Novel citrate synthase mutant and method for producing L-amino acids using the same

Abstract

The present application relates to a novel citrate synthase mutant, a microorganism containing said mutant, and a method for producing an L-amino acid using said microorganism.

Description

[Technical Field]

[0001] The present application relates to a novel citrate synthase mutant, a microorganism containing the mutant, and a method for producing L-amino acids using the microorganism. [Background technology]

[0002] In order to produce L-amino acids and other useful substances, various studies have been conducted to develop highly efficient production microorganisms and fermentation process technologies. For example, target substance-specific approaches such as increasing the expression of genes encoding enzymes involved in L-valine biosynthesis or deleting genes unnecessary for biosynthesis have been mainly used (Patent Documents 1 and 2).

[0003] On the other hand, citrate synthase (CS) is an enzyme that polymerizes acetyl-CoA and oxaloacetate, which are produced during glycolysis in microorganisms, to produce citric acid, and is also an important enzyme that determines the carbon flow into the TCA pathway.

[0004] The phenotypic changes of L-lysine-producing strains due to the deletion of the gltA gene, which encodes citrate synthase, have been reported in a previous paper (Non-Patent Document 1). However, in the case of gltA gene-deficient strains, not only is the growth of the strain inhibited, but the sugar consumption rate is significantly reduced, resulting in low lysine production per unit time. Therefore, there is still a need for research that takes into account both the effective increase in L-amino acid productivity and the growth of the strain. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 8,465,962 [Patent Document 2] Korean Patent Registration No. 10-2153534 [Patent Document 3] Korean Patent Registration No. 10-1992-0007401 [Patent Document 4] U.S. Patent No. 7662943 [Patent Document 5] U.S. Patent No. 10584338 [Patent Document 6] U.S. Patent No. 10273491 [Patent Document 7] U.S. Patent No. 8426171 [Patent Document 8] Korean Patent Publication No. 10-2020-0136813 [Patent Document 9] Korean Patent Registration No. 10-1947945

Non-licensed literature

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[0007] The present inventors have made extensive efforts to produce L-amino acids at high yields, and have confirmed that a novel citrate synthase mutant increases L-amino acid productivity, thereby completing the present application.

[0008] One object of the present application is to provide a citrate synthase mutant in which the lysine amino acid corresponding to position 415 of the amino acid sequence of SEQ ID NO: 1 is substituted with histidine.

[0009] Another object of the present application is to provide polynucleotides encoding the variants of the present application.

[0010] Another object of the present application is to provide a microorganism of the genus Corynebacterium comprising a mutant of the present application or a polynucleotide encoding said mutant.

[0011] Another object of the present application is to provide a method for producing L-amino acids using the microorganism of the present application.

[0012] Another object of the present application is to provide a composition for producing L-amino acids, comprising the microorganism of the present application; a medium in which the microorganism of the present application has been cultured; or a combination thereof.

[0013] Effect of the invention When the citrate synthase mutant of the present application is used, L-amino acids can be produced in high yields. [Means for solving the problem]

[0014] This will be explained in more detail as follows. Meanwhile, each description and embodiment disclosed in this application may also be applied to different descriptions and embodiments. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the specific descriptions set forth below are not intended to limit the scope of this application. Furthermore, numerous papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the state of the art to which this application pertains and the contents of this application.

[0015] One aspect of the present application is to provide a citrate synthase mutant in which the lysine amino acid corresponding to position 415 of the amino acid sequence of SEQ ID NO: 1 is substituted with histidine.

[0016] The variant of the present application may be a variant in which the amino acid corresponding to position 415 in the amino acid sequence of SEQ ID NO: 1 is histidine, and which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or more homology or identity to the amino acid sequence of SEQ ID NO: 1. For example, the variant of the present application may have, include, or consist of, or essentially consist of, an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity to the amino acid sequence of SEQ ID NO: 1, where the amino acid corresponding to position 415 is histidine. It is also clear that variants having such homology or identity and exhibiting the efficacy of the variant of the present application, including partial deletions, modifications, substitutions, conservative substitutions, or additions, are also included within the scope of the present application.

[0017] For example, the above amino acid sequence may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally that do not alter the function of the variant of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0018] The term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

[0019] As used herein, the term "variant" refers to a polypeptide in which one or more amino acids have been conservatively substituted and / or modified, resulting in a difference from the amino acid sequence of the variant but maintaining its functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the variant. That is, the performance of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. Some variants may also include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been deleted. Other variants may include variants in which portions have been deleted from the N- and / or C-termini of the mature protein. The term "variant" can be interchangeable with terms such as "mutated type," "modified," "mutated polypeptide," "mutated protein," "mutant," "mutein," and "divergent" (e.g., English terms such as "modification," "modified polypeptide," "modified protein," "mutant," "mutein," and "divergent"), as long as the term is used in the sense of "mutated." These terms are not limited to these. For purposes of this application, the above mutant may be a mutant in which lysine (Lys, K), the amino acid corresponding to position 415 of the amino acid sequence of SEQ ID NO: 1, is replaced with histidine (His, H).

[0020] The variants may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be conjugated to a signal (or leader) sequence involved in co- or post-translational protein translocation. The variants may also be conjugated to other sequences or linkers to allow identification, purification, or synthesis.

[0021] 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 are often used interchangeably.

[0022] Sequence homology or identity of conserved polynucleotides or polypeptides may be determined by standard sequence algorithms, with default gap penalties established by the program being used. Substantially homologous or identical sequences generally can hybridize to all or part of the sequence under moderately or highly stringent conditions. Hybridization obviously includes hybridization to polynucleotides containing common codons in polynucleotides or codons that take codon degeneracy into account.

