Mutant L-threonine export protein and method for producing L-threonine using the same

Abstract

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

Description

[Technical field]

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

[0002] Corynebacterium is a gram-positive microorganism that is widely used for the production of L-amino acids. L-Amino acids, especially L-threonine, are essential amino acids that are used in animal feed, human pharmaceutical and cosmetic industries, and are produced by fermentation using Corynebacterium strains.

[0003] Many attempts have been made to improve the method for producing L-amino acids using Corynebacterium strains. Among them, there have been studies that have attempted to improve Corynebacterium strains that produce L-amino acids by disrupting or attenuating the expression of specific genes using recombinant DNA technology. There have also been many studies that have attempted to improve L-amino acid-producing Corynebacterium strains by amplifying genes related to the biosynthesis of each L-amino acid and studying the effect on L-amino acid production. There have also been cases where foreign genes derived from other bacteria are introduced. However, even with the above conventional methods, there is still a demand for strains with improved L-threonine production ability.

[0004] Meanwhile, expression of specific amino acid export genes has led to improved productivity of the amino acid in microorganisms. Enhancement of expression of the L-lysine export gene (lysE) in Corynebacterium genus microorganisms has improved lysine productivity (WO1997-023597). In addition, a patent (EP1016710B1) has disclosed that enhancement of the yeaS, yahN, yfiK, and yggA genes, the functions of which have not been elucidated in Escherichia coli, has improved L-lysine, L-alanine, L-glutamic acid, L-threonine, L-histidine, L-valine, L-arginine, L-proline, and L-isoleucine productivity. [Prior art documents]

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed Document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

[0007] The present inventors have identified mutants of L-threonine export proteins and confirmed that when microorganisms expressing mutants of L-threonine export proteins are cultured, higher yields of L-threonine are produced than when microorganisms having existing wild-type export proteins are cultured, thereby completing the present application. [Means for solving the problem]

[0008] The present application provides a mutant L-threonine excretion protein in which the amino acid corresponding to position 143 in the amino acid sequence of SEQ ID NO:1 has been substituted with another amino acid. The present application provides polynucleotides encoding the mutant L-threonine excretion proteins of the present application.

[0009] The present application provides a microorganism comprising a mutant L-threonine excretion protein of the present application; or a polynucleotide encoding same. The present application provides a method for producing L-threonine, comprising the step of culturing in a medium a microorganism containing a mutant L-threonine excretion protein of the present application or a polynucleotide encoding same.

[0010] The present application provides a composition for producing L-threonine, comprising a mutant L-threonine export protein of the present application; a polynucleotide encoding the same; a vector comprising the polynucleotide; a microorganism comprising the mutant L-threonine export protein or a polynucleotide encoding the same; a medium in which the same is cultured; or a combination of two or more of them.

[0011] The present application provides use of the mutant L-threonine export protein of the present application; a polynucleotide encoding the same; a vector containing the polynucleotide; or a microorganism containing the mutant L-threonine export protein or a polynucleotide encoding the same for producing L-threonine. Effect of the Invention

[0012] When an L-threonine-producing microorganism having a mutant of the L-threonine export protein of the present invention is cultured, it is possible to produce L-threonine in a higher yield than when a microorganism has an existing wild-type export protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] This will be specifically explained as follows. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. That is, all combinations of various elements disclosed in this application belong to the scope of this application. In addition, the following specific description is not considered to limit the category of this application. In addition, throughout this specification, numerous papers and patent documents are referenced and citations thereof are displayed. The disclosure contents of the cited papers and patent documents are incorporated by reference in their entirety into this specification to more clearly explain the state of the art to which this application pertains and the contents of this application.

[0014] One aspect of the present application provides a mutant L-threonine excretion protein in which the amino acid corresponding to position 143 in the amino acid sequence of SEQ ID NO:1 has been substituted with another amino acid. The mutant L-threonine export protein of the present application may be one in which the amino acid at the position corresponding to the 143rd position in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid different from the amino acid before the replacement, or the mutant L-threonine export protein may be, but is not limited to, a mutant L-threonine export protein in which the amino acid at the position corresponding to the 143rd position in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid having an uncharged side chain, which is different from the amino acid before the replacement.

[0015] Specifically, the mutant L-threonine excretion protein may be one in which the amino acid corresponding to the 143rd position in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid selected from the group consisting of arginine, lysine, histidine, aspartic acid, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, glutamic acid and glutamine, but is not limited thereto.

[0016] More specifically, the mutant L-threonine excretion protein may have the amino acid corresponding to the 143rd position in the amino acid sequence of SEQ ID NO: 1 substituted with an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline, for example, with one of the branched-chain amino acids including valine, leucine and isoleucine, an example of which may be isoleucine, but is not limited thereto.

[0017] More specifically, the mutant L-threonine excretion protein of the present application may have, comprise, consist of, or be essentially composed of the amino acid sequence set forth in SEQ ID NO:3.

[0018] The protein to be subjected to the mutation of the present application may be a protein having L-threonine export activity. Specifically, the protein may include the amino acid sequence of SEQ ID NO: 1 and have threonine export activity, but is not limited thereto. This does not exclude the addition of meaningless sequences before or after the amino acid sequence of SEQ ID NO: 1, or naturally occurring mutations, or silent mutations thereof, and if the protein has the same or corresponding activity as the protein containing the amino acid sequence of SEQ ID NO: 1, it corresponds to the protein to be subjected to the mutation of the present application. For example, the protein to be subjected to the mutation of the present application may be a protein composed of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. It is also obvious that a protein having an amino acid sequence with such homology or identity and exhibiting an efficacy corresponding to the protein, in which a part of the sequence is deleted, modified, substituted, or added, is also included in the scope of the protein to be subjected to the mutation of the present application.

[0019] The mutant L-threonine export protein of the present application may include an amino acid sequence in which the amino acid corresponding to position 143 in the amino acid sequence of SEQ ID NO: 1 is an amino acid other than asparagine, for example, isoleucine, and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% or more, but less than 100%, homology or identity with the amino acid sequence described in SEQ ID NO: 1. It is also obvious that mutant L-threonine export proteins having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted, or added are also included within the scope of the present application, so long as the amino acid sequence has such homology or identity and exhibits an efficacy corresponding to the mutant L-threonine export protein of the present application.

