NOVEL CITRATE SYNTHASE VARIANT AND METHOD FOR PRODUCING L-VALINE USING THE SAME

Abstract

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

Description

[Technical field]

[0001] The present application relates to a novel citrate synthase mutant, a microorganism containing said mutant, and a method for producing L-valine using said 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 are 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 flux into the TCA pathway.

[0004] The phenotype change of L-lysine producing strains due to the deletion of the gltA gene encoding citrate synthase has been reported in a prior literature (Non-Patent Document 1). However, in the case of a gltA gene deleted strain, 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 considers 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 No. 10-2153534 [Patent Document 3] Korea Registration Patent 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] Korean Patent Publication No. 10-2020-0136813 [Patent Document 8] Korea Registration Patent No. 10-1947945

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-patent document 6

Non-patent document 7

Non-patent document 8

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

[0007] The present inventors have made extensive efforts to produce L-valine in a high yield, and have confirmed that a novel citrate synthase mutant increases L-valine productivity, thereby completing the present application.

[0008] One object of the present application is to provide a citrate synthase mutant in which the amino acid glycine corresponding to position 413 of the amino acid sequence of SEQ ID NO:1 is replaced with aspartic acid.

[0009] It is another object of the present application to provide polynucleotides encoding the variants of the present application. Another object of the present application is to provide a microorganism of the genus Corynebacterium comprising a variant of the present application or a polynucleotide encoding said variant.

[0010] Another object of the present application is to provide a method for producing L-valine using the microorganism of the present application. Another object of the present application is to provide a composition for producing L-valine, comprising the microorganism of the present application; a medium in which the microorganism of the present application is cultured; or a combination thereof.

[0011] Effect of the Invention When the citrate synthase mutant of the present application is used, L-valine can be produced in high yield. [Means for solving the problem]

[0012] This will be specifically explained as follows. Meanwhile, each description and embodiment disclosed in this application may also be applied to each different description and embodiment. That is, all combinations of the various elements disclosed in this application are within the scope of this application. Moreover, the specific description described below is not considered to limit the scope of this application. Furthermore, numerous papers and patent documents are referenced and citations are provided throughout this specification. The disclosure contents 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.

[0013] One aspect of the present application is to provide a citrate synthase mutant in which the amino acid glycine corresponding to position 413 of the amino acid sequence of SEQ ID NO:1 is substituted with aspartic acid.

[0014] The variant of the present application may be a variant in which the amino acid corresponding to the 413th position in the amino acid sequence of SEQ ID NO: 1 is aspartic acid and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.7% 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 be composed of an amino acid sequence in which the amino acid corresponding to the 413th position in the amino acid sequence of SEQ ID NO: 1 is aspartic acid and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.7% or more homology or identity to the amino acid sequence of SEQ ID NO: 1. It is also obvious that variants having amino acid sequences in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application, so long as the variant has such homology or identity and exhibits efficacy corresponding to the variant of the present application.

[0015] 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 change the function of the variant of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0016] 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.

[0017] In the present application, the term "variant" refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, and which differs from the amino acid sequence of the variant before the mutation, but maintains its functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the variant. That is, the ability 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 removed. Other variants may include variants in which a portion has been removed from the N- and / or C-terminus of a mature protein. The term "variant" may be used interchangeably with terms such as mutant, modified, mutant polypeptide, mutant protein, mutation, and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited to these terms as long as they are used in the sense of being mutated. 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).

[0018] The variants may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be conjugated to a signal (or leader) sequence involved in co-translationally or post-translationally protein translocation. The variants may also be conjugated to other sequences or linkers to allow identification, purification, or synthesis.

[0019] In this application, the term "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, which can be expressed as a percentage. The terms homology and identity can often be used interchangeably.

[0020] 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 can generally hybridize to all or a portion of the sequence under moderate or high stringent conditions. Hybridization obviously includes hybridization to polynucleotides containing common codons in polynucleotides or codons that take into account codon degeneracy.

[0021] 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 versions) (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.

