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, research is being conducted on improving Corynebacterium strains that produce L-amino acids by disrupting or attenuating the expression of specific genes using recombinant DNA technology. In addition, research is being actively conducted on improving L-amino acid-producing Corynebacterium strains by amplifying genes involved in the biosynthesis of each L-amino acid and studying the effect on L-amino acid production. Furthermore, foreign genes from other bacteria may be introduced. However, even if the conventional methods described above are used, there is still a need for strains with improved L-threonine production ability.

[0004] On the other hand, expression of a specific amino acid excretion gene improves the productivity of the amino acid in a microorganism. Enhancement of expression of the L-lysine excretion gene (lysE) in a microorganism of the genus Corynebacterium improves the productivity of lysine (Patent Document 1). Patent Document 2 discloses that enhancement of the yeaS gene, yahN gene, yfiK gene, and yggA gene, the functions of which are unknown, in Escherichia coli improves the productivity of L-lysine, L-alanine, L-glutamic acid, L-threonine, L-histidine, L-valine, L-arginine, L-proline, and L-isoleucine. [Prior art documents]

Chartered Documents

[0005] [Patent Document 1] International Publication No. 1997 / 023597 [Patent Document 2] European Patent No. 1016710 [Patent Document 3] Korean Patent Registration No. 10-2126951 [Patent Document 4] U.S. Patent No. 10590446 [Patent Document 5] Japanese Patent Publication No. 2005-227977 [Patent Document 6] U.S. Patent No. 7662943 [Patent Document 7] U.S. Patent No. 10584338 [Patent Document 8] U.S. Patent No. 10273491 [Patent Document 9] Korea Registration Patent No. 2278000

Non-licensed 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 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 mutant L-threonine export proteins and confirmed that when microorganisms expressing mutant L-threonine export proteins are cultured, L-threonine is produced in higher yields than when microorganisms having conventional 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 133 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 the 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 microorganism has been cultured, or a combination of two or more thereof.

[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 a microorganism that produces L-threonine is cultured using the mutant of the L-threonine export protein of the present application, it is possible to produce L-threonine in a higher yield than when a microorganism has a conventional wild-type export protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] These will be described in detail below. Each description and embodiment disclosed in this application is also applicable to other descriptions and embodiments. That is, all combinations of various elements disclosed in this application are included in this application. In addition, this application is not limited to the following specific description. Furthermore, many papers and patent documents are referenced throughout this specification, and citations are provided. The disclosure contents of the cited papers and patent documents are incorporated in their entirety into this specification by reference, thereby more clearly explaining 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 133 in the amino acid sequence of SEQ ID NO:1 is substituted with another amino acid. The mutant L-threonine excretion protein of the present application may be one in which the amino acid at the position corresponding to the 133rd 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. Alternatively, the mutant L-threonine excretion protein is one in which the amino acid at the position corresponding to the 133rd position in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid that is a polar or hydrophilic amino acid having an uncharged side chain, but is not limited thereto.

[0015] Specifically, the mutant L-threonine excretion protein has the amino acid corresponding to the 133rd position in the amino acid sequence of SEQ ID NO: 1 replaced with an amino acid selected from the group consisting of arginine, lysine, histidine, aspartic acid, asparagine, 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 has the amino acid corresponding to the 133rd position in the amino acid sequence of SEQ ID NO:1 replaced with an amino acid selected from the group consisting of serine, threonine, cysteine, tyrosine, asparagine and glutamine, for example, but not limited to, serine.

[0017] More specifically, the mutant L-threonine excretion protein of the present application may have the amino acid sequence shown in SEQ ID NO: 3, may contain said amino acid sequence, may consist of said amino acid sequence, or may essentially consist of said amino acid sequence.

[0018] The protein to be subjected to the mutagenesis of the present application may be a protein having L-threonine export activity. Specifically, the protein includes the amino acid sequence of SEQ ID NO: 1 and has threonine export activity, but is not limited thereto. Meaningless sequence addition before or after the amino acid sequence of SEQ ID NO: 1, naturally occurring mutations, and silent mutations thereof are not excluded, and any protein having the same or corresponding activity as a protein containing the amino acid sequence of SEQ ID NO: 1 is included in the protein to be subjected to the mutagenesis of the present application. For example, the protein to be subjected to the mutagenesis of the present application may be a protein consisting 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 goes without saying that a protein having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, or added is also included in the protein to be subjected to the mutagenesis of the present application, so long as it has such homology or identity and shows an efficacy corresponding to the above protein.

[0019] The mutant L-threonine export protein of the present application may be one in which the amino acid corresponding to the 133rd position in the amino acid sequence of SEQ ID NO: 1 is an amino acid other than glycine, for example serine, and which 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 represented by SEQ ID NO: 1. Needless to say, the present application also includes mutant L-threonine export proteins having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted or added, so long as the amino acid sequence has such homology or identity and exhibits an efficacy equivalent to that of the mutant L-threonine export protein of the present application.