[0023] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity may 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 may 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 from the National Database Center for Biotechnology Information, or ClustalW can be used to determine homology, similarity, or identity.

[0024] Homology, similarity, or identity of polynucleotides or polypeptides may be determined by comparing sequence information using a GAP computer program, such as that of Needleman et al. (1970), J Mol Biol. 48:443, as known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines a match as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a binary 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.

[0025] As used herein, the term "corresponding to" refers to the amino acid residue at the recited position in the polypeptide, or an amino acid residue that is similar, identical, or homologous to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position may be determining the specific amino acid of a sequence that references a particular sequence. As used herein, a "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0026] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue in the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described in 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 where a variation such as a substitution, insertion, or deletion occurs.

[0027] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) or the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) can be used, but is not limited to these. Sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the art can also be used as appropriate.

[0028] In this application, the term "citrate synthase" refers to an enzyme that polymerizes acetyl-CoA and oxaloacetate, which are produced in the glycolysis process of microorganisms, to produce citric acid. The enzyme also catalyzes the condensation reaction of acetyl-CoA with a 2-carbon acetate residue from a 4-carbon oxaloacetate molecule to produce a 6-carbon oxaloacetate. citric acid In the present application, the term "citrate synthase" may refer to citrate synthase, CS, GltA protein, or GltA. In the present application, the sequence of GltA can be obtained from the publicly known database, GenBank, of NCBI. In addition, GltA may be, but is not limited to, a polypeptide having citrate synthase activity encoded by the gltA gene.

[0029] The variants of the present application may have an activity that increases L-valine productivity compared to the wild-type polypeptide.

[0030] The variants of the present application may have a sequence identity of 80% or more with the amino acid sequence of SEQ ID NO:1.

[0031] Furthermore, the variant of the present application may include a polypeptide having the amino acid sequence of SEQ ID NO: 3. The amino acid sequence of SEQ ID NO: 3 may be an amino acid sequence of the amino acid sequence of positions 362 to 415 from the N-terminus of the amino acid sequence of SEQ ID NO: 1, in which the lysine at position 415 is substituted with histidine.

[0032] The variants of the present application may comprise an amino acid sequence of general formula 1:

[0033] [General formula 1] X1N HGGDATX2FMN KVKNKEDGVR LMGFGHRVYK NYDPRAAIVK ETAHEILEHL GGDDLLDLAI KLEEIALADD X3FISRKLYPN VDFYTGLIYR AMGFPTDFFT VLFAIGRLPG WIAHYREQLG AAGNH (SEQ ID NO: 51)

[0034] wherein X1 in the above general formula 1 is asparagine or serine; X2 is alanine or glutamic acid, X3 is tyrosine or cysteine.

[0035] A variant of the present application may have 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 8, 10, or 12. Alternatively, a variant of the present application may comprise, consist of, or consist essentially of an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 8, 10, or 12. As an example, a variant of the present application may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.7% or more sequence identity to the amino acid sequence of SEQ ID NO: 8, 10, or 12, or may comprise, consist of, or consist essentially of an amino acid sequence having the above sequence identity.

[0036] Another aspect of the present application is to provide polynucleotides encoding the variants of the present application.

[0037] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, a DNA or RNA chain of a certain length or more, and more specifically, a polynucleotide fragment encoding the above-mentioned variant.

[0038] The polynucleotide of the present application may include a polynucleotide described by a nucleic acid base sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more, but less than 100%, homology or identity to the nucleic acid base sequence described in SEQ ID NO: 2, in which the bases corresponding to positions 1243 to 1245 are CAC. It is also clear that the scope of the present application also includes polynucleotides described by nucleic acid base sequences in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, as long as the sequence encodes a polypeptide or protein having such homology or identity and exhibiting the efficacy corresponding to the variant of the present application.

[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 variant of the present application, taking into consideration codon degeneracy or preferred codons in the organism in which the variant of the present application is to be expressed. In this case, in the above-mentioned homologous or identical sequence, the codon encoding the amino acid corresponding to position 415 of SEQ ID NO: 1 may be one of the codons encoding histidine.

[0040] Furthermore, the polynucleotides of the present application include, without limitation, sequences that can hybridize under stringent conditions to probes prepared from known gene sequences, for example, sequences complementary to all or part of the polynucleotide sequences 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; F.M. Ausubel et al., "Current Protocols in Molecular Biology," John Wiley & Sons, Inc., New York, pp. 9.50-9.51, 11.7). For example, conditions include conditions under which polynucleotides with high homology or identity, such as polynucleotides with 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 with lower homology or identity do not hybridize with each other, or conditions for washing once, specifically two to three times, at a salt concentration and temperature equivalent to those for Southern hybridization, such as 60°C, 1×SSC, and 0.1% SDS, specifically 60°C, 0.1×SSC, and 0.1% SDS, more specifically 68°C, 0.1×SSC, and 0.1% SDS.

[0041] 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 are capable of hybridizing to one another. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, polynucleotides of the present application may also include isolated nucleic acid fragments that are complementary to entire sequences, as well as substantially similar nucleic acid base 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, 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 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] As an example, the polynucleotide of the present application may include a polynucleotide described by the nucleic acid base sequence of positions 1084 to 1245 based on the nucleic acid base sequence of SEQ ID NO: 9, 11, or 13, or a polynucleotide described by the nucleic acid base sequence of SEQ ID NO: 9, 11, 13, or 15.

[0045] In the polynucleotide of the present application, the variants are as described above in other aspects.

[0046] Another aspect of the present application is to provide a vector comprising the polynucleotide of the present application. The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0047] The vector of the present application may comprise a DNA construct comprising a base sequence of a polynucleotide encoding the target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide in a suitable host. The expression control region may comprise a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector can replicate or function independently of the host genome, or may be integrated into the genome itself.