[0022] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, e.g., Needleman et al. (1970), J Mol Biol. 48: 443, as known in, e.g., Smith and Waterman, Adv. Appl. Math (1981) 2: 482. In summary, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a 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.

[0023] In this application, the term "corresponding to" refers to an amino acid residue at a recited position in a polypeptide, or an amino acid residue that is similar or identical or homologous to a recited residue in a polypeptide. Identifying an amino acid at a corresponding position may be determining the particular amino acid of a sequence that references a particular sequence. As used in this application, a "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0024] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, based on which each amino acid residue in the amino acid sequence is numbered with reference to the numeric position of the corresponding amino acid residue in SEQ ID NO: 1. For example, sequence alignment algorithms such as those described in this application can identify the position of an amino acid relative to a query sequence (also referred to as a "reference sequence"), or the position at which a modification such as a substitution, insertion or deletion occurs.

[0025] 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 thereto. Sequence alignment programs and pairwise sequence comparison algorithms known in the art can be used as appropriate.

[0026] In this application, the term "citrate synthase" refers to an enzyme that polymerizes acetyl-CoA and oxaloacetate, which are produced during glycolysis in microorganisms, to produce citric acid. The enzyme also catalyzes the condensation reaction of acetyl-CoA with the 2-carbon acetate residue from a 4-carbon oxaloacetate molecule to produce a 6-carbon oxaloacetate. Quen In the present application, the term "citrate synthase" may be used interchangeably with citrate synthase, CS, GltA protein, or GltA. In the present application, the sequence of GltA can be obtained from the public database GenBank of NCBI. In addition, GltA may be a polypeptide having citrate synthase activity encoded by the gltA gene, but is not limited thereto.

[0027] The variants of the present application may have an activity that results in increased L-valine producing ability compared to the wild-type polypeptide. A variant of the present application may have 80% or more sequence identity with the amino acid sequence of SEQ ID NO:1.

[0028] 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 362nd to 413th amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 1, in which the glycine at the 413th position is substituted with aspartic acid.

[0029] The variants of the present application may comprise an amino acid sequence of general formula 1: [General formula 1] X1N HGGDATX2FMN KVKNKEDGVR LMGFGHRVYK NYDPRAAIVK ETAHEILEHL GGDDLLDLAI KLEEIALADD X3FISRKLYPN VDFYTGLIYR AMGFPTDFFT VLFAIGRLPG WIAHYREQLG AADNX4 (SEQ ID NO: 24) wherein X1 in the above general formula 1 is asparagine or serine; X2 is alanine or glutamic acid, X3 is tyrosine or cysteine, X4 is lysine or histidine.

[0030] The variants of the present application may have 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 8, 10, 12 or 14. The variants of the present application may also comprise, consist of, or essentially consist of an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 8, 10, 12 or 14. As an example, the variants 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, 12 or 14, may comprise, consist of, or essentially consist of an amino acid sequence having the above sequence identity.

[0031] Another aspect of the present application is to provide a polynucleotide encoding the variant of the present application. 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 mutant.

[0032] The polynucleotide of the present application may include a polynucleotide described in 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 with the nucleic acid base sequence described in SEQ ID NO: 2, where the bases corresponding to positions 1237 to 1239 are GAC. It is also self-evident that a polynucleotide described in a nucleic acid base sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted, or added is also included within the scope of the present application, so long as it is a sequence that has such homology or identity and encodes a polypeptide or protein that exhibits an efficacy corresponding to the variant of the present application.

[0033] 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 the degeneracy of codons or the codons preferred 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 the 413th position of SEQ ID NO: 1 may be one of the codons encoding aspartic acid.