[0020] For example, the amino acid sequence may include those having 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 having electrically charged side chains, positively charged (basic) amino acids include arginine, lysine, and histidine, negatively charged (acidic) amino acids include glutamic acid and aspartic acid, and amino acids having uncharged side chains include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0023] In the present application, a "mutated protein" or "variant" refers to a polypeptide that differs from the amino acid sequence of the variant by conservative substitution and / or modification of at least one amino acid, but maintains its functions or properties. Such variants can generally be identified by modifying at least one amino acid in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the performance of the variant may be improved, unchanged, or decreased compared to the polypeptide of the variant. Some variants also include variants in which at least one portion, such as an N-terminal leader sequence or a transmembrane domain, has been removed. Other variants also include variants in which a portion has been removed from the N- and / or C-terminus of the mature protein. The "mutant protein" is used interchangeably with mutant, alteration, mutant polypeptide, mutated protein, mutation, variant, etc. (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), but may be any term meaning mutation. For the purposes of this application, the mutant may be a polypeptide in which the amino acid at position 133 of the amino acid sequence of SEQ ID NO: 1 is substituted with serine. Specifically, the mutant is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3, but is not limited thereto.

[0024] The variant 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 linked to a signal (or leader) sequence involved in co-translationally or post-translationally protein translocation. The variant may also be linked to other sequences or linkers to allow identification, purification, or synthesis.

[0025] In this application, "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often 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 will generally hybridize to all or a portion of the sequence under moderate or high stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides having common codons in the polynucleotide 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 with default parameters as in, for example, Non-Patent Document 1. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 3), as implemented in the Needle program (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) of the EMBOSS package (including the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, FASTA (Non-Patent Documents 5, 6 and 7)). For example, BLAST or Clustal W from the National 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 a GAP computer program such as that disclosed in Non-Patent Document 3, as disclosed in Non-Patent Document 8. In summary, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include (1) a unitary matrix (identity takes the value 1 and non-identity takes the value 0) and a weighted comparison matrix of Non-Patent Document 10 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9, (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps.

[0029] As an example of the present application, the mutant L-threonine export protein of the present application may have an activity that improves 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 is used interchangeably with RhtC. The amino acid sequence of RhtC can be obtained from a publicly known database, such as NCBI Genebank.

[0031] For example, the RhtC of the present application is derived from a microorganism, specifically, from a prokaryotic microorganism, more specifically, from Escherichia coli, etc., and includes proteins having L-threonine export activity of various origins. In the present application, the pre-modification amino acid corresponding to the 133rd position of SEQ ID NO: 1 in the pre-modification amino acid sequence of the RhtC to be mutated may be glycine (G).

[0032] "Corresponding to" in this application means the amino acid residue at the recited position in the polypeptide, or an amino acid residue similar, identical or equivalent to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position will determine the particular amino acid of the sequence to which the particular sequence is referenced. "Corresponding region" in this application 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, and based thereon, each amino acid residue of the amino acid sequence can be numbered with reference to the number and position of the amino acid residues corresponding to the amino acid residues in SEQ ID NO: 1. For example, the sequence alignment algorithm in the present application can identify the positions of amino acids or positions where modifications such as substitutions, insertions, deletions, etc. occur when compared to a query sequence (also referred to as a "reference sequence").

[0034] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Document 3) or the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) can be used, but is not limited to these. Sequence alignment programs and pairwise sequence comparison algorithms known in the technical field can be used as appropriate.

[0035] Another aspect of the present application provides a polynucleotide encoding a variant L-threonine excretion protein of the present application. The rhtC gene of the present application may be 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. For example, the rhtC gene of the present application may be a polynucleotide encoding WP_000928824.1 derived from E. coli.

[0037] The term "polynucleotide" in this application means a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and refers to a DNA or RNA chain longer than a certain length, 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 133rd 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 397th to 399th 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 represented by SEQ ID NO: 3. More specifically, as an example of the present application, the polynucleotide of the present application may have the sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or may include said sequence. In addition, the polynucleotide of the present application may consist of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or may be substantially composed of said sequence.

[0039] The polynucleotide of the present application may be modified in various ways in the coding region, taking into consideration the codon degeneracy or the codons preferred in the organism in which the mutant L-threonine export protein of the present application is to be expressed, as long as the amino acid sequence of the mutant L-threonine export protein of the present application is not changed. Specifically, the polynucleotide of the present application has 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 includes the base sequence, or consists 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, or is substantially composed of the base sequence, but is not limited thereto. Here, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 133rd position of SEQ ID NO: 1 may be one of the codons encoding an amino acid other than glycine, for example, serine.

[0040] Furthermore, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a complementary sequence to all or a part of the 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 Non-Patent Documents 11 and 12). 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 than that 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 normal Southern hybridization washing conditions of 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 hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may include isolated nucleic acid fragments that are complementary to the entire sequence, 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 hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and may be appropriately adjusted by those skilled in the art depending on the purpose.

[0043] The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables known in the art (eg, Non-Patent Document 11).

[0044] Yet another aspect of the present application provides a vector comprising a polynucleotide of the present application, the vector being, but not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0045] The vector of the present application includes a DNA product comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. The expression control region includes a promoter for initiating transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate and 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 may 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, Charon21A, etc. may be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. may be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. may be used.

[0047] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome by a vector for intracellular chromosome introduction. The polynucleotide can be inserted into a chromosome by any method known in the art, such as, but not limited to, homologous recombination. A selection marker for confirming whether or not it has been inserted into the chromosome may be further included. The selection marker is for selecting cells transformed with the vector, i.e., for confirming whether or not the target nucleic acid molecule has been inserted, 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 a different phenotype, so that transformed cells can be selected.