[0048] The vectors used in the present application are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117 (Biotechnology Letters vol. 13, No. 10, pp. 721-726 (1991), Korean Patent Registration No. 10-1992-0007401), pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0049] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a vector for chromosomal integration in a cell. The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of the insertion into the chromosome may also be included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the insertion of the target nucleic acid molecule. A marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide, is used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0050] The term "transformation" as used herein refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, thereby enabling the expression of the polypeptide encoded by the polynucleotide in the host cell. A transformed polynucleotide may be either integrated into the host cell's chromosome or extrachromosomally located, as long as it is expressible 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 that can be introduced and expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form, operably linked to sequences necessary for expression in the host cell, but is not limited thereto.

[0051] 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 of the present application.

[0052] In the vectors of the present application, the variants and polynucleotides are as described in other aspects above.

[0053] Another aspect of the present application is to provide a microorganism of the genus Corynebacterium comprising a variant of the present application or a polynucleotide of the present application.

[0054] The microorganism of the present application may comprise a variant of the present application, a polynucleotide encoding said variant, or a vector comprising a polynucleotide of the present application.

[0055] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, including 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 a microorganism that contains genetic modifications for the production of a desired polypeptide, protein, or product.

[0056] The microorganism of the present application may be, but is not limited to, a microorganism comprising one or more of the variants of the present application, the polynucleotides of the present application, and the vectors comprising the polynucleotides of the present application; a microorganism that has been modified to express the variants of the present application or the polynucleotides of the present application; a microorganism (e.g., a recombinant strain) that expresses the variants of the present application or the polynucleotides of the present application; or a microorganism (e.g., a recombinant strain) that has the activity of the variants of the present application.

[0057] The microorganism of the present application may be a strain capable of producing an L-amino acid. Specifically, the L-amino acid producing ability of the microorganism of the present application may be the ability to produce L-valine or O-acetyl-L-homoserine.

[0058] The microorganism of the present application may be, but is not limited to, a microorganism that naturally has the ability to produce GltA or an L-amino acid, or a parent strain that does not have the ability to produce GltA or an L-amino acid, into which the mutant of the present application or a polynucleotide encoding it (or a vector containing the polynucleotide) has been introduced, and / or the ability to produce GltA or an L-amino acid has been imparted.

[0059] For example, the microorganism of the present application is a cell or microorganism transformed with the polynucleotide of the present application or a vector containing the polynucleotide of the present application and expressing the variant of the present application. For purposes of this application, the microorganism of the present application may include all microorganisms capable of producing L-amino acids, including the variant of the present application. For example, the strain of the present application may be a naturally occurring wild-type microorganism, or a recombinant strain in which the ability to produce L-amino acids has been increased by introducing a polynucleotide encoding the variant of the present application into an L-amino acid-producing microorganism. The recombinant strain with increased L-amino acid production ability may be, but is not limited to, a naturally occurring wild-type microorganism or a microorganism that is not citrate synthase-modified (i.e., a microorganism that expresses the wild-type (SEQ ID NO: 1) protein or a microorganism that does not express the variant of the present application). For example, the non-citrate synthase-transformed microorganism, which is the subject strain for comparing the increase in L-amino acid production ability, may be, but is not limited to, ATCC14067 strain, ATCC13032 strain, ATCC13869 strain, Corynebacterium glutamicum CJ7V strain, Corynebacterium glutamicum CJ8V strain, or CA08-0072 strain.

[0060] For example, a recombinant strain with increased L-amino acid production ability may have an increased L-amino acid production ability of about 1% or more, 5% or more, 7% or more, about 10% or more, about 20% or more, or about 30% or more (the upper limit is not particularly limited, and may be, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, or about 30% or less) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto as long as there is an increase in the + value compared to the L-amino acid production ability of the parent strain or unmodified microorganism before mutation. As another example, the recombinant strain with increased production ability may have an L-valine production ability that is increased by about 1.01-fold or more, about 1.05-fold or more, about 1.07-fold or more, about 1.1-fold or more, about 1.2-fold or more, or about 1.3-fold or more (the upper limit is not particularly limited, and 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 or untransformed microorganism before mutation.

[0061] The term "about" refers to a range that includes, but is not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values in a range that is equal to or similar to the numerical value following the term about.

[0062] 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 naturally occurring strain itself, or a strain before its traits are changed due to genetic mutations caused by natural or artificial factors. For example, the non-modified microorganism refers to a strain before the protein mutants described herein are introduced or have been introduced. The term "non-modified microorganism" may be used interchangeably with "strain before modification," "microorganism before mutation," "non-mutated strain," "non-modified strain," "non-mutated microorganism," or "reference microorganism."

[0063] In another example of the present application, the microorganism of the present application is Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

[0064] The microorganism of the present application may be a microorganism in which the NCgl2335 protein is further weakened.The microorganism of the present application may be a microorganism in which the activity of a protein selected from the group consisting of acetolactate synthase isozyme 1 small subunit (IlvN) and L-methionine / branched-chain amino acid exporter (YjeH) is further enhanced.

[0065] Specifically, the L-valine-producing microorganism of the present application may further have enhanced I1vN activity and / or attenuated NCgl2335 activity, and the O-acetyl-L-homoserine-producing microorganism of the present application may further have enhanced YjeH (L-methionine / branched-chain amino acid exporter) activity.