[0020] For example, the amino acid sequence may have additions or deletions of sequences that do not change the function of the mutant L-threonine excretion protein of the present application, naturally occurring mutations, silent mutations or conservative substitutions at the N-terminus, C-terminus and / or internally.

[0021] The term "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0022] For example, among amino acids with electrically charged side chains, the positively charged (basic) amino acids include arginine, lysine, and histidine, the negatively charged (acidic) amino acids include glutamic acid and aspartic acid; and the amino acids with uncharged side chains can be classified as including glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0023] In the present application, the term "mutated protein" or "variant" refers to a polypeptide in which one or more amino acids have been conservatively substituted and / or modified, such that the amino acid sequence of the variant differs from that of the variant, but functions or properties are maintained. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the variant. That is, the ability of the variant may be increased, unchanged, or decreased compared to the variant. Some variants may also include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other variants may include variants in which portions have been removed from the N- and / or C-termini of the mature protein. The term "mutated protein" may be used interchangeably with terms such as mutation, modification, mutant polypeptide, mutated protein, mutation, and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited thereto as long as it is a term used to mean mutated. For the purposes of this application, the mutant may be a polypeptide in which the amino acid corresponding to the 143rd position of the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine. Specifically, the mutant may be a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3, but is not limited thereto.

[0024] The variants may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be conjugated to a signal (or leader) sequence involved in co-translationally or post-translationally protein translocation. The variants may also be conjugated to other sequences or linkers to allow identification, purification, or synthesis.

[0025] In this application, the term "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, which can be expressed as a percentage. The terms homology and identity can often be used interchangeably.

[0026] Sequence homology or identity of conserved polynucleotides or polypeptides may be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences are generally capable of hybridizing to all or a portion of the sequence under moderate or high stringent conditions. Hybridization obviously includes hybridization to polynucleotides containing common codons in polynucleotides or codons that take into account codon degeneracy.

[0027] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example, as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later), the GCG program package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO et al.] (1988) SIAM J Applied Math 48:1073. For example, BLAST or ClustalW from the National Database Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0028] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, e.g., Needleman et al. (1970), J Mol Biol. 48: 443, as known in, e.g., Smith and Waterman, Adv. Appl. Math (1981) 2: 482. In summary, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a unitary comparison matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0029] As an example of the present application, the mutant L-threonine export protein of the present application can have an activity that increases L-threonine producing ability compared to a wild-type polypeptide having L-threonine export protein activity.

[0030] In the present application, the term "L-Threonine efflux protein" refers to a membrane protein having L-Threonine efflux activity, and may be used interchangeably with RhtC. The amino acid sequence of RhtC can be obtained from a known database, such as NCBI Genebank.

[0031] For example, the RhtC of the present application may be derived from a microorganism, specifically, from a prokaryotic microorganism, more specifically, from Escherichia coli, etc., but may also be a protein having L-threonine export activity of various origins. In the amino acid sequence of RhtC before modification to be mutated in the present application, the amino acid before modification corresponding to the 143rd amino acid of SEQ ID NO: 1 may be asparagine (N).

[0032] In this application, the term "corresponding to" refers to an amino acid residue at a recited position in a polypeptide, or an amino acid residue that is similar or identical or homologous to a recited residue in a polypeptide. Identifying the amino acid at a corresponding position can also determine the particular amino acid of a sequence that references a particular sequence. As used in this application, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0033] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, based on which each amino acid residue in the amino acid sequence is numbered with reference to the numeric position of the corresponding amino acid residue in SEQ ID NO: 1. For example, sequence alignment algorithms such as those described in this application can identify the position of an amino acid relative to a query sequence (also referred to as a "reference sequence"), or the position at which a variation, such as a substitution, insertion or deletion, occurs.

[0034] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), etc. can be used, but are not limited thereto, and any sequence alignment program, pairwise sequence comparison algorithm, etc. known in the art can be appropriately used.

[0035] Another aspect of the present application is to provide a polynucleotide encoding a mutant L-threonine excretion protein of the present application. The rhtC gene of the present application can include any gene known to encode a protein having RhtC activity.

[0036] Specifically, the rhtC gene of the present application may be an rhtC gene derived from E. coli. As an example, the rhtC gene of the present application may be a polynucleotide encoding WP_000928824.1 derived from E. coli.

[0037] In the present application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and a DNA or RNA chain of a certain length or more, and more specifically, refers to a polynucleotide fragment encoding the mutant L-threonine excretion protein.

[0038] The polynucleotide encoding the mutant L-threonine export protein of the present application may include a base sequence encoding a mutant L-threonine export protein in which the amino acid corresponding to the 143rd position in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid, or may include a base sequence in which the codons corresponding to the 427th to 429th positions in the nucleotide sequence of SEQ ID NO: 2 are replaced with codons encoding other amino acids. Specifically, the polynucleotide of the present application may include a base sequence encoding the amino acid sequence described in SEQ ID NO: 3. As a more specific example of the present application, the polynucleotide of the present application may have or include the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In addition, the polynucleotide of the present application may consist of or essentially consist of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

[0039] The polynucleotide of the present application may be modified in various ways in the coding region without changing the amino acid sequence of the mutant L-threonine export protein of the present application, taking into consideration the degeneracy of codons or the codons preferred in the organism in which the mutant L-threonine export protein of the present application is to be expressed. Specifically, the polynucleotide of the present application may have or include a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 2, or may consist of or essentially consist of a base sequence having 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity to the sequence of SEQ ID NO: 2, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 143rd position of SEQ ID NO: 1 may be one of the codons encoding an amino acid other than asparagine, for example, isoleucine.

[0040] Furthermore, the polynucleotide of the present application may include, without limitation, a sequence that can hybridize under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to the entire or partial polynucleotide sequence of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; FMA Usubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, the conditions include conditions under which polynucleotides having high homology or identity, such as polynucleotides having a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, but polynucleotides having lower homology or identity do not hybridize with each other, or conditions under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions for normal Southern hybridization, such as 60°C, 1 x SSC, 0.1% SDS, specifically 60°C, 0.1 x SSC, 0.1% SDS, more specifically 68°C, 0.1 x SSC, 0.1% SDS.