[0048] In the present application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or a microorganism, thereby expressing the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide that is expressed in the host cell, regardless of whether it is inserted into the chromosome of the host cell or located extrachromosomally. The polynucleotide may include DNA and / or RNA that encodes the target polypeptide. The polynucleotide may be introduced in any form as long as it is introduced into the host cell and expressed. 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. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may 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 target mutant L-threonine excretion protein of the present application.

[0050] Yet another aspect of the present application provides 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, either naturally or artificially, and is a microorganism in which a specific mechanism has been weakened or enhanced by, for example, inserting an exogenous gene or enhancing or inactivating the activity of an endogenous gene, and is a microorganism that has been genetically modified to produce a desired polypeptide, protein, or product.

[0052] The strain of the present application may be a strain naturally having L-threonine producing ability, or a microorganism in which L-threonine producing ability has been imparted to a strain that does not have L-threonine producing ability, such as, but not limited to, a microorganism in which the L-threonine export ability has been improved by introducing the mutant L-threonine export protein of the present application or a polynucleotide encoding the same.

[0053] The strain of the present application may be a microorganism having improved L-threonine production ability compared to a parent strain not containing the mutant of the present application or a wild-type Corynebacterium strain. The microorganism may have improved L-threonine production ability by introducing the mutant of the present application, thereby improving L-threonine secretion ability.

[0054] For example, the subject strains for comparing whether the L-threonine production ability is improved or not include L-threonine producing strains such as Corynebacterium glutamicum CA09-0903 (Accession No. KCCM12502P, Patent Document 3) and Corynebacterium glutamicum KFCC10881-THR (Accession No. KCCM11222P, Patent Document 4), but are not limited thereto.

[0055] As an example, the recombinant strain with improved production ability has an L-threonine production ability improved by about 1% or more, specifically about 1% or more, about 2.5% or more, about 5% or more, about 7.5% or more, about 10% or more, about 12.5% ​​or more, about 15% or more, about 17.5% or more, about 20% or more, about 22.5% or more, about 25% or more, about 27.5% or more, about 30% or more, about 32.5% or more, or about 33% or more (there is no particular upper limit, 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) compared to the parent strain or unmodified microorganism before mutation, but may be any that shows an increase in + value compared to the production ability of the parent strain, unmodified microorganism, or unmodified microorganism of L-threonine excretion protein before mutation. In other examples, the recombinant strain with improved L-threonine producing ability has an L-threonine producing ability that is improved by about 1.01 times or more, about 1.025 times or more, about 1.05 times or more, about 1.075 times or more, about 1.10 times or more, about 1.125 times or more, about 1.15 times or more, about 1.175 times or more, about 1.20 times or more, about 1.225 times or more, about 1.25 times or more, about 1.275 times or more, about 1.30 times or more, about 1.325 times or more, or about 1.33 times or more (there is no particular upper limit, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less) compared to the parent strain before mutation, the unmodified microorganism, or the unmodified microorganism with an L-threonine excretion protein, but is not limited thereto.

[0056] In the present application, the term "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but refers to wild-type or natural strains themselves, or strains before their characteristics are changed due to genetic mutations caused by natural or artificial factors. In addition, in the present application, the term "unmodified L-threonine export protein microorganism" refers to a strain into which the L-threonine export protein mutant described herein has not been introduced or has not been introduced. In the present application, the unmodified L-threonine export protein microorganism does not exclude strains into which other proteins or other genes have been modified other than the modification of the L-threonine export protein or the polynucleotide encoding it have been modified.

[0057] In this application, the term "unmodified microorganism" is used interchangeably with "strain before modification", "microorganism before modification", "non-mutated strain", "unmodified strain", "non-mutated microorganism" or "reference microorganism". The microorganisms of the present application are, but are not limited to, microorganisms containing a mutant L-threonine export protein or a polynucleotide encoding the same, or microorganisms genetically modified to contain a mutant L-threonine export protein or a polynucleotide encoding the same (e.g., recombinant microorganisms). The term "endogenous activity" refers to the activity of a specific polypeptide that a parent strain, wild type, or unmodified microorganism originally had before the trait change when the trait is changed by genetic mutation due to natural or artificial factors. This term is used interchangeably with "activity before modification."

[0058] 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.

[0059] As yet another example, the recombinant microorganism of the present application may be a microorganism having enhanced L-threonine production ability by further enhancing the activity of a part of a protein in the L-threonine biosynthetic pathway or by further weakening the activity of a part of a protein in the L-threonine degradative pathway.

[0060] Specifically, in order to enhance the biosynthetic pathway of L-threonine, the microorganism of the present application may be one in which expression of, for example, the thrC gene encoding threonine synthase, the ppc gene encoding phosphoenolpyruvate carboxykinase, the galP gene involved in glucose uptake, the lysC gene encoding lysine-sensitive aspartokinase 3, the hom gene encoding homoserine dehydrogenase, the pyc gene inducing an increase in the oxaloacetate pool, etc. is enhanced or increased in the microorganism.

[0061] In order to release the feedback inhibition against L-threonine, gene mutations may be introduced into, for example, 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).