[0034] Furthermore, the polynucleotide of the present application includes, without limitation, a sequence that can hybridize under stringent conditions with a probe prepared from a known gene sequence, for example, a sequence complementary to the entire or partial polynucleotide sequence of the present application. The above "stringent conditions" refer 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; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7). For example, conditions include conditions under which polynucleotides having high homology or identity, such as polynucleotides having a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, but polynucleotides having lower homology or identity do not hybridize with each other, or conditions for washing in Southern hybridization, such as 60°C, 1 x SSC, 0.1% SDS, specifically, 60°C, 0.1 x SSC, 0.1% SDS, more specifically, 68°C, 0.1 x SSC, 0.1% SDS, at a salt concentration and temperature equivalent thereto, in which washing is performed once, specifically 2 to 3 times.

[0035] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to one another. For example, with respect to DNA, adenine is complementary to thymine and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may also include isolated nucleic acid fragments that are complementary to entire sequences, as well as substantially similar nucleobase sequences.

[0036] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the above-mentioned conditions, using hybridization conditions including a hybridization step at a Tm value of 55° C. The Tm value may be 60° C., 63° C. or 65° C., but is not limited thereto, and may be appropriately adjusted by those skilled in the art depending on the purpose.

[0037] 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).

[0038] As an example, the polynucleotide of the present application may include a polynucleotide described by the nucleic acid base sequence of positions 1084 to 1239 based on the nucleic acid base sequence of SEQ ID NO: 9, 11, or 13, 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: 15, or a polynucleotide described by the nucleic acid base sequence of SEQ ID NO: 9, 11, 13, or 15. In the polynucleotide of the present application, the above-mentioned variants are as described in the other aspects above.

[0039] 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.

[0040] The vector of the present application may comprise a DNA construct comprising a base sequence of a polynucleotide encoding the above-mentioned target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to be able to express the target polypeptide in a suitable host. The expression control region may comprise a promoter capable of initiating transcription, an optional operator sequence for controlling such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling the termination of transcription and decoding. After being transformed into a suitable host cell, the vector may be capable of replicating or functioning independently of the host genome, or may be integrated into the genome itself.

[0041] The vector used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ, 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, p. 721-726 (1991), Korean Patent Registration No. 10-1992-0007401), pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0042] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome through a vector for chromosomal insertion in a cell. The polynucleotide can be inserted into a chromosome by any method known in the art, for example, but not limited to, homologous recombination. A selection marker for checking the presence or absence of insertion into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to check the presence or absence of insertion of the target nucleic acid molecule, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide is used. In an environment treated with a selective agent, only cells expressing the selection marker survive or show other expression traits, so that transformed cells can be selected.

[0043] The term "transformation" in this application means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include both, regardless of whether it is inserted and located within the chromosome of the host cell or located extrachromosomally, as long as it can be expressed in the host cell. The polynucleotide may also include DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form as long as it can be introduced and expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all elements necessary for its own expression. The expression cassette may usually include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self-replicating. The polynucleotide may also be introduced into the host cell in its own form and operably linked to sequences necessary for expression in the host cell, but is not limited thereto.

[0044] 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.

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

[0046] 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. 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 may be microorganisms 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 may also be microorganisms that contain genetic modifications for the production of a desired polypeptide, protein, or product.

[0047] The microorganism of the present application may be, but is not limited to, a microorganism comprising one or more of a variant of the present application, a polynucleotide of the present application, and a vector comprising a polynucleotide of the present application; a microorganism that has been modified to express a variant of the present application or a polynucleotide of the present application; a microorganism (e.g., a recombinant strain) that expresses a variant of the present application or a polynucleotide of the present application; or a microorganism (e.g., a recombinant strain) that has an activity of a variant of the present application.

[0048] The microorganism of the present application may have the ability to produce L-valine. The microorganism of the present application may be, but is not limited to, a microorganism that naturally has the ability to produce GltA or L-valine, or a parent strain lacking the ability to produce GltA or L-valine into which a mutant of the present application or a polynucleotide encoding the mutant (or a vector containing the above-mentioned polynucleotide) has been introduced and / or which has been imparted the ability to produce GltA or L-valine.