[0041] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to one another. For example, for DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can also include isolated nucleic acid fragments that are complementary to entire sequences, as well as substantially similar nucleic acid sequences.

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

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

[0044] Another aspect of the present application is to provide a vector comprising a polynucleotide of the present application, which may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0045] The vector of the present application may comprise 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 be able to express the target polypeptide in a suitable host. The expression control region may comprise a promoter capable of initiating transcription, an optional operator sequence for controlling such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and decoding. After being transformed into a suitable host cell, the vector can replicate or function independently of the host genome, and is integrated into the genome itself.

[0046] 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 or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0047] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome through a vector for chromosomal insertion in a cell. The polynucleotide can be inserted into a chromosome by any method known in the art, for example, but not limited to, homologous recombination. A selection marker for checking the presence or absence of insertion into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to check the presence or absence of insertion of a target nucleic acid molecule, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide is used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other phenotypes, so that transformed cells can be selected.

[0048] The term "transformation" in the present application 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 may include all of the above, regardless of whether it is inserted and located in the chromosome of the host cell or located extrachromosomally, as long as it can be expressed in the host cell. The polynucleotide may also include DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form as long as it can be introduced and expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all elements necessary for its own expression. The expression cassette may usually include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replicating. The polynucleotide may also be introduced into the host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0049] In addition, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the mutant L-threonine excretion protein of interest of the present application.

[0050] Another aspect of the present application is to provide a microorganism comprising a mutant L-threonine excretion protein of the present application or a polynucleotide of the present application. The strain of the present application may comprise a mutant L-threonine excretion protein of the present application, a polynucleotide encoding said polypeptide, or a vector comprising a polynucleotide of the present application.

[0051] In the present application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially, and may be microorganisms in which a specific mechanism has been weakened or enhanced by inserting an exogenous gene or enhancing or inactivating the activity of an endogenous gene, and may also be microorganisms that contain genetic modifications for the production of a desired polypeptide, protein, or product.

[0052] The strain of the present application may be a microorganism in which L-threonine-producing ability has been imparted to a strain that naturally has L-threonine-producing ability or a strain that does not have L-threonine-producing ability. As an example, the strain may be a microorganism in which the mutant L-threonine export protein of the present application or a polynucleotide encoding the same has been introduced to improve L-threonine export ability, but is not limited thereto.

[0053] The strain of the present application may be a microorganism having an increased L-threonine-producing ability compared to a parent strain or a wild-type Corynebacterium strain not containing the mutant of the present application. The microorganism may have an improved L-threonine-producing ability due to the introduction of the mutant of the present application, by increasing the L-threonine-secreting ability.

[0054] For example, the L-threonine excretion protein non-modified microorganism, which is the subject strain for comparing the increase in L-threonine production ability, may be, but is not limited to, L-threonine producing strains, Corynebacterium glutamicum CA09-0903 strain (Accession No. KCCM12502P, Korean Patent Registered No. 10-2126951) and Corynebacterium glutamicum KFCC10881-THR (Accession No. KCCM11222P, US 10590446 B2).

[0055] For example, the recombinant strain having increased L-threonine production ability may be increased by about 1% or more, specifically, about 1% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 32.5% or more, about 35% or more, about 37.5% or more, about 40% or more, about 42.5% or more, about 45% or more, about 46% or more, or about 47% or more (there is no particular upper limit, and it may be, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 15% or less), as long as there is an increase in the L-threonine production ability compared to the parent strain, the unmodified microorganism, or the unmodified microorganism before the mutation, but is not limited thereto. In another example, the recombinant strain having increased L-threonine producing ability may have an increased L-threonine producing ability of about 1.01-fold or more, about 1.05-fold or more, about 1.10-fold or more, about 1.15-fold or more, about 1.20-fold or more, about 1.25-fold or more, about 1.30-fold or more, about 1.325-fold or more, about 1.35-fold or more, about 1.375-fold or more, about 1.40-fold or more, about 1.425-fold or more, about 1.45-fold or more, about 1.46-fold or more, or about 1.47-fold or more (there is no particular upper limit, and it may be, for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, or about 2-fold or less) compared to the parent strain before mutation, the unmodified microorganism, or the unmodified microorganism having an L-threonine excretion protein, but is not limited thereto.

[0056] In the present application, the term "non-modified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type or natural strain itself, or a strain before its traits are changed by genetic mutations caused by natural or artificial factors. In addition, the term "L-threonine export protein non-modified microorganism" in the present application refers to a strain into which the L-threonine export protein mutants described herein have not been introduced or have not been introduced. The L-threonine export protein non-modified microorganism in the present application does not exclude strains containing modifications of other proteins or other genes other than the modification of the L-threonine export protein or the polynucleotide encoding it.

[0057] In this application, the term "unaltered microorganism" may be used interchangeably with "pre-altered strain", "pre-altered microorganism", "non-mutated strain", "non-mutated strain", "non-mutated microorganism" or "reference microorganism".

[0058] The microorganism of the present application may be, but is not limited to, a microorganism containing a mutant L-threonine export protein or a polynucleotide encoding the same; or a microorganism genetically modified to contain a mutant L-threonine export protein or a polynucleotide encoding the same (e.g., a recombinant microorganism). The term "intrinsic activity" refers to the activity of a specific polypeptide that a parent strain, a wild type, or a non-modified microorganism originally had before the trait change when the trait has been changed due to a genetic mutation caused by natural or artificial factors. This term may be used interchangeably with "activity before the change".

[0059] As yet another example of the present application, the microorganism of the present application is Corynebacterium stationis, Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium The bacterium may be Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

[0060] As another example, the recombinant microorganism of the present application may be a microorganism in which the activity of a part of a protein in the L-threonine biosynthetic pathway is additionally enhanced, or the activity of a part of a protein in the L-threonine decomposition pathway is additionally weakened, thereby enhancing L-threonine production ability.