[0066] In the present application, the term "attenuation" of a polypeptide activity is a concept that encompasses a decrease in activity or no activity compared to the endogenous activity. The term "attenuation" may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0067] The attenuation can also 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, for example, a mutation in the polynucleotide encoding the polypeptide; where the overall polypeptide activity and / or concentration (expression level) in the cell is lower than that of a wild-type strain due to, for example, inhibition of gene expression of the encoding polynucleotide or inhibition of translation into the polypeptide; where the polynucleotide is not expressed at all; and / or where the polypeptide activity is absent despite the expression of the polynucleotide. The term "endogenous activity" refers to the activity of a specific polypeptide originally possessed by a parent strain, wild-type, or unaltered microorganism before the trait has been altered due to genetic mutation caused by natural or artificial factors. This term may be used interchangeably with "activity before the transformation." The term "inactivation, deficiency, reduction, downregulation, decrease, or attenuation" of a polypeptide activity compared to the endogenous activity means that the activity of a specific polypeptide is reduced compared to that originally possessed by a parent strain or unaltered microorganism before the trait has been altered.

[0068] The activity of such polypeptides can be attenuated by any method known in the art, including, but not limited to, by applying 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 attenuation of the polypeptide of the present application is 1) Deletion of all or part of a gene encoding a polypeptide; 2) modification of the expression control region (or expression control sequence) so that expression of the gene encoding the 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 as to eliminate or attenuate the activity of the polypeptide; 4) modification of the gene sequence encoding the polypeptide so that the activity of the polypeptide is eliminated or attenuated (e.g., deletion / substitution / addition of one or more nucleic acid bases in the nucleic acid base sequence of the polypeptide gene so as to encode a polypeptide that has been altered so that the activity of the polypeptide is eliminated or attenuated); 5) a modification of the nucleotide sequence encoding the initiation codon or 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 above gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide in order to form a secondary structure to which ribosomes cannot attach; 8) 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); or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited to this.

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

[0071] Furthermore, the above-mentioned 2) modification of the expression regulatory region (or expression regulatory sequence) may be mutation of the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacement with a sequence having 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] Furthermore, the modifications of the amino acid sequence or polynucleotide sequence in 3) and 4) above may be, but are not limited to, mutations in the amino acid sequence of the polypeptide or the sequence of the polynucleotide encoding the polypeptide, such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to attenuate the activity of the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence that has been improved to have weaker activity or no activity. For example, but not limited to, introducing a mutation into the polynucleotide sequence to form a stop codon may inhibit or attenuate gene expression.

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

[0074] 6) For the introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide, see, for example, 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 before the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure that prevents ribosome attachment may disable or slow down mRNA translation.

[0076] 8) 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 attenuating the activity.

[0077] As used herein, the term "enhancement" of polypeptide activity means that the activity of a 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 may all encompass the display of an activity not inherently possessed, or an improved activity compared to the endogenous activity or the activity prior to transformation. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or an unmodified microorganism prior to transformation, in cases where a trait has been altered by genetic mutation due to natural or artificial factors. This term may be used interchangeably with "activity prior to transformation." The term "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 specific polypeptide is improved compared to the activity and / or concentration (expression level) of a specific polypeptide inherently possessed by a parent strain or an unmodified microorganism prior to 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 thereto, as long as the activity of the polypeptide of interest can be enhanced compared to that of the microorganism before modification. Specifically, the enhancement may be achieved by utilizing genetic engineering and / or protein engineering, which are routine methods in molecular biology and well known to those of ordinary skill 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 polypeptide activity of the present application is achieved by: 1) an increase in the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the expression regulatory region of the gene on the chromosome that encodes the polypeptide with a sequence with strong activity; 3) a modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the polypeptide activity is enhanced; 5) modifying a polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide's activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide's activity); 6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same; 7) codon optimization of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of a polypeptide and selectively modifying or chemically modifying exposed sites; or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0081] More specifically, The 1) increase in the intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a host cell a vector capable of replicating and functioning independently of the host, to which a polynucleotide encoding the polypeptide is operably linked. 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 replacement of the expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide with a sequence with stronger activity may involve, for example, mutation of the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or replacement with a sequence with stronger activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. One example is, but is not limited to, replacing the original promoter with a strong promoter.

[0083] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (U.S. Pat. No. 7,662,943), 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 (U.S. Pat. No. 10,584,338), the O2 promoter (U.S. Pat. No. 10,273,491), the tkt promoter, and the yccA promoter.

[0084] The modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide (3) may be, for example, but is not limited to, substituting a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon.

[0085] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above may be, but is not limited to, a sequence mutation such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, or an amino acid sequence or polynucleotide sequence modified to have stronger activity or to have increased activity. The replacement can be performed, for example, by inserting the polynucleotide into a chromosome via homologous recombination, but is not limited to this. The vector used in this case may further contain a selection marker for detecting the presence or absence of insertion into the chromosome. The selection marker is as described above.

[0086] The introduction of an exogenous polynucleotide that exhibits the activity of a polypeptide (6) may be the introduction of an exogenous polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide into a host cell. The exogenous polynucleotide is not limited in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be any known transformation method appropriately selected by those skilled in the art, and the introduced polynucleotide may be expressed in a host cell to produce a polypeptide, and its activity may 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] In addition, the above 8) analyzing the tertiary structure of a polypeptide and selecting and deforming or chemically modifying exposed sites may involve, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and deforming or chemically modifying exposed sites.