[0062] In order to inactivate genes that weaken the L-threonine biosynthetic pathway, the expression of, for example, the pckA gene, which is involved in the conversion of oxaloacetate (OAA), an intermediate in L-threonine biosynthesis, to phosphoenolpyruvate (PEP), the tyrR gene, which represses the lysC gene, the galR gene, which represses the expression of the galP gene, which is involved in glucose uptake, and the mcbR gene, which is a DNA-binding transcriptional dual regulator, may be weakened or inactivated in a microorganism.

[0063] In order to increase the activity of the L-threonine operon, a plasmid containing a threonine operon consisting of genes encoding aspartokinase, homoserine dehydrogenase, homoserine kinase, and threonine synthase (Patent Document 5), a threonine operon derived from Escherichia coli, or the like may be introduced into a microorganism (Non-Patent Document 13), thereby increasing the expression of the threonine operon in the microorganism.

[0064] Resistance to L-threonine analogs such as α-amino-β-hydroxyvaleric acid and D,L-threonine hydroxamate may also be imparted. Furthermore, 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.

[0065] However, the L-threonine-producing ability can be enhanced by any gene expression regulation method known in the art, without being limited thereto. In the present application, the term "attenuation" of the activity of a polypeptide (including, for example, proteins specified by the name of each enzyme) is a concept that includes all of the following: a decrease in activity compared to the endogenous activity, or the absence of activity. The term "attenuation" is also used interchangeably with other terms such as inactivation, deficiency, down-regulation, decrease, reduction, and attenuation.

[0066] The attenuation includes at least one of the following: 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.; the overall level and / or concentration (expression amount) of the polypeptide activity in the cell is reduced compared to the natural strain due to inhibition of expression of the gene of the encoding polynucleotide or inhibition of translation into the polypeptide, etc.; no expression of the polynucleotide at all; and no activity of the polypeptide even if the polynucleotide is expressed. The activity of a polypeptide being "inactivated", "deficient", "reduced", "down-regulated", "decreased" or "attenuated" compared to the endogenous activity means that the activity of a specific polypeptide is reduced compared to the activity of the parent strain or unmodified microorganism before the transformation.

[0067] Such attenuation of the activity of a polypeptide can be achieved by applying various methods well known in the art, including, but not limited to, those described in Non-Patent Documents 14 and 15.

[0068] Specifically, the activity of a polypeptide of the present application can be attenuated by 1) deleting all or a part of a gene encoding the polypeptide, 2) modifying an expression regulatory region (or an expression regulatory sequence) so as to reduce the expression of a gene encoding the polypeptide, 3) modifying an amino acid sequence constituting the polypeptide so as to delete or attenuate the activity of the polypeptide (e.g., deleting / substituting / adding one or more amino acids in the amino acid sequence), or 4) modifying a gene sequence encoding the polypeptide so as to delete or attenuate the activity of the polypeptide (e.g., modifying the gene sequence encoding the polypeptide so as to encode a polypeptide modified so as to delete or attenuate the activity of the polypeptide). The modification can be carried out by, but is not limited to, one or more of the following: deleting / substituting / adding one or more nucleic acid bases in the nucleic acid base sequence of a gene; 5) modifying the base sequence encoding the start codon or 5'UTR region of the gene transcript encoding a polypeptide; 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding a polypeptide; 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding a polypeptide so that a secondary structure that makes ribosome attachment impossible is formed; 8) adding a promoter to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide so that it is reverse transcribed (reverse transcription engineering, RTE); or 9) a combination of two or more selected from 1) to 8) above.

[0069] For example, 1) deleting a part or the whole of the gene encoding the polypeptide may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide in a chromosome, or by replacing it with a polynucleotide lacking some nucleotides or a marker gene.

[0070] In addition, the expression regulatory region (or expression regulatory sequence) may be modified by generating a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting a sequence having a lower 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.

[0071] The modification of the amino acid sequence or polynucleotide sequence of 3) and 4) above can be performed by, but is not limited to, generating a mutation in the sequence 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 by replacing the sequence with an amino acid sequence or polynucleotide sequence improved to have a lower activity, or an amino acid sequence or polynucleotide sequence improved to lose activity. For example, gene expression can be inhibited or weakened by, but is not limited to, introducing a mutation into a polynucleotide sequence to form a stop codon.

[0072] The nucleotide sequence encoding the start codon or 5'UTR region of the gene transcription product encoding the polypeptide (5) can be modified, for example, by substituting a nucleotide sequence encoding another start codon that has a lower expression rate of the polypeptide compared to the endogenous start codon, but is not limited to this.

[0073] 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcription product encoding the polypeptide may be carried out, for example, as described in Non-Patent Document 16.

[0074] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide so that a secondary structure is formed that makes ribosome attachment impossible may be achieved by making mRNA translation impossible or slowing down the rate.

[0075] In addition, the above 8) adding a promoter to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide so as to reverse transcribe the ORF (reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the gene transcription product encoding the polypeptide to weaken the activity.