[0049] As an 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, and for the purposes of this application, the microorganism of the present application may include all microorganisms capable of producing L-valine, 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 a polynucleotide encoding the variant of the present application is introduced into an L-valine producing microorganism. The recombinant strain with increased L-valine production ability may be, but is not limited to, a naturally occurring wild-type microorganism, or a microorganism that does not express the citrate synthase-modified microorganism (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 citrate synthase non-transformed microorganism, which is the subject strain for comparing the increase in L-valine 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.

[0050] As an example, the recombinant strain with increased production ability may have an L-valine production ability increased by about 1% or more, 5% or more, about 10% or more, about 13% or more, about 15% or more, about 17% or more, about 20% or more, about 30% or more, or about 40% 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, or about 40% 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 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 1.1-fold or more, about 1.2-fold or more, about 1.3-fold or more, or about 1.4-fold or more (there is no particular upper limit, and it may be, for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, or about 2-fold or less) compared to the parent strain or non-transformed microorganism before the mutation.

[0051] The term "about" refers to a range including, but not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​in a range equal to or similar to the numerical value following the term about.

[0052] In the present application, the term "non-altered 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 by genetic mutations due to natural or artificial factors. For example, the non-altered microorganism refers to a strain before the protein mutants described herein are introduced or before they are introduced. The term "non-altered microorganism" may be used interchangeably with "strain before modification," "microorganism before mutation," "non-mutated strain," "non-altered strain," "non-mutated microorganism," or "reference microorganism."

[0053] As 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 The bacterium may be Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens.

[0054] The microorganism of the present application may be a microorganism in which the activity of acetolactate synthase isozyme 1 small subunit (IlvN) is further enhanced.

[0055] In the present application, the term "attenuation" of a polypeptide activity is a concept that includes all of a decrease in activity or no activity compared to the endogenous activity. The above attenuation may be mixed with terms such as inactivation, deficiency, down-regulation, decrease, reduction, and attenuation.

[0056] The attenuation may include cases where the activity of the polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by the microorganism due to a mutation in the polynucleotide encoding the polypeptide, where the overall polypeptide activity and / or concentration (expression amount) in the cell is lower than that of a wild-type strain due to inhibition of gene expression of the encoding polynucleotide or inhibition of translation into the polypeptide, where the polynucleotide is not expressed at all, and / or where the polypeptide activity is absent despite the expression of the polynucleotide. The "intrinsic activity" refers to the activity of a specific polypeptide originally possessed by a parent strain, a wild-type or a non-transformed microorganism before the transformation, when the trait is changed due to a genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before transformation." The activity of a polypeptide being "inactivated, deficient, reduced, down-regulated, decreased, attenuated" compared to the intrinsic activity means that the activity of a specific polypeptide is decreased compared to that of a parent strain or a non-transformed microorganism before the transformation.

[0057] Such attenuation of the activity of a polypeptide can be achieved by any method known in the art, including but not limited to, the application of various methods well known in the art (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014; 15(2): 2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

[0058] Specifically, the attenuation of the polypeptide of the present application is 1) A deletion of all or part of a gene encoding a polypeptide; 2) modification of an expression control region (or expression control sequence) so that expression of a gene encoding a polypeptide is reduced; 3) modification of the amino acid sequence constituting the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence) so that the activity of the polypeptide is eliminated or weakened; 4) modification of a gene sequence encoding a polypeptide such that the activity of the polypeptide is eliminated or attenuated (e.g., deletion / substitution / addition of one or more nucleic acid bases on the nucleic acid base sequence of the polypeptide gene to encode a polypeptide that has been altered such that the activity of the polypeptide is eliminated or attenuated); 5) a modification of the nucleotide sequence encoding the initiation codon or the 5'-UTR region of a gene transcript encoding a polypeptide; 6) introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the 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) Reverse transcription engineering (RTE) of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide; or 9) A combination of two or more selected from the above 1) to 8) may be used, but is not particularly limited thereto.

[0059] for example, The deletion of a part or the whole of the gene encoding the polypeptide (1) above may be removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacement with a polynucleotide lacking some nucleotides, or replacement with a marker gene.