[0061] Specifically, in order to enhance the biosynthetic pathway of L-threonine in the microorganism of the present application, the expression of, for example, the thrC gene encoding threonine synthase, the ppc gene encoding phosphoenolpyruvate carboxylase, the galP gene involved in glucose influx, the lysC gene encoding lysine-sensitive aspartokinase 3, the hom gene encoding homoserine dehydrogenase, or the pyc gene inducing an increase in the oxaloacetate pool can be enhanced or increased in the microorganism.

[0062] In order to release the feedback inhibition of L-threonine, for example, a gene mutation can be introduced into the lysC gene, the hom gene, or the thrA gene having the bifunctional properties of aspartokinase and homoserine dehydrogenase 1 (bifunctional aspartokinase / homoserine dehydrogenase 1).

[0063] In order to inactivate genes that weaken the L-threonine biosynthetic pathway, for example, the expression of the pckA gene involved in converting oxaloacetate (OAA), an L-threonine biosynthetic intermediate, to phosphoenolpyruvate (PEP), the tyrR gene that represses the lysC gene, the galR gene that represses the expression of the galP gene involved in glucose influx, or the mcbR gene, which is a DNA-binding transcriptional dual regulator, may be weakened or inactivated in a microorganism.

[0064] In order to increase the activity of the L-threonine operon, a plasmid containing the threonine operon (Japanese Patent Publication No. 2005-227977) composed of genes encoding aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine synthase, or a threonine operon derived from Escherichia coli, may be introduced into a microorganism (TURBA E, et al., Agric. Biol. Chem. 53:2269-2271, 1989), thereby increasing expression of the threonine operon in the microorganism.

[0065] Resistance to L-threonine analogs such as α-amino-β-hydroxyvaleric acid or D,L-threonine hydroxamate may also be imparted. In addition, the dapA (dihydrodipicolinate synthase), lysA (diaminopimelate decarboxylase), and ddh (diaminopimelate dehydrogenase) genes, which act in the biosynthetic pathway of L-lysine, which has a common precursor with L-threonine, may be weakened.

[0066] However, the present invention is not limited thereto, and L-threonine producing ability can be enhanced by any gene expression regulation method known in the art. In the present application, the term "attenuation" of the activity of a polypeptide (including, as an example, the proteins specified in the name of each enzyme) is a concept that includes all of the following: reduced activity or no activity compared to the endogenous activity. The attenuation may be mixed with terms such as inactivation, deficiency, down-regulation, decrease, reduction, and attenuation.

[0067] The attenuation may include cases where the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by the microorganism due to a mutation in the polynucleotide encoding the polypeptide, etc., where the overall polypeptide activity and / or concentration (expression level) in the cell is lower than that of a wild-type strain due to inhibition of gene expression of the encoding polynucleotide or inhibition of translation into the polypeptide, etc., where the expression of the polynucleotide does not occur at all, and / or where the polypeptide activity is absent despite the expression of the polynucleotide. The term "inactivated, depleted, reduced, down-regulated, decreased, attenuated" of a polypeptide activity compared to its endogenous activity means that the activity of a specific polypeptide is reduced compared to that originally possessed by a parent strain or a non-transformed microorganism before transformation.

[0068] Such attenuation of the activity of a polypeptide can be achieved by any method known in the art, but is not limited to these, and can be achieved by application of various methods well known in the art (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014; 15(2): 2773-2793, Sambrook et al., Molecular Cloning 2012, etc.).

[0069] Specifically, the activity of the polypeptide of the present application can be attenuated by: 1) A deletion of all or part of a gene encoding a polypeptide; 2) modification of an expression control region (or expression control sequence) so that expression of a gene encoding a polypeptide is reduced; 3) modification of the amino acid sequence constituting the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence) so that the activity of the polypeptide is eliminated or weakened; 4) modification of a gene sequence encoding a polypeptide such that the activity of the polypeptide is eliminated or attenuated (e.g., deletion / substitution / addition of one or more nucleic acid bases on the nucleic acid base sequence of the polypeptide gene to encode a polypeptide that has been altered such that the activity of the polypeptide is eliminated or attenuated); 5) a modification of the nucleotide sequence encoding the initiation codon or the 5'-UTR region of a gene transcript encoding a polypeptide; 6) introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence to the front end of a gene encoding a polypeptide in order to form a secondary structure to which ribosomes cannot attach; 8) Reverse transcription engineering (RTE) of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide; or 9) A combination of two or more selected from the above 1) to 8) may be used, but is not particularly limited thereto.

[0070] for example, The 1) deletion of a part or the whole of the gene encoding the polypeptide may be removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacement with a polynucleotide lacking some nucleotides, or replacement with a marker gene.

[0071] The modification of the expression regulatory region (or expression regulatory sequence) in 2) above may be a mutation in the expression regulatory region (or expression regulatory sequence) caused by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or an exchange with a sequence having a weaker activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0072] The modification of the amino acid sequence or polynucleotide sequence in 3) and 4) above may be, but is not limited to, the occurrence of sequence mutations by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, so as to weaken the activity of the polypeptide, or the exchange with an amino acid sequence or polynucleotide sequence improved to have a weaker activity or an amino acid sequence or polynucleotide sequence improved to have no activity. For example, the expression of a gene can be inhibited or weakened by introducing a mutation in a polynucleotide sequence to form a stop codon, but is not limited to this.

[0073] The 5) modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, a replacement with a base sequence encoding another start codon that has a lower polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0074] The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide (6) can be carried out with reference to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol. 1(1) 1986].

[0075] 7) Addition of a sequence complementary to the Shine-Dalgarno sequence to the front end of a gene encoding a polypeptide in order to form a secondary structure to which ribosomes cannot attach may disable or slow down mRNA translation.

[0076] In addition, the addition of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE) may create an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide, thereby weakening the activity.

[0077] In the present application, the term "enhancement" of a polypeptide activity means that the activity of the polypeptide is increased compared to its endogenous activity. The term "enhancement" may be used interchangeably with terms such as "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and increase can all include the display of an activity that was not originally possessed, or the display of an activity that is improved compared to the endogenous activity or the activity before modification. "Enhancement," "up-regulation," "overexpression," or "increase" of a polypeptide activity compared to its endogenous activity means that the activity and / or concentration (expression level) of a particular polypeptide is improved compared to the activity and / or concentration (expression level) that the parent strain or non-modified microorganism originally possessed before the transformation.