[0076] In the present application, "enhancing" a polypeptide activity means improving the activity of the polypeptide compared to its endogenous activity. The term "enhancing" is used interchangeably with terms such as "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancing, up-regulation, overexpression, and increase all include exhibiting an activity that was not originally present, and improving an activity compared to the endogenous activity or the activity before modification. "Enhancing," "up-regulating," "overexpressing," or "improving" a polypeptide activity compared to its endogenous activity means improving the activity and / or concentration (expression level) of a specific polypeptide that was originally possessed by a parent strain or an unmodified microorganism before the transformation.

[0077] The enhancement may be achieved by introducing a foreign polypeptide, or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide has been enhanced can be confirmed by an increase in the level of activity of the polypeptide, the expression level, or the amount of a product secreted from the polypeptide.

[0078] To enhance the activity of the polypeptide, various methods well known in the art can be applied, and any method can be used as long as it can enhance the activity of the target polypeptide compared to the unmodified microorganism. Specifically, the method is a conventional method in molecular biology that uses genetic engineering and / or protein engineering well known to those skilled in the art, but is not limited to these (e.g., Non-Patent Documents 15, 17, etc.).

[0079] Specifically, the enhancement of the polypeptide activity of the present application is carried out by 1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide, 2) replacing the expression regulatory region of a gene on a chromosome encoding the polypeptide with a sequence having a stronger activity, 3) modifying the base sequence encoding the start codon or 5'UTR region of a gene transcript encoding the polypeptide, 4) modifying the amino acid sequence of the polypeptide so as to enhance the polypeptide activity, 5) modifying the polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide activity), 6) introducing a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same, 7) optimizing the codons of the polynucleotide encoding the polypeptide, 8) analyzing the tertiary structure of the polypeptide and selecting and modifying or chemically modifying exposed portions, or 9) a combination of two or more selected from 1) to 8) above, but is not particularly limited thereto.

[0080] More specifically, the above 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide may be achieved by introducing into the host cell a vector to which the polynucleotide encoding the polypeptide is operably linked, the vector replicating and functioning independently of the host. Alternatively, the above 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 the chromosome is achieved by introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell, but is not limited thereto. The vector is as described above.

[0081] The expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide is replaced with a sequence having a stronger activity, for example, by generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so that the activity of the expression regulatory region is further enhanced, or by replacing the expression regulatory region with a sequence having a higher activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating the termination of transcription and translation, and the like. For example, the expression regulatory region is replaced with a strong promoter, but is not limited to this.

[0082] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (Patent Document 6), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (Patent Document 7), the O2 promoter (Patent Document 8), the tkt promoter, and the yccA promoter.

[0083] The nucleotide sequence encoding the start codon or 5'UTR region of the gene transcription product encoding the polypeptide (3) can be modified, for example, by substituting it with a nucleotide sequence encoding another start codon that has a higher expression rate of the polypeptide compared to the endogenous start codon, but is not limited to this.

[0084] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above can be performed by, but is not limited to, generating a sequence mutation 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 enhance the activity of the polypeptide, or by replacing the sequence with an improved amino acid sequence or polynucleotide sequence having a higher activity or an improved amino acid sequence or polynucleotide sequence having an improved activity. Specifically, the replacement can be performed by, but is not limited to, inserting the polynucleotide into a chromosome by homologous recombination. Here, the vector used may further include a selection marker for checking whether it has been inserted into the chromosome.

[0085] The above 6) introduction of a foreign polynucleotide exhibiting the activity of the polypeptide may be carried out by introducing into a host cell a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide. The foreign polynucleotide may be of any origin or sequence as long as it exhibits the same / similar activity as the polypeptide. The introduction can be carried out by a person skilled in the art by appropriately selecting a known transformation method, and the introduced polynucleotide is expressed in the host cell as described above, thereby producing a polypeptide and improving its activity.

[0086] 7) Optimizing the codons of a polynucleotide encoding a polypeptide may be carried out by optimizing the codons of an endogenous polynucleotide so as to increase transcription or translation in a host cell, or by optimizing the codons of an exogenous polynucleotide so as to perform optimized transcription or translation in a host cell.

[0087] 8) Analyzing the tertiary structure of a polypeptide and selecting and altering or chemically modifying an exposed portion may be carried out, for example, by comparing sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, determining candidates for template proteins depending on the degree of sequence similarity, confirming the structure based on the candidates, and selecting and altering or modifying the exposed portion to be altered or chemically modified.

[0088] Such enhancement of polypeptide activity can be achieved by, but is not limited to, improving the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in a wild-type or unmodified microbial strain, or by increasing the amount of a product produced from the polypeptide.

[0089] In the microorganism of the present application, modification of a part or the whole of a polynucleotide can be induced by (a) homologous recombination using a vector for chromosomal introduction in the microorganism, or genome editing using engineered nuclease (e.g., CRISPR-Cas9), and / or (b) light such as ultraviolet light or radiation and / or chemical treatment, but is not limited thereto. Methods for modifying a part or the whole of the gene include methods using DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene is injected into the microorganism to cause homologous recombination, thereby deleting a part or the whole of the gene. The introduced nucleotide sequence or vector contains a dominant selection marker, but is not limited thereto.

[0090] In the microorganism of the present application, the mutant L-threonine excretion protein, the polynucleotide, L-threonine, etc. are as described above. Yet 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.

[0091] 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.