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

[0090] Modification of a portion or all of a polynucleotide in the microorganism of the present application (e.g., modification to encode the above-described protein mutant) 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 nucleases (e.g., CRISPR-Cas9) and / or (b) treatment with light and / or chemicals, such as ultraviolet light and radiation. Methods for modifying a portion or all of the gene include methods using DNA recombination technology. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene may be injected into the microorganism to cause homologous recombination, thereby deleting a portion or all of the gene. The injected nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

[0091] More specifically, the L-valine-producing microorganism of the present application may be a microorganism comprising a polypeptide set forth in the amino acid sequence of SEQ ID NO: 27 and / or a polynucleotide set forth in the nucleic acid sequence of SEQ ID NO: 28. Additionally, the O-acetyl-L-homoserine-producing microorganism of the present application may be a microorganism comprising a mutation selected from the group consisting of: a polypeptide set forth in the amino acid sequence of SEQ ID NO: 47 and / or a polynucleotide set forth in the nucleic acid sequence of SEQ ID NO: 48; an inactive polypeptide set forth in the amino acid sequence of SEQ ID NO: 37 and / or a deletion in the polynucleotide set forth in the nucleic acid sequence of SEQ ID NO: 38.

[0092] In the microorganisms of the present application, the mutants, polynucleotides, etc. are as described above in other aspects.

[0093] Another aspect of the present application provides a method for producing an L-amino acid, comprising the step of culturing a Corynebacterium microorganism containing a mutant of the present application or a polynucleotide of the present application in a medium.

[0094] The method for producing an L-amino acid of the present application may include culturing a Corynebacterium glutamicum strain containing a mutant of the present application, a polynucleotide of the present application, or a vector of the present application in a medium.

[0095] Additionally, in the method for producing an L-amino acid of the present application, the L-amino acid may be L-valine, O-acetyl-L-homoserine, or L-methionine.

[0096] In the present application, the term "culturing" refers to growing the Corynebacterium microorganism of the present application under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using appropriate media and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be, but is not limited to, a batch, continuous, and / or fed-batch culture.

[0097] In the present application, the term "culture medium" refers to a mixture of nutrients required for culturing the microorganism of the genus Corynebacterium 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 genus Corynebacterium of the present application are not particularly limited as long as they are media used for culturing conventional microorganisms, and the microorganism of the genus Corynebacterium of the present application can be cultured under aerobic conditions while controlling the temperature, pH, etc. in a conventional culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins, etc.

[0098] 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)].

[0099] 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.; and amino acids such as glutamic acid, methionine, lysine, etc. Natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, bacus, and corn steeping liquid may also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used. A variety of other suitable carbon sources may also be used without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0100] 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.; or an organic nitrogen source such as an amino acid such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its degradation products, defatted soybean cake or its degradation products, etc. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0101] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, 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 other compounds, including amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, the present invention is not limited thereto.

[0102] During the cultivation of the Corynebacterium glutamicum strain of the present application, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner. Furthermore, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, to maintain an aerobic state in the medium, oxygen or an oxygen-containing gas can be injected into the medium, and to maintain an anaerobic or microaerobic state, no gas can be injected or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limiting.

[0103] 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 to this.

[0104] The L-amino acids produced by the culture of the present application are either secreted into the medium or remain intracellularly.

[0105] The method for producing an L-amino acid of the present application may further include a step of preparing a Corynebacterium microorganism of the present application, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example, before the culturing step.

[0106] The method for producing an L-amino acid of the present application may further include a step of recovering the L-amino acid from the culture medium (cultured medium) or the Corynebacterium microorganism of the present application. The recovery step can be performed after the culturing step.

[0107] The recovery may involve collecting the desired L-amino acid using a suitable method known in the art based on the microbial culture method of the present application, such as a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination of these methods can be used to recover the desired L-amino acid from the medium or the microorganism using a suitable method known in the art.

[0108] Furthermore, the method for producing an L-amino acid of the present application may further include a purification step. The purification can be performed using an appropriate method known in the art. For example, when the method for producing an L-amino acid of the present application includes both a recovery step and a purification step, the recovery step and the purification step can be performed continuously or discontinuously in any order, or can be performed simultaneously or integrated into one step, but this is not limiting.

[0109] Additionally, the method for producing L-methionine of the present application may further include a step of converting the O-acetyl-L-homoserine to L-methionine. In the method for producing L-methionine of the present application, the converting step may be further included after the culturing step or the recovering step. The converting step may be carried out using a suitable method known in the art (U.S. Patent No. 8,426,171). In one embodiment, the method for producing L-methionine of the present application may include a step of producing L-methionine by contacting O-acetyl-L-homoserine, methyl mercaptan, and O-acetylhomoserine sulfhydrylase, cystathionine gamma-synthase, or O-succinyl homoserine sulfhydrylase.

[0110] In the methods of the present application, the mutant, polynucleotide, vector, microorganism, etc. are as described in other aspects above.

[0111] Another aspect of the present application is to provide a composition for producing an L-amino acid, comprising a Corynebacterium microorganism containing a variant of the present application, a polynucleotide encoding the variant of the present application, or a vector containing the polynucleotide of the present application; a medium in which the microorganism is cultured; or a combination thereof.

[0112] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing L-amino acids, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonicity agents.

[0113] In the composition for producing an L-amino acid of the present application, the L-amino acid may be L-valine, O-acetyl-L-homoserine, or L-methionine.

[0114] In the composition of the present application, the mutant, polynucleotide, vector, strain, medium, etc. are as described in the other embodiments above. DETAILED DESCRIPTION OF THE INVENTION

[0115] The present application will be described in more detail below through experimental examples. However, the following examples are merely preferred embodiments for illustrating the present application, and are not intended to limit the scope of the present application. Meanwhile, technical matters not described in this specification can be fully understood and easily implemented by those skilled in the technical field of the present application or a similar technical field.

[0116] Example 1: Citrate synthase (GltA) mutant vector construction The inventors identified the 415th amino acid residue of GltA as the acetyl-coA binding site and predicted that substituting this residue with another amino acid would increase the Km value of acetyl-coA while weakening citrate synthase activity. To address this issue, we constructed vectors that replace the lysine at position 415 of GltA with other amino acids. Specifically, we constructed vectors containing mutations to replace the lysine at position 415 with histidine (K415H), tryptophan (K415W), and glycine (K415G).