[0078] The enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. The enhancement of the activity of the polypeptide can be confirmed by an increase in the activity, expression level, or amount of a product secreted from the polypeptide.

[0079] The activity of the polypeptide can be enhanced by various methods well known in the art, and is not limited as long as the activity of the polypeptide of interest can be enhanced compared to that of the microorganism before transformation. Specifically, the activity of the polypeptide can be enhanced by genetic engineering and / or protein engineering, which are routine methods in molecular biology and well known to those skilled in the art, but is not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes, Advances in Cell Biology, 2010, Vol. 2.1-16, Sambrook et al., Molecular Cloning, 2012, etc.).

[0080] Specifically, the enhancement of the activity of the polypeptide of the present application is achieved by: 1) increasing the intracellular copy number of a polynucleotide encoding a polypeptide; 2) replacing the expression regulatory region of the gene on the chromosome that encodes the polypeptide with a sequence with stronger activity; 3) a modification of the nucleotide sequence encoding the initiation codon or the 5'-UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the activity of the polypeptide is enhanced; 5) modifying a polynucleotide sequence encoding a polypeptide so as to enhance the polypeptide's activity (e.g., modifying the polynucleotide sequence of the polypeptide gene to encode a polypeptide that has been modified so as to enhance the polypeptide's activity); 6) introduction of a foreign polypeptide exhibiting a polypeptide activity or a foreign polynucleotide encoding the same; 7) codon optimization of a polynucleotide encoding a polypeptide; 8) analyzing the tertiary structure of a polypeptide and selectively deforming or chemically modifying exposed sites; or 9) A combination of two or more selected from the above 1) to 8) may be used, but is not particularly limited thereto.

[0081] More specifically, The 1) increase in intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a host cell a vector to which a polynucleotide encoding the polypeptide is operably linked, the vector being capable of replicating and functioning independently of a host. Alternatively, the increase may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into a chromosome in the host cell. The introduction into a chromosome can be achieved by, but is not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell. The vector is as described above.

[0082] The 2) exchange of the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the polypeptide with a sequence having a stronger activity may be, for example, the occurrence of a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to further enhance the activity of the expression regulatory region, or the exchange with a sequence having a stronger activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and decoding. As an example, but not limited to, the original promoter may be exchanged with a strong promoter.

[0083] Examples of known strong promoters include, but are not limited to, CJ1 to CJ7 promoters (U.S. Patent US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (U.S. Patent US 10584338 B2), O2 promoter (U.S. Patent US 10273491 B2), tkt promoter, and yccA promoter.

[0084] The modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, a replacement with a base sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0085] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) may be, but is not limited to, a mutation in the sequence of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, caused by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to enhance the activity of the polypeptide, or an amino acid sequence or polynucleotide sequence improved to have stronger activity or an amino acid sequence or polynucleotide sequence improved to increase activity. Specifically, the replacement can be performed by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited to these. The vector used in this case may further include a selection marker for checking whether or not the chromosome has been inserted.

[0086] The introduction of a foreign polynucleotide exhibiting the activity of the polypeptide 6) may be the introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide into a host cell. The foreign polynucleotide is not limited in origin or sequence as long as it exhibits the same / similar activity as the polypeptide. The method used for the introduction can be a known transformation method appropriately selected by those skilled in the art, and the introduced polynucleotide is expressed in a host cell to produce a polypeptide, and its activity can be increased.

[0087] The codon optimization of the polynucleotide encoding the polypeptide (7) may be that of an endogenous polynucleotide that has been codon-optimized to increase transcription or translation in a host cell, or that of an exogenous polynucleotide that has been codon-optimized to optimize transcription or translation in a host cell.

[0088] The above 8) analyzing the tertiary structure of a polypeptide and selecting and modifying or chemically modifying exposed sites may involve, for example, comparing sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining template protein candidates according to the degree of sequence similarity, confirming the structure based on the template protein candidates, and selecting and modifying exposed sites to be modified or chemically modified.

[0089] Such enhanced polypeptide activity may be, but is not limited to, an increase in the activity, concentration, or expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in a wild-type or unaltered microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0090] In the microorganism of the present application, modification of a part or the whole of a polynucleotide may be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal insertion in the microorganism or genome editing using engineered nuclease (e.g., CRISPR-Cas9) and / or (b) treatment with light and / or chemicals such as ultraviolet light and radiation. The method of modifying a part or the whole of the gene may include a method using DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene may be injected into the microorganism to cause homologous recombination, thereby deleting a part or the whole of the gene. The injected nucleotide sequence or vector may contain a dominant selection marker, but is not limited to these.

[0091] In the microorganism of the present application, the mutant L-threonine excretion protein, the polynucleotide, and L-threonine are as described in the other aspects above. Another aspect of the present application provides a method for producing L-threonine, comprising the step of culturing in a medium a microorganism comprising a mutant L-threonine excretion protein of the present application or a polynucleotide of the present application.

[0092] The method for producing L-threonine of the present application may comprise the step of culturing in a medium a microorganism comprising a mutant L-threonine excretion protein of the present application, a polynucleotide of the present application, or a vector of the present application.

[0093] In the present application, the term "culture" means growing the microorganism of the present application under appropriately controlled environmental conditions. The culture process of the present application can be carried out according to suitable media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected microorganism. Specifically, the culture may be, but is not limited to, a batch, continuous and / or fed-batch culture.

[0094] In the present application, the term "culture medium" refers to a mixture of nutrients required for culturing the microorganism of the present application as the main components, and supplies nutrients such as water essential for survival and growth, growth factors, etc. Specifically, the culture medium and other culture conditions used for culturing the microorganism of the present application can be any medium used for culturing ordinary microorganisms without any particular restrictions, and the microorganism of the present application can be cultured under aerobic conditions in an ordinary medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, etc., while controlling the temperature, pH, etc.

[0095] Specifically, culture media for Corynebacterium microorganisms can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0096] 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 nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and other appropriate amounts of carbon sources may be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0097] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; an organic nitrogen source such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more kinds, and are not limited thereto.