[0092] "Culturing" in the present application means growing the microorganism of the present application under appropriately adjusted environmental conditions. The culturing process of the present application can be carried out in a suitable medium and under suitable culture conditions known in the art. Such a culturing process can be easily adjusted and used by a person skilled in the art depending on the microorganism selected. Specifically, the culturing can be batch, continuous and / or fed-batch culture, but is not limited thereto.

[0093] In the present application, the term "culture medium" refers to a substance that is a mixture of nutrients necessary for culturing the microorganism of the present application as the main components, and that 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 may be any medium that is used for culturing a normal microorganism, and the microorganism of the present application can be cultured in a normal culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, etc., under aerobic conditions by adjusting the temperature, pH, etc.

[0094] Specifically, a culture medium for Corynebacterium microorganisms is disclosed in Non-Patent Document 18. In the present application, the carbon source may be 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. Also, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, corn steeping liquid, etc. may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and any other suitable carbon source may be used. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0095] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, or ammonium nitrate, or an organic nitrogen source such as an amino acid such as glutamic acid, methionine, or glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition product, or defatted soybean cake or its decomposition product. These nitrogen sources may be used alone or in combination of two or more kinds, but are not limited thereto.

[0096] The phosphorus source may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a sodium-containing salt equivalent thereto. The inorganic compound may be sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, or other suitable compounds, such as amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, they are not limited thereto.

[0097] In addition, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be added to the medium in a suitable manner during the cultivation of the microorganism of the present application to adjust the pH of the medium. Furthermore, foam generation may be suppressed during the cultivation using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, and in order to maintain the anaerobic and microaerobic states, no gas may be injected, and nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.

[0098] In the culture of the present application, the culture temperature is maintained at 20 to 45° C., specifically 25 to 40° C., and the culture is carried out for about 10 to 160 hours, but is not limited thereto. The L-threonine produced by the culture of the present application is either secreted into the medium or remains intracellularly.

[0099] The L-threonine production method of the present application may further comprise the step of providing a microorganism of the present application, the step of providing a medium for culturing said microorganism, or a combination thereof (in any order), e.g., prior to said culturing step.

[0100] The method for producing L-threonine of the present application may further include a step of recovering L-threonine from the medium used in the culture (the medium in which the culture was carried out) or the Corynebacterium glutamicum strain. The recovery step may be further included after the culture step.

[0101] The recovery may be a method of collecting the target L-threonine using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, fed-batch culture, etc. For example, centrifugation, filtration, crystallization, treatment with a 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 thereof may be used, and the target L-threonine can be collected from the medium or the microorganism using a suitable method known in the art.

[0102] In addition, the L-threonine production method of the present application may further include a purification step. Said purification can be carried out by any suitable method known in the art. For example, when the L-threonine production method of the present application includes both a recovery step and a purification step, said recovery step and said purification step may be carried out continuously or discontinuously regardless of the order, and may be carried out simultaneously or integrated into one step, but is not limited thereto.

[0103] In the method of the present application, the mutant L-threonine excretion protein, polynucleotide, vector, strain, etc. are as described above. Yet another aspect of 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 microorganism has been cultured, or a combination of two or more thereof.

[0104] The composition of the present application may further contain any suitable excipient that is commonly used in compositions for producing L-amino acids, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0105] Yet another aspect of the present application provides use of the mutant L-threonine export protein of the present application, a polynucleotide encoding the same, a vector comprising the polynucleotide, or a microorganism comprising the mutant L-threonine export protein or a polynucleotide encoding the same, for producing L-threonine.

[0106] The L-threonine export protein, mutant L-threonine export protein, polynucleotide, vector, strain, medium, L-threonine, etc. are as described above. Working Example The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the present application is not limited thereto. Note that technical matters not described in this specification are fully understood and easily implemented by a skilled engineer in the technical field of the present application or a similar technical field.

[0107] Example 1 Construction of L-threonine excretion protein mutation library and plasmid To prepare a template for error-prone PCR, first, PCR was performed using the primer pair of SEQ ID NO: 7 and SEQ ID NO: 8 from the genomic DNA of Escherichia coli W3110 strain to obtain a base sequence fragment of PrhtC (SEQ ID NO: 6).

[0108] The primers used here are shown in Table 1.

[0109] [Table 1]

[0110] Next, the recombinant plasmid pCC1BAC-PrhtC was obtained by cloning the DNA fragment obtained as described above into pCC1BAC(EPICENTRE) cut with SmaI restriction enzyme using the Gibson assembly method. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated molar amounts, and then storing at 50°C for 1 hour.

[0111] In order to induce random mutagenesis in wild-type rhtC (rhtC WT) (SEQ ID NO: 2) encoding an L-threonine excretion protein, error-prone PCR was performed using Diversify PCR random mutagenesis kit (Takara). To select the conditions for the mutation rate, error-prone PCR was performed under the following two conditions with different MnSO4 concentrations. As a DNA template for introducing mutations, genomic DNA of E. coli W3110 strain was used. The composition of the reactants for error-prone PCR is shown in Table 2. The PCR conditions were denaturation at 95°C for 30 seconds, followed by 25 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 68°C for 30 seconds, followed by polymerization at 68°C for 60 seconds. As primers for PCR, a primer pair of SEQ ID NO: 9 and SEQ ID NO: 10 was used.

[0112] The primers used here are shown in Table 3.