[0117] Using wild-type Corynebacterium glutamicum ATCC14067 gDNA (genomic DNA) as a template, PCR was performed using the primer pair of SEQ ID NOs: 15 and 17 with the primer pair of SEQ ID NOs: 16 and 18, the primer pair of SEQ ID NOs: 15 and 20 with the primer pair of SEQ ID NOs: 18 and 19, and the primer pair of SEQ ID NOs: 15 and 22 with the primer pair of SEQ ID NOs: 18 and 21. Of the six fragments obtained above, overlapping PCR was performed again using a mixture of both fragments as a template and the primer pair of SEQ ID NOs: 15 and 18 to obtain three fragments. PCR was performed at 94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute 30 seconds, followed by 72°C for 5 minutes. The pDCM2 vector (SEQ ID NO: 14, Korean Patent Publication No. 10-2020-0136813) was digested with smaI, and the three PCR products obtained above were each fusion cloned. Fusion cloning was performed using the In-Fusion® HD Cloning Kit (Clontech). The resulting plasmids were named pDCM2-gltA(K415H), pDCM2-gltA(K415W), and pDCM2-gltA(K415G), respectively. The sequences of the primers used in this example are listed in Table 1 below.

[0118] [Table 1]

[0119] Example 2: Introduction of GltA mutations into L-valine-producing strains and evaluation 2-1. Construction and evaluation of L-valine-producing strains A single mutation [ilvN(A42V); Biotechnology and Bioprocess Engineering, June 2014, Volume 19, Issue 3, pp. 456-467] (SEQ ID NO: 27) was introduced into the acetolactate synthase isozyme 1 subunit (IlvN) of wild-type Corynebacterium glutamicum ATCC14067 and ATCC13869 to create strains with improved L-valine production (Korean Patent Registration No. 10-1947945).

[0120] Specifically, PCR was performed using wild-type Corynebacterium glutamicum ATCC14067 DNA (genomic DNA) as a template and the primer pair of SEQ ID NOs: 29 and 31, and the primer pair of SEQ ID NOs: 30 and 32, respectively. Using a mixture of the two fragments obtained above as a template, overlapping PCR was performed again using the primer pair of SEQ ID NOs: 29 and 32, to obtain three fragments. PCR was performed after denaturation at 94°C for 5 minutes, followed by 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute 30 seconds, followed by 5 minutes at 72°C. The pDCM2 vector was digested with smaI, and the three PCR products obtained above were fusion cloned. The resulting plasmids were designated pDCM2-ilvN(A42V). The pDCM2-ilvN(A42V) was transformed into wild-type Corynebacterium glutamicum strains ATCC14067 and ATCC13869, respectively, and homologous recombination was induced in the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector had been integrated into the chromosome via homologous recombination were selected in a medium containing 25 mg / L of kanamycin. The selected Corynebacterium glutamicum transformants were subjected to PCR amplification of gene fragments using the primer pair of SEQ ID NOs: 33 and 34, and accurate introduction of the mutation was confirmed through gene sequence analysis. The recombinant strains were designated Corynebacterium glutamicum CJ7V and CJ8V, respectively. The sequences of the primers used in this example are listed in Table 2 below.

[0121] [Table 2]

[0122] Fermentation titer experiments were then conducted on wild-type Corynebacterium glutamicum ATCC14067 and ATCC13869 strains, as well as the CJ7V and CJ8V strains constructed above. Each strain, subcultured in nutrient medium, was inoculated into a 250ml corner-baffled flask containing 25ml of production medium and cultured at 30°C for 72 hours with shaking at 200 rpm. The L-valine concentration was then analyzed using HPLC, and is shown in Table 3 below.

[0123] <Nutrient medium (pH 7.2)> Glucose 10g, meat juice 5g, polypeptone 10g, sodium chloride 2.5g, yeast extract 5g, agar 20g, urea 2g (based on 1 liter of distilled water)

[0124] <Production medium (pH 7.0)> Glucose 100g, ammonium sulfate 40g, soy protein 2.5g, corn steep solids 5g, urea 3g, dipotassium phosphate 1g, magnesium sulfate heptahydrate 0.5g, biotin 100μg, thiamine HCl 1mg, calcium pantothenate 2mg, nicotinamide 3mg, calcium carbonate 30g (based on 1 liter of distilled water)

[0125] [Table 3]

[0126] As can be seen from the above results, it was confirmed that the L-valine productivity was increased in the CJ7V and CJ8V strains into which the ilvN (A42V) gene mutation was introduced, compared to the wild-type Corynebacterium glutamicum strains ATCC14067 and 13869.

[0127] 2-2. Introduction and evaluation of GltA-weakened mutants (K415H, K415W, K415G) into L-valine-producing strains The GltA mutant was introduced into an L-valine-producing strain to evaluate its L-valine-producing ability. The pDCM2-gltA(K415H), pDCM2-gltA(K415W), and pDCM2-gltA(K415G) vectors constructed in Example 1 were transformed into the L-valine-producing strains CJ7V, CJ8V, and CA08-0072 (KCCM11201P, U.S. Patent No. 8,465,962) by homologous recombination. Strains in which the vector had been integrated into the chromosome via recombination of the homologous sequences were selected on a medium containing 25 mg / L kanamycin.

[0128] After the second recombination, the gene fragment was amplified by PCR using the primer pair of SEQ ID NOs: 23 and 24 (Table 4) from the Corynebacterium glutamicum transformant, and the mutant-inserted strain was identified through gene sequence analysis. The recombinant strain was named Corynebacterium glutamicum as follows, and its titer was evaluated in the same manner as in Example 2-1, and the results are shown in Table 5 below.