[0098] The phosphorus source may include potassium monophosphate, potassium diphosphate, or the corresponding sodium-containing salts. The inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and may further include amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, they are not limited thereto.

[0099] 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, foam generation may be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. 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 states, no gas may be injected or nitrogen, hydrogen or carbon dioxide gas may be injected, but is not limited thereto.

[0100] 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.

[0101] The L-threonine produced by the culture of the present application is either secreted into the medium or remains intracellularly. The L-threonine production method 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 microorganism, or a combination thereof (in any order), for example, prior to the culturing step.

[0102] The method for producing L-threonine of the present application may further include a step of recovering L-threonine from the culture medium or the Corynebacterium glutamicum strain. The recovering step may be further included after the culturing step.

[0103] Said recovery may be to collect the target L-threonine using a suitable method known in the art by the microbial culture method of the present application, for example, batch, continuous or fed-batch culture method, etc. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination of these methods can be used, and the target L-threonine can be recovered from the medium or the microorganism using a suitable method known in the art.

[0104] In addition, the L-threonine production method of the present application may further include a purification step. The purification can be carried out by using a suitable method known in the art. In one example, when the L-threonine production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step can be carried out continuously or discontinuously regardless of the procedure, or can be carried out simultaneously or integrated into one step, but is not limited thereto.

[0105] In the method of the present application, the mutant L-threonine excretion protein, polynucleotide, vector, strain, etc. are as described in the other aspects above. Another aspect of the present application is to provide a composition for producing L-threonine, comprising a mutant L-threonine export protein of the present application; a polynucleotide encoding the same; a vector comprising the polynucleotide; a microorganism comprising the mutant L-threonine export protein or a polynucleotide encoding the same; a medium in which the microorganism is cultured; or a combination of two or more of them.

[0106] The composition of the present application may further contain any suitable excipient that is commonly used in compositions for producing L-amino acids, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonicity agents.

[0107] Another aspect of the present application provides use of a mutant L-threonine export protein of the present application; a polynucleotide encoding the same; a vector containing the polynucleotide; or a microorganism containing the mutant L-threonine export protein or a polynucleotide encoding the same, for producing L-threonine.

[0108] The L-threonine export protein, mutant L-threonine export protein, polynucleotide, vector, strain, medium, and L-threonine are the same as those described in the other aspects.

[0109] Working Example The present application will be described in more detail with reference to the following examples. However, the following examples are merely preferred embodiments for illustrating the present application, and are not intended to limit the scope of the present application. Meanwhile, technical matters not described in this specification can be fully understood and easily performed by ordinary skilled artisans in the technical field of the present application or a similar technical field.

[0110] Example 1: Construction of L-threonine excretion protein mutation library and plasmid To prepare a template for error-prone PCR, a PrhtC base sequence fragment (SEQ ID NO: 6) was obtained by PCR using the primer pair of SEQ ID NO: 7 and SEQ ID NO: 8 from the genomic DNA of Escherichia coli W3110 strain.

[0111] The primers used here are as shown in Table 1 below.

[0112] [Table 1]

[0113] The recombinant plasmid pCC1BAC-PrhtC was obtained by cloning the DNA fragment obtained above into pCC1BAC(EPICENTRE) cut with SmaI restriction enzyme using the Gibson assembly method. The cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated molar amount and storing it at 50℃ for 1 hour.

[0114] To induce random mutagenesis in wild-type rhtC (rhtC WT) (SEQ ID NO: 2) encoding an L-threonine excretion protein, error-induced PCR was performed using a diversify PCR random mutagenesis kit (Takara). To select the mutation rate condition, error-induced PCR was performed under the following two conditions according to the concentration of MnSO4. Genomic DNA of E. coli W3110 strain was used as a DNA template to introduce mutations. The composition of the reactants for error-induced PCR was as shown in Table 2 below. The PCR conditions were denaturation at 95°C for 30 seconds, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 68°C for 30 seconds, repeated 25 times, and polymerization at 68°C for 60 seconds. A primer pair of SEQ ID NO: 9 and SEQ ID NO: 10 was used as primers in PCR.

[0115] The primers used here are as shown in Table 3 below.

[0116] [Table 2]

[0117] [Table 3]

[0118] The products obtained by performing error-induced PCR under the conditions in Table 2 were treated with DpnI and cloned into pCC1BAC-PrhtC digested with BamHI restriction enzyme by Gibson assembly method to obtain recombinant mutant plasmid pCC1BAC-PrhtC_rhtC(mt) library. The mutant library obtained by the above method was transformed into E. coli K12 strain and spread on LB plate medium containing 50μg / L spectinomycin.

[0119] Fifty colonies were selected from the E. coli K12 (K12 / pCC1BAC-PrhtC-rhtC(mt)) strain library transformed with the mutation library, and sequencing was performed to determine the mutation rate and whether mutations occurred at various positions. The sequencing results showed that the mutation rate was one per 1.2 kb in case #1 and one per 2.0 kb in case #2. Both cases #1 and #2 were determined to have mutation rates suitable for obtaining a mutant library, and the library constructed under the above conditions was used to select effective mutations.

[0120] In addition, to prepare a control vector pCC1BAC-PrhtC-rhtC(WT), PCR was performed using the primer pair of SEQ ID NO:9 and SEQ ID NO:10 from the genomic DNA of E. coli W3110 strain in a conventional manner to obtain a base sequence fragment (SEQ ID NO:2) encoding RhtC WT. The PCR conditions were denaturation at 95°C for 30 seconds, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 68°C for 30 seconds, repeated 25 times, and polymerization at 68°C for 60 seconds. The resulting product was treated with DpnI and cloned into pCC1BAC-PrhtC cleaved with BamHI restriction enzyme by the Gibson assembly method to prepare a recombinant mutant plasmid pCC1BAC-PrhtC_rhtC(WT).

[0121] Example 2: Identification of mutations in L-threonine excretion proteins through library screening 300μl of M9 minimal media containing 60g / L L-threonine was dispensed into a 96-deep-well plate, and the K12 / pCC1BAC-PrhtC-rhtC(mt) colonies transformed with the mutant library prepared in Example 1 and K12 / pCC1BAC and K12 / pCC1BAC-PrhtC-rhtC(WT) colonies as controls were inoculated. After culturing at 1200rpm / 15hr / 37℃, the OD was measured at a wavelength of 600nm to select strains that did not show growth inhibition in the M9 minimal media containing 60g / L L-threonine, unlike the wild-type E. coli K12 strain.