[0113] [Table 2]

[0114] [Table 3]

[0115] The products obtained by performing error-prone PCR under the conditions in Table 2 were treated with DpnI and cloned into pCC1BAC-PrhtC cleaved with BamHI restriction enzyme by the Gibson assembly method to obtain a 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 an LB plate medium containing 50 μg / L spectinomycin.

[0116] 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 the presence or absence of mutations at various positions. The results of sequencing showed that the mutation rate under case #1 conditions was 1 per 1.2 kb, and under case #2 conditions it was 1 per 2.0 kb. Both cases #1 and #2 were determined to have mutation rates suitable for securing a mutant library, and effective mutation selection work was performed using the library prepared under the above conditions.

[0117] In addition, to prepare a control pCC1BAC-PrhtC-rhtC(WT) vector, PCR was performed from the genomic DNA of E. coli W3110 strain using a primer pair of SEQ ID NO: 9 and SEQ ID NO: 10 in a conventional manner to obtain a nucleotide sequence fragment (SEQ ID NO: 2) encoding RhtC WT. The PCR conditions were denaturation at 95°C for 30 seconds, followed by 25 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 68°C for 30 seconds, followed by 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) vector.

[0118] Example 2 Identification of mutations in L-threonine excretion proteins by library screening 300 μl of M9 minimal media containing 60 g / L L-threonine was dispensed into a 96-deep-well plate, and colonies of K12 / pCC1BAC-PrhtC-rhtC (mt) transformed with the mutant library prepared in Example 1, and K12 / pCC1BAC and K12 / pCC1BAC-PrhtC-rhtC (WT) as controls were inoculated. Next, the medium was cultured at 1200 rpm / 15 hr / 37° C., and the OD was measured at a wavelength of 600 nm. From the M9 minimal media containing 60 g / L L-threonine, strains that did not show growth inhibition were selected, unlike wild-type E. coli K12 strains.

[0119] As a result, most of the mutant strains, like the control group strains (K12 / pCC1BAC, K12 / pCC1BAC-PrhtC_rhtC (WT)), showed almost no growth in deep well plates, so approximately 4,000 strains were selected primarily based on the measured OD. Of these, secondary screening was performed on approximately the top 5% (150 strains), and colonies with high average values ​​were selected in triplicate. Colonies with large deviations were excluded, and six strains that showed growth in contrast to the control group strains were finally selected and named K12 pCC1BAC-PrhtC_rhtC (m1-m6). The recorded ODs are shown in Table 4.

[0120] [Table 4]

[0121] In order to confirm the base sequence of the rhtC gene contained in the six selected strains K12 / pCC1BAC-PrhtC_rhtC (m1 to m6), a DNA fragment containing the rhtC gene was amplified by PCR under the same conditions as in Example 1 using the primer pair of sequence numbers 9 and 10.

[0122] Analysis of the nucleotide sequence of the amplified gene confirmed that the K12 / pCC1BAC-PrhtC-rhtC(m1-m6) strains all had one or two mutations introduced into the nucleotide sequence located 397-399 bp downstream of the ORF initiation codon of the rhtC gene. That is, the K12 / pCC1BAC-PrhtC_rhtC(m1-m6) strains were confirmed to contain a sequence encoding a mutant RhtC(G133S) protein in which the nucleotide sequence at positions 397-399 in SEQ ID NO:2 was mutated from the conventional GGC to AGC or AGT, and the glycine (G), which is the 133rd amino acid from the N-terminus of the amino acid sequence of SEQ ID NO:1, was replaced with serine (S).

[0123] Example 3 Construction of plasmids for expression of mutant L-threonine efflux proteins In order to introduce the mutant gene that expresses 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.

[0124] First, to prepare a deletion vector for Ncgl1762, the genome DNA of Corynebacterium glutamicum ATCC13032 strain was used as a template, and PCR was performed under the same conditions as in Example 1 using 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 for fragments of the homologous region upstream and downstream of Ncgl1762, respectively, to amplify the base sequence. The amplified product was then treated with the restriction enzyme SmaI, and then ligated to pDCM2 vector (Patent Document 9) that had been heat-treated at 65°C for 20 minutes using the Infusion Cloning Kit, and then transformed into Escherichia coli DH5α. The transformed strain was smeared on LB solid medium containing kanamycin (25 mg / l) as described above, and the selected colonies were cultured to obtain a plasmid, which was named pDCM2-△Ncgl1762.

[0125] The primers used here are shown in Table 5.

[0126] [Table 5]

[0127] Next, vectors for inserting the wild-type rhtC and mutant rhtC genes at the position of Ncgl1762 were prepared. PCR was performed under the same conditions as in Example 1 from the genomic DNA of E. coli W3110 strain using the primer pair of SEQ ID NO: 15 and SEQ ID NO: 16 to obtain an rhtC (WT) fragment. In addition, a mutant rhtC (G133S) fragment was amplified using the primer pair of SEQ ID NO: 15 and SEQ ID NO: 16, using as a template a fragment of rhtC containing a sequence encoding a mutant protein in which the 133rd glycine was replaced with serine, as confirmed in Example 2. Thereafter, each fragment and the pDCM2-ΔNcgl1762 vector were treated with the restriction enzyme XhoI, heat-treated at 65° C. for 20 minutes, and then ligated using the Infusion Cloning Kit, and transformed into E. coli DH5α strain. The strain was spread on LB solid medium containing kanamycin (25 mg / l), and selected colonies were cultured to obtain plasmids, which were named pDCM2-△Ncgl1762::PrhtC-rhtC(WT) and pDCM2-△Ncgl1762::PrhtC-rhtC(G133S), respectively.