[0129] [Table 4]

[0130] [Table 5]

[0131] As can be seen from the above results, it was confirmed that the K415H mutant increased L-valine productivity without reducing growth.

[0132] The above CA08-0072:gltA(K415H) was designated CA08-1688 and deposited with the Korea Microorganism Collection, a depository under the Budapest Treaty, on September 28, 2020, and was assigned the accession number KCCM12795P.

[0133] Example 3: Construction of strains enhanced for O-acetyl-L-homoserine production and evaluation of O-acetyl-L-homoserine productivity 3-1 Construction of a strain carrying the exogenous membrane protein mutant YjeH To assess the effectiveness of the YjeH mutant, an exogenous membrane protein and an O-acetylhomoserine efflux protein, introduced into Corynebacterium glutamicum ATCC13032, a chromosomal introduction vector containing the gene (SEQ ID NO: 48) encoding the YjeH mutant (SEQ ID NO: 47) was constructed.

[0134] Specifically, to construct a transposase-deficient vector, a primer pair (SEQ ID NOs: 39 and 40) was designed to amplify the 5'-end region and a primer pair (SEQ ID NOs: 41 and 42) was designed to amplify the 3'-end region of the gene encoding transposase (SEQ ID NO: 38, gene ID NCgl2335). The primer pair of SEQ ID NOs: 39 and 42 inserted an XbaI restriction enzyme site at each end, while the primer pair of SEQ ID NOs: 40 and 41 was designed to cross over each other, so that the restriction enzyme SmaI sequence was located at this site. The primer sequences are listed in Table 6 below.

[0135] [Table 6]

[0136] PCR was performed using the ATCC13032 wild-type (WT) chromosome as a template and the primer pair of SEQ ID NOs: 39 and 40 and the primer pair of SEQ ID NOs: 41 and 42. The PCR conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 7 minutes. As a result, an 851-bp DNA fragment at the 5'-top and an 847-bp DNA fragment at the 3'-bottom were obtained, centered on the deletion site of the NCgl2335 gene.

[0137] Using the two amplified DNA fragments as templates, PCR was performed with the primer pair of SEQ ID NOs: 39 and 42. The PCR conditions were as follows: denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 90 seconds, followed by polymerization at 72°C for 7 minutes. As a result, a 1648 bp DNA fragment containing a site that could be deleted for the gene encoding transposase (SEQ ID NO: 38, gene number NCgl2335) was amplified.

[0138] The resulting PCR product was fusion cloned with pDCM2 vector digested with SmaI restriction enzyme using the Infusion HD Cloning Kit. The cloned vector was transformed into E. coli DH5α, and the transformed E. coli was plated on LB solid medium containing 25 mg / L kanamycin. Colonies transformed with the plasmid containing the target gene were selected through PCR, and the plasmid was isolated using the plasmid extraction method. Finally, the pDCM2-△NCgl2335 recombinant vector into which the NCgl2335 deletion cassette was cloned was constructed.

[0139] To assess the effectiveness of the O-acetylhomoserine efflux protein, a chromosomal vector containing a gene encoding the E. coli-derived YjeH mutant (SEQ ID NO: 48) was constructed. To this end, a vector expressing the yjeH gene using the CJ7 promoter (U.S. Patent No. 7,662,943) was constructed. A primer pair (SEQ ID NOs: 43 and 44) for amplifying the CJ7 promoter region and a primer pair (SEQ ID NOs: 45 and 46) for amplifying the E. coli yjeH region were designed. The primer sequences are listed in Table 7 below.

[0140] [Table 7]

[0141] PCR was performed using pECCG117-PCJ7-gfp (U.S. Patent No. 7,662,943, p117-Pcj7-gfp) as a template and the primer pairs SEQ ID NOs: 43 and 44, and wild-type E. coli chromosome as a template and SEQ ID NOs: 45 and 46, respectively. The PCR conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 90 seconds, followed by polymerization at 72°C for 7 minutes. As a result, a 360 bp DNA fragment from the CJ7 promoter region and a 1297 bp DNA fragment from the E. coli yjeH gene region were obtained.

[0142] Using the two amplified DNA fragments as templates, PCR was performed with primers SEQ ID NO: 43 and SEQ ID NO: 46. The PCR conditions were denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 90 seconds, followed by polymerization at 72°C for 7 minutes. As a result, a 1614 bp DNA fragment containing the CJ7 promoter and the yjeH gene insertion site was amplified.

[0143] The gene-deleted DNA fragment obtained through PCR was cloned into the pDCM2-ΔNCgl2335 vector digested with the restriction enzyme SmaI using the Infusion HD cloning kit to construct the pDCM2-ΔNCgl2335::PCJ7-yjeH(eco,WT) recombinant vector.

[0144] In addition, a recombinant vector for introducing the mutant yjeH(eco,F351L) gene was constructed.

[0145] Specifically, using the pDCM2-ΔNCgl2335::PCJ7-yjeH(eco,WT) plasmid as a template, the 351st amino acid in the YjeH amino acid sequence, phenylalanine, was replaced with leucine (F351L) using primers set forth in SEQ ID NOs: 49 and 50. The resulting plasmid containing the gene encoding the mutant YjeH(F351L) was designated pDCM2-ΔNCgl2335::PCJ7-yjeH(eco,F351L). The primer sequences are listed in Table 8 below.