[0122] As a result, most of the mutant strains were observed to barely grow like the control strains (K12 / pCC1BAC, K12 / pCC1BAC-PrhtC_rhtC(WT)) in the deep well plate, and about 4000 strains were initially selected based on the measured OD. Of these, the top 5% (150 strains) were subjected to secondary screening, and colonies with high average values ​​were selected in triplicate. After excluding colonies with large deviations, four strains that showed growth in contrast to the control strains were finally selected and named K12 pCC1BAC-PrhtC_rhtC(m1~m4), and the OD was recorded and shown in Table 4 below.

[0123] [Table 4]

[0124] In order to confirm the base sequence of the rhtC gene contained in the four selected strains K12 / pCC1BAC-PrhtC_rhtC (m1 to m4), a DNA fragment containing the rhtC gene was amplified by PCR under the same conditions as in Example 1 using the primer pair of SEQ ID NO: 9 and SEQ ID NO: 10.

[0125] Analysis of the base sequence of the amplified gene confirmed that the K12 / pCC1BAC-PrhtC-rhtC(m1-m4) strains have one or two mutations introduced into the base sequence located at 427-429 bp downstream from the start codon of the rhtC gene ORF. That is, it was confirmed that the K12 / pCC1BAC-PrhtC_rhtC(m1-m4) strains contain a sequence encoding a mutant RhtC(N143I) protein in which the base sequence at 427-429 bp based on SEQ ID NO:2 has been mutated from the existing AAC to ATT or ATC, and the 143rd amino acid from the N-terminus of the amino acid sequence of SEQ ID NO:1, asparagine (N), has been replaced with isoleucine (I).

[0126] Example 3: Construction of plasmids for expression of mutant L-threonine excretion proteins In order to introduce the mutant gene for expressing the mutant L-threonine excretion protein confirmed in Example 2 into the chromosome of a Corynebacterium sp. strain, a recombinant vector was constructed as follows.

[0127] First, to prepare a deletion vector for Ncgl1762, fragments of the upstream and downstream homologous regions of Ncgl1762 were amplified by PCR under the same conditions as in Example 1 using the genomic DNA of Corynebacterium glutamicum ATCC13032 strain as a template and the primer pair of SEQ ID NO: 11 and SEQ ID NO: 12 and the primer pair of SEQ ID NO: 13 and SEQ ID NO: 14, respectively. The amplified product was then treated with the restriction enzyme SmaI, and ligated to pDCM2 vector (Korea Patent No. 2278000) that had been heat-treated at 65°C for 20 minutes using an Infusion Cloning Kit, and then transformed into E. coli DH5α. The transformed strain was smeared on LB solid medium containing kanamycin (25 mg / l), and the selected colonies were cultured to obtain a plasmid, which was named pDCM2-△Ncgl1762.

[0128] The primers used here are as shown in Table 5 below.

[0129] [Table 5]

[0130] Next, vectors for inserting wild-type rhtC and mutant rhtC genes at the Ncgl1762 position were constructed. PCR was performed under the same conditions as in Example 1 using the primer pair of SEQ ID NO: 15 and SEQ ID NO: 16 from the genomic DNA of E. coli W3110 strain to obtain an rhtC (WT) fragment. In addition, a mutant rhtC (N143I) fragment was amplified using the primer pair of SEQ ID NO: 15 and SEQ ID NO: 16, using the fragment of rhtC containing a sequence encoding a mutant protein in which the 143rd asparagine was replaced with isoleucine as confirmed in Example 2 as a template. Then, each fragment and the pDCM2-△Ncgl1762 vector were treated with the restriction enzyme XhoI, heat-treated at 65℃ for 20 minutes, and then ligated using the Infusion Cloning Kit to transform E. coli DH5α strain. The strain was spread on LB solid medium containing kanamycin (25 mg / l), and the selected colonies were cultured to obtain plasmids, which were named pDCM2-△Ncgl1762::PrhtC-rhtC(WT) and pDCM2-△Ncgl1762::PrhtC-rhtC(N143I), respectively.

[0131] The primers used here are as shown in Table 6 below.

[0132] [Table 6]

[0133] Example 4: Construction of Corynebacterium glutamicum KCCM12502P strain carrying mutant L-threonine export protein and analysis of L-threonine production ability The pDCM2-△Ncgl1762, pDCM2-△Ncgl1762::PrhtC_rhtC(WT), and pDCM2-△Ncgl1762::PrhtC_rhtC(N143I) vectors prepared in Example 3 were transformed by electroporation into Corynebacterium glutamicum CA09-0903 strain (accession number KCCM12502P, Korean Patent Registration No. 10-2126951) conferred with L-threonine production ability, and then a second crossover process was performed to obtain a Ncgl1762-deficient strain, a strain in which the wild-type rhtC gene or a mutant rhtC gene was inserted at the Ncgl1762 position on the chromosome, and each of the strains was obtained. The genetic manipulation was confirmed through PCR and genome sequencing using the primer pair of SEQ ID NO: 17 to SEQ ID NO: 23, which can amplify the external sites of the upstream and downstream regions of homologous recombination into which the gene was inserted.

[0134] The primers used here are as shown in Table 7 below.

[0135] [Table 7]

[0136] The obtained Ncgl1762-deficient strain, and the strain in which the wild-type rhtC gene or the mutant rhtC gene was inserted at the Ncgl1762 site were designated 12502P-del, 12502P-WT, and 12502P-N143I, respectively.

[0137] To analyze the L-threonine production ability of the three mutants, flask culture was performed using the following medium. As a control, the parent strain Corynebacterium glutamicum KCCM12502P was used and cultured in the following manner to measure the L-threonine production.

[0138] First, each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of seed medium, and cultured with shaking at 200 rpm at 30° C. for 20 hours. Then, 1 ml of the seed culture was inoculated into a 250 ml corner baffle flask containing 24 ml of production medium, and cultured with shaking at 200 rpm at 32° C. for 72 hours. The compositions of the seed medium and production medium are as follows, and the L-threonine production concentration by the culture is shown in Table 8.

