhtA P119K, pDCM2 -PgapA_rhtA P119L, pDCM2-PgapA_rhtA P119M, pDCM2-PgapA_rhtA P119N, The vectors pDCM2-PgapA_rhtA P119Q, pDCM2-PgapA_rhtA P119R, pDCM2-PgapA_rhtA P119S, pDCM2-PgapA_rhtA P119V, pDCM2-PgapA_rhtA P119W, and pDCM2-PgapA_rhtA P119Y were electroporated into the Cgl-HS-3 strain using the method of Example 2-3. After transformation by the law, each strain with 18 mutant rhtA genes inserted on the chromosome was obtained through a second crossover process. The external regions of the upstream and downstream regions of the homologous recombination region where the gene was inserted were amplified, respectively. The genetic manipulation was confirmed by PCR using primer pairs of sequence numbers 83 and 84 and genome sequencing.

The transformed strains obtained above were Cgl-HS-3(P119A), Cgl-HS-3(P119C), Cgl-HS-3(P119D), Cgl-HS-3(P119E), Cgl-HS-3(P119F), Cgl-HS-3(P119G), Cgl-HS-3(P119H), Cgl-HS-3(P119I), Cgl-HS-3(P119K), Cgl-HS-3(P119L), Cgl-HS-3(P119M), Cgl-HS-3(P119N), Cgl-HS-3(P119Q), Cgl-HS-3(P119R), Cgl-HS-3(P119S), Cgl-HS-3(P119V), They were named Cgl-HS-3(P119W) and Cgl-HS-3(P119Y).

Example 4. L-homoserine production ability of Corynebacterium strains containing RhtA mutations

In order to compare the L-homoserine production of Cgl-HS-2 of the above Example 1-2, Cgl-HS-3, Cgl-HS-3(m7) of the above Example 2-3, and 18 strains produced in the above Example 3, they were cultured using the method of the above Example 2-4. After the culture was completed, the production of L-homoserine was measured by HPLC, and the results are shown in Table 10 below.

Strain name rhtA type L-homoserine production (g/L) L-homoserine yield (g/g, %) Cgl-HS-2 - 0.3 0.6 Cgl-HS-3 rhtA wild type 0.9 1.8 Cgl-HS-3(m7) rhtA P119T 3.0 6.0 Cgl-HS-3(P119A) rhtA P119A 3.2 6.4 Cgl-HS-3(P119C) rhtA P119C 3.1 6.2 Cgl-HS-3(P119D) rhtA P119D 2.7 5.4 Cgl-HS-3(P119E) rhtA P119E 2.0 4.0 Cgl-HS-3(P119F) rhtA P119F 2.5 5.0 Cgl-HS-3(P119G) rhtA P119G 2.0 4.0 Cgl-HS-3(P119H) rhtA P119H 1.5 3.0 Cgl-HS-3(P119I) rhtA P119I 4.0 8.0 Cgl-HS-3(P119K) rhtA P119K 2.8 5.6 Cgl-HS-3(P119L) rhtA P119L 3.9 7.8 Cgl-HS-3(P119M) rhtA P119M 2.1 4.2 Cgl-HS-3(P119N) rhtA P119N 2.1 4.2 Cgl-HS-3(P119Q) rhtA P119Q 2.2 4.4 Cgl-HS-3(P119R) rhtA P119R 1.4 2.8 Cgl-HS-3(P119S) rhtA P119S 1.8 3.6 Cgl-HS-3(P119V) rhtA P119V 3.8 7.6 Cgl-HS-3(P119W) rhtA P119W 2.3 4.6 Cgl-HS-3(P119Y) rhtA P119Y 1.7 3.4

As shown in Table 10 above, all 19 Cgl-HS-3 strains with mutations of rhtA(P119A), rhtA(P119C), rhtA(P119D), rhtA(P119E), rhtA(P119F), rhtA(P119G), rhtA(P119H), rhtA(P119I), rhtA(P119K), rhtA(P119L), rhtA(P119M), rhtA(P119N), rhtA(P119Q), rhtA(P119R), rhtA(P119S), rhtA(P119V), rhtA(P119W), and rhtA(P119Y) had the PgapA_rhtA wild-type trait introduced. The L-homoserine fermentation yield increased compared to the Cgl-HS-3 strain. Accordingly, it was confirmed that the strain expressing RhtA in which proline, the 119th amino acid in the amino acid sequence of RhtA, was mutated to 19 other amino acids other than proline, had a superior L-homoserine production compared to the strain expressing wild-type RhtA.

Example 5. Threonine production capacity of Corynebacterium strains introducing RhtA mutation

In order to compare the threonine production of the Cgl-HS-2 strain of Example 1-2, the Cgl-HS-3 strain of Example 2-3, and the Cgl-HS-3(m7) strain, they were cultured using the method of Example 2-4. After the culture was completed, the threonine production was measured by HPLC, and the results are shown in Table 11 below.

Strain name rhtA type Threonine production (g/L) Threonine yield (g/g, %) Cgl-HS-2 - 0.2 0.4 Cgl-HS-3 rhtA wild type 1.4 2.8 Cgl-HS-3(m7) rhtA P119T 2.2 4.4

As shown in Table 11 above, the Cgl-HS-3(m7) strain into which the P119T mutation was introduced showed an increased threonine fermentation yield compared to the Cgl-HS-3 strain into which the PgapA_rhtA wild-type trait was introduced. Accordingly, it was confirmed that the strain expressing RhtA in which proline, the 119th amino acid in the amino acid sequence of RhtA, was mutated to another amino acid had a superior threonine production compared to the strain expressing wild-type RhtA.

From the above description, those skilled in the art to which the present application belongs will be able to understand that the present application can be implemented in other specific forms without changing the technical idea or essential features thereof. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the present application should be interpreted as including all changes or modified forms derived from the meaning and scope of the patent claims described below rather than the above detailed description and their equivalent concepts within the scope of the present application.

Attach electronic file of sequence list

Claims (10)

A mutant threonine/homoserine exporter protein in which the amino acid corresponding to position 119 in the amino acid sequence of sequence number 1 is substituted with another amino acid.
A mutant threonine/homoserine excretion protein, wherein in claim 1, the other amino acid is an amino acid selected from the group consisting of threonine, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, glutamine, arginine, serine, valine, tryptophan, and tyrosine.
A polynucleotide encoding a mutant threonine/homoserine exporter protein of any one of claims 1 or 2.
A microorganism comprising at least one selected from the group consisting of a mutant threonine/homoserine exporter protein in which the amino acid corresponding to position 119 in the amino acid sequence of sequence number 1 is substituted with another amino acid; a polynucleotide encoding the same; or a vector comprising the same.
In claim 4, the microorganism has increased L-amino acid production ability compared to a microorganism comprising a wild-type threonine/homoserine excretion protein having the amino acid sequence of sequence number 1 or a polynucleotide encoding the same.
A microorganism, in claim 4, 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.
In claim 4, the microorganism is a microorganism of the genus Corynebacterium.
In claim 7, the microorganism of the genus Corynebacterium is Corynebacterium glutamicum.
A method for producing L-amino acid, comprising the step of culturing a microorganism including at least one selected from among a mutant threonine/homoserine export protein in which an amino acid corresponding to position 119 in the amino acid sequence of sequence number 1 is substituted with another amino acid; a polynucleotide encoding the same; or a vector including the same;
A method in claim 9, 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.