[0146] [Table 8]

[0147] The constructed pDCM2-ΔNCgl2335 and pDCM2-ΔNCgl2335::PCJ7-yjeH(eco,F351L) were transformed into ATCC13032 by electroporation, and a second crossover process was performed to obtain ATCC13032 ΔNCgl2335 and ATCC13032 ΔNCgl2335::PCJ7-yjeH(eco,F351L), which lacked the NCgl2335 gene on the chromosome. The presence or absence of the NCgl2335 gene deletion and the insertion of the gene encoding the YjeH mutant was confirmed by PCR using the primer pair of SEQ ID NOs: 39 and 42, followed by comparison with ATCC13032.

[0148] 3-2. Evaluation of O-acetylhomoserine production ability To compare the O-acetylhomoserine (O-AH) production abilities of ATCC13032 △NCgl2335 and ATCC13032 △NCgl2335::PCJ7-yjeH(eco,F351L) constructed in Example 3-1 with the wild-type strain ATCC13032, the cultures were cultured as follows, and the O-acetylhomoserine in the culture medium was analyzed. A loopful of the strain was inoculated into a 250 ml corner-baffled flask containing 25 ml of the O-acetylhomoserine production medium described below, and cultured at 33°C for 20 hours with shaking at 200 rpm. The O-acetylhomoserine concentration was analyzed using HPLC, and the analyzed concentrations are shown in Table 9.

[0149] O-acetylhomoserine production medium (pH 7.2) Glucose 30g, KH2PO42g, Urea 3g, (NH4)2SO440g, Peptone 2.5g, CSL (Corn steep liquor, Sigma) 5g (10ml), MgSO 4. 7H2O 0.5g, CaCO320g (based on 1 liter of distilled water)

[0150] [Table 9]

[0151] As a result, as shown in Table 9, when the control strain ATCC13032 was cultured, O-acetyl-L-homoserine accumulated at 0.3 g / L, confirming that deletion of the transposase NCgl2335 gene did not affect O-acetyl-L-homoserine production. In particular, when the mutant yjeH gene was expressed, O-acetyl-L-homoserine accumulated at a level of 1.0 g / L.

[0152] 3-3. Introduction and evaluation of the GltA mutant (K415H) into an O-acetyl-L-homoserine-producing strain The GltA mutant was introduced into the O-acetyl-L-homoserine-producing strain of Example 3-2, and the ability to produce O-acetyl-L-homoserine was evaluated. The pDCM2-gltA(K415H) vector constructed in Example 1 was transformed into the wild-type strains ATCC13032 and ATCC13032 △NCgl2335 and the O-acetyl-L-homoserine-producing strain ATCC13032 △NCgl2335::PCJ7-yjeH(eco,F351L) by homologous recombination on the chromosome. Strains in which the vector had been integrated into the chromosome by recombination of the homologous sequences were selected on a medium containing 25 mg / L of kanamycin.

[0153] After the secondary recombination, the transformed strain of Corynebacterium glutamicum was subjected to PCR using the primer pair of SEQ ID NOs: 23 and 24 to amplify the gene fragment, and the gltA(K415H) mutation-inserted strain was identified through gene sequence analysis. The recombinant strain was named Corynebacterium glutamicum as follows, and its titer was evaluated in the same manner as in Example 3-2. The results are shown in Table 10 below.

[0154] [Table 10]

[0155] As seen from the above results, it was confirmed that the strains into which the GltA K415H mutant was introduced had increased O-acetyl-L-homoserine productivity compared to the parent strain into which the mutant was not introduced.

[0156] The above ATCC13032 △NCgl2335::PCJ7-yjeH(eco,F351L)gltA(K415H) was designated CM04-1006 and deposited with the Korea Microorganism Collection, a depository under the Budapest Treaty, on October 21, 2020, and was assigned the accession number KCCM12809P.

[0157] From the above description, those skilled in the art to which the present invention pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including within the meaning and scope of the claims below, and any modifications or variations derived from the equivalent concepts thereof, rather than the above detailed description.

[0158] [Table 11]

[0159] [Table 12]

Claims

1. A citrate synthase mutant having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, in which the lysine amino acid corresponding to position 415 of the amino acid sequence of SEQ ID NO: 1 is replaced with histidine.

2. The variant of claim 1, wherein the variant comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:

3.

3. The variant of claim 1, comprising a polypeptide having an amino acid sequence represented by the following general formula 1: [General formula 1] X １ N HGGDATX ２ FMN KVKNKEDGVR LMGFGHRVYK NYDPRAAIVK ETAHEILEHL GGDDLLDLAI KLEEIALADD X ３ FISRKLYPN VDFYTGLIYR AMGFPTDFFT VLFAIGRLPG WIAHYREQLG AAGNH (SEQ ID NO: 51); Here, X in the general formula 1 １ is asparagine or serine, X ２ is alanine or glutamic acid, X ３ is tyrosine or cysteine.

4. The variant of claim 1, wherein the variant has 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 8, 10 or 12.

5. A polynucleotide encoding the variant according to any one of claims 1 to 4.

6. A Corynebacterium microorganism comprising the citrate synthase mutant of claim 1 or a polynucleotide encoding said mutant.

7. The microorganism according to claim 6, which has the ability to produce L-valine or O-acetyl-L-homoserine.

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

9. A method for producing an L-amino acid, comprising culturing the Corynebacterium microorganism of claim 6 in a medium.

10. 10. The method for producing an L-amino acid according to claim 9, further comprising recovering the L-amino acid from the culture medium or the microorganism.

11. 10. The method for producing an L-amino acid according to claim 9, wherein the L-amino acid is L-valine, O-acetyl-L-homoserine, or L-methionine.

12. A composition for producing an L-amino acid, comprising the Corynebacterium microorganism according to claim 6; a medium in which the microorganism is cultured; or a combination thereof.

13. 13. The composition for producing an L-amino acid according to claim 12, wherein the L-amino acid is L-valine, O-acetyl-L-homoserine, or L-methionine.

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