[0128] The primers used here are shown in Table 6.

[0129] [Table 6]

[0130] Example 4 Construction of Corynebacterium glutamicum KCCM12502P strain expressing mutant L-threonine efflux protein and analysis of L-threonine production ability The pDCM2-△Ncgl1762, pDCM2-△Ncgl1762::PrhtC_rhtC(WT), and pDCM2-△Ncgl1762::PrhtC_rhtC(G133S) vectors prepared in Example 3 were transformed by electroporation into Corynebacterium glutamicum CA09-0903 strain (Accession No. KCCM12502P, Patent Document 3) to which L-threonine production ability had been imparted, and then a secondary crossover process was performed to obtain a strain lacking Ncgl1762 on the chromosome, and a strain having a wild-type rhtC or mutant-type rhtC gene inserted at the position of Ncgl1762. The genetic manipulation was confirmed by PCR and genome sequencing using the primer pairs of SEQ ID NO: 17 to SEQ ID NO: 23, which respectively amplify the external sites of the upstream and downstream regions of the homologous recombination where the gene was inserted.

[0131] The primers used here are shown in Table 7.

[0132] [Table 7]

[0133] 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-G133S, respectively.

[0134] To analyze the L-threonine production ability of the three mutants, they were cultured in flasks using the following media. As a control, the parent strain, Corynebacterium glutamicum KCCM12502P, was used. They were cultured as follows, and the amount of L-threonine produced was measured.

[0135] First, each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of seed medium, and cultured with shaking at 30°C and 200 rpm for 20 hours. Next, 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 32°C and 200 rpm for 72 hours. The compositions of the seed medium and production medium are as follows. The L-threonine production concentration by culture is shown in Table 8.

[0136] <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 (in 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 (in 1 liter of distilled water)

[0137] [Table 8]

[0138] As a result, as shown in Table 8, the control KCCM12502P produced 3.26 g / L of L-threonine, and KCCM12502P-del produced 3.27 g / L of L-threonine. In addition, KCCM12502P-G133S, which had the mutant rhtC(G133S) introduced therein, produced 4.36 g / L of L-threonine.

[0139] The L-threonine fermentation yield of KCCM12502P-WT showed a 5% increase compared to KCCM12502P, whereas the KCCM12502P-G133S strain into which mutant rhtC (G133S) had been introduced showed a 33% increase.

[0140] Example 5 Construction of Corynebacterium glutamicum KCCM11222P strain expressing mutant L-threonine efflux protein and analysis of L-threonine production ability In order to confirm whether introduction of mutant RhtC increases L-threonine production in other Corynebacterium glutamicum strains that produce L-threonine, the pDCM2-△Ncgl1762, pDCM2-△Ncgl1762::PrhtC_rhtC(WT), and pDCM2-△Ncgl1762::PrhtC_rhtC(G133S) vectors prepared in Example 3 were transformed into the L-threonine-producing Corynebacterium glutamicum KFCC10881-THR (deposit number KCCM11222P, Patent Document 4) in the same manner as in Example 4, and the transformed strains were named 11222P-del, 11222P-WT, and 111222P-G133S, respectively.

[0141] To analyze the L-threonine production ability of the three mutants, they were cultured in flasks using the following media. The parent strain, Corynebacterium glutamicum KCCM11222P, was used as a control. They were cultured as follows, and the amount of L-threonine produced was measured.

[0142] First, each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of seed medium, and cultured with shaking at 30°C and 200 rpm for 20 hours. Next, 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 32°C and 200 rpm for 72 hours. The compositions of the seed medium and production medium are as follows. The L-threonine production concentration by culture is shown in Table 9.

[0143] <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 (in 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 (in 1 liter of distilled water)

[0144] [Table 9]

[0145] As a result, as shown in Table 9, it was confirmed that the L-threonine producing ability was increased by an average of 25% when the mutant rhtC (G133S) was introduced into the L-threonine producing strain Corynebacterium glutamicum KCCM11222P.

[0146] From the above description, a person skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. This application should be interpreted as including all modifications or alterations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A mutant L-threonine excretion protein in which the amino acid at position 133 in the amino acid sequence of SEQ ID NO:1 has been 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 serine, threonine, cysteine, tyrosine, asparagine and glutamine.

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

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 mutant L-threonine excretion protein in which the amino acid at position 133 in the amino acid sequence of SEQ ID NO:1 has been replaced with another amino acid, or a microorganism containing a polynucleotide encoding the same.

7. The microorganism according to claim 6, wherein the microorganism has improved L-threonine producing ability compared to 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 the 133rd position in the amino acid sequence of SEQ ID NO:1 has been 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 the 133rd position in the amino acid sequence of SEQ ID NO:1 has been replaced with another amino acid, 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 has been cultured, or a combination of two or more of these.

12. A mutant L-threonine export protein in which the amino acid corresponding to the 133rd 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.

Images