[0139] <Seed medium (pH 7.0)> Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO48g, MgSO4·7H2O 0.5g, biotin 0.1mg, thiamine HCl 1mg, calcium pantothenate 22mg, nicotinamide 2mg (based on 1 liter of distilled water) <Production medium (pH 7.0)> Glucose 63g, (NH4)2SO4 28g, soy protein 20g, molasses 14g, KH2PO4 1.1g, MgSO4·7H2O 1.2g, biotin 1.8mg, thiamine hydrochloride 9mg, calcium pantothenate 9mg, MnSO4 180mg, FeSO4 200mg, ZnSO4 1mg, CuSO4 1mg, CaCO3 30g (based on 1 liter of distilled water)

[0140] [Table 8]

[0141] As a result, as shown in Table 8, the control KCCM12502P produced 3.26 g / L of L-threonine, KCCM12502P-del produced 3.24 g / L of L-threonine, and KCCM12502P-N143I, into which the mutant trait was inserted, produced 4.78 g / L of L-threonine.

[0142] The L-threonine fermentation yield of KCCM12502P-WT was increased by 3% compared to KCCM12502P, whereas the KCCM12502P-N143I strain into which the N143I mutation was introduced showed an increased rate of 47%.

[0143] Example 5: Construction of Corynebacterium glutamicum KCCM11222P strain carrying mutant L-threonine export protein and analysis of L-threonine production ability In order to confirm whether the introduction of mutant RhtC increases L-threonine production in other L-threonine-producing Corynebacterium glutamicum strains, the pDCM2-△Ncgl1762, pDCM2-△Ncgl1762::PrhtC_rhtC(WT), and pDCM2-△Ncgl1762::PrhtC_rhtC(N143I) vectors prepared in Example 3 were transformed into L-threonine-producing Corynebacterium glutamicum KFCC10881-THR (accession number KCCM11222P, US 10590446 B2) in the same manner as in Example 4, and the transformed vectors were named 11222P-del, 11222P-WT, and 111222P-N143I, respectively.

[0144] To analyze the L-threonine production ability of the three mutants, flask culture was performed using the following medium. As a control, the parent strain Corynebacterium glutamicum KCCM11222P was used and cultured as follows to measure the L-threonine production.

[0145] First, each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of seed medium, and cultured with shaking at 200 rpm at 30° C. for 20 hours. Then, 1 ml of the seed culture was inoculated into a 250 ml corner baffle flask containing 24 ml of production medium, and cultured with shaking at 200 rpm at 32° C. for 72 hours. The compositions of the seed medium and production medium are as follows, and the L-threonine production concentration by the culture is shown in Table 9.

[0146] <Seed medium (pH 7.0)> Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO48g, MgSO4·7H2O 0.5g, biotin 0.1mg, thiamine HCl 1mg, calcium pantothenate 22mg, nicotinamide 2mg (based on 1 liter of distilled water) <Production medium (pH 7.0)> Glucose 63g, (NH4)2SO4 28g, soy protein 20g, molasses 14g, KH2PO4 1.1g, MgSO4·7H2O 1.2g, biotin 1.8mg, thiamine hydrochloride 9mg, calcium pantothenate 9mg, MnSO4 180mg, FeSO4 200mg, ZnSO4 1mg, CuSO4 1mg, CaCO3 30g (based on 1 liter of distilled water)

[0147] [Table 9]

[0148] As a result, as shown in Table 9, it was confirmed that when mutant RhtC(N143I) was introduced into the L-threonine producing strain Corynebacterium glutamicum KCCM11222P, the L-threonine producing ability increased by an average of 35%.

[0149] From the above description, a person skilled in the art to which the present application pertains will understand that the present application may be implemented in other specific forms without changing its technical ideas or essential features. In this regard, it should be understood that the above described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including any modified or altered forms derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

Claims

1. A mutant L-threonine excretion protein in which the amino acid corresponding to the 143rd position in the amino acid sequence of SEQ ID NO:1 is replaced with another amino acid.

2. 2. The mutant L-threonine excretion protein according to claim 1, wherein the other amino acid is an amino acid selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline.

3. The mutant L-threonine excretion protein according to claim 2, wherein the other amino acid is isoleucine.

4. The mutant L-threonine excretion protein according to claim 1, which has a sequence identity of 70% or more and less than 100% with the amino acid sequence of SEQ ID NO:

1.

5. A polynucleotide encoding the mutant L-threonine excretion protein according to any one of claims 1 to 4.

6. A microorganism comprising a mutant L-threonine excretion protein in which the amino acid corresponding to the 143rd position in the amino acid sequence of SEQ ID NO:1 is substituted with another amino acid; or a polynucleotide encoding the same.

7. 7. The microorganism according to claim 6, wherein the microorganism has an increased ability to produce L-threonine, as compared with a Corynebacterium microorganism containing a wild-type L-threonine excretion protein having the amino acid sequence of SEQ ID NO:1 or a polynucleotide encoding the same.

8. The microorganism according to claim 6 , wherein the microorganism is a Corynebacterium microorganism.

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

10. A method for producing L-threonine, comprising the step of culturing in a medium a microorganism containing a mutant L-threonine excretion protein in which the amino acid corresponding to position 143 in the amino acid sequence of SEQ ID NO:1 is replaced with another amino acid; or a polynucleotide encoding the same.

11. A composition for producing L-threonine, comprising: a mutant L-threonine export protein in which the amino acid corresponding to position 143 in the amino acid sequence of SEQ ID NO:1 is replaced with another amino acid; a polynucleotide encoding the same; a vector containing the polynucleotide; a microorganism containing the mutant L-threonine export protein or a polynucleotide encoding the same; a medium in which the microorganism is cultured; or a combination of two or more of them.

12. A mutant L-threonine export protein in which the amino acid corresponding to the 143rd position in the amino acid sequence of SEQ ID NO:1 is replaced with another amino acid; a polynucleotide encoding the same; a vector containing the polynucleotide; or use of the mutant L-threonine export protein or a microorganism containing the polynucleotide encoding the same in producing L-threonine.

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