[0060] The above 2) modification of the expression regulatory region (or expression regulatory sequence) may be a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or an exchange with a sequence having a weaker activity. The above 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.

[0061] The modification of the amino acid sequence or polynucleotide sequence in 3) and 4) above may be, but is not limited to, the occurrence of mutations in the sequence of the amino acid sequence of the polypeptide or the sequence of the polynucleotide encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacement with an amino acid sequence or polynucleotide sequence improved to have a weaker activity or an amino acid sequence or polynucleotide sequence improved to have no activity, so as to weaken the activity of the polypeptide. For example, but not limited to, the expression of a gene may be inhibited or weakened by introducing a mutation in the polynucleotide sequence to form a stop codon.

[0062] The modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, but is not limited to, a replacement with a nucleotide sequence encoding another start codon that has a lower polypeptide expression rate than the endogenous start codon.

[0063] The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide (6) can be carried out by referring to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0064] 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 may disable or slow down mRNA translation.

[0065] 8) The addition of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE) may create an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide, thereby weakening the activity.

[0066] In the present application, the term "enhancement" of a polypeptide activity means that the activity of the polypeptide is increased compared to the endogenous activity. The term "enhancement" may be used interchangeably with terms such as "activation," "up-regulation," "overexpression," and "increase." Here, the terms "activation," "enhancement," "up-regulation," "overexpression," and "increase" may all include the display of an activity that was not originally possessed, or the display of an activity that is improved compared to the endogenous activity or the activity before transformation. The term "endogenous activity" refers to the activity of a specific polypeptide that was originally possessed by a parent strain or a non-transformed microorganism before transformation, when a trait has been changed by a genetic mutation due to natural or artificial factors. This term may be used interchangeably with "activity before transformation." The term "enhancement," "up-regulation," "overexpression," or "increase" of a polypeptide activity compared to the 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 that was originally possessed by a parent strain or a non-transformed microorganism before transformation.

[0067] 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.

[0068] The activity of the polypeptide can be enhanced by various methods well known in the art, and is not limited as long as the activity of the target polypeptide can be enhanced compared to that of the microorganism before modification. Specifically, the method may utilize genetic engineering and / or protein engineering, which are routine methods in molecular biology and are well known to ordinary technicians 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.).

[0069] Specifically, the enhancement of the polypeptide activity of the present application is achieved by: 1) increasing the intracellular copy number of a polynucleotide encoding a polypeptide; 2) replacing the expression regulatory region of a gene on a chromosome that encodes a polypeptide with a sequence with a strong activity; 3) a modification of the nucleotide sequence encoding the initiation codon or the 5'-UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the activity of the polypeptide is enhanced; 5) modifying a polynucleotide sequence encoding a polypeptide so as to enhance the activity of the polypeptide (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the activity of the polypeptide); 6) introduction of a foreign polypeptide exhibiting a polypeptide activity or a foreign polynucleotide encoding the same; 7) codon optimization of a polynucleotide encoding a polypeptide; 8) analyzing the tertiary structure of a polypeptide and selectively modifying or chemically modifying exposed sites; or 9) A combination of two or more selected from the above 1) to 8) may be used, but is not particularly limited thereto.

[0070] More specifically, The 1) increase in intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a host cell a vector to which a polynucleotide encoding the polypeptide is operably linked, the vector being capable of replicating and functioning independently of a host. Alternatively, the increase may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into a chromosome in the host cell. The introduction into a chromosome can be achieved by, but is not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell. The vector is as described above.

[0071] The replacement of the gene expression regulatory region (or expression regulatory sequence) on the chromosome encoding the polypeptide with a sequence having a stronger activity may be, for example, the occurrence of a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to further enhance the activity of the expression regulatory region, or the replacement with a sequence having a stronger activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and decoding. As an example, the original promoter may be replaced with a strong promoter, but is not limited to these.

[0072] Examples of known strong promoters include, but are not limited to, CJ1 to CJ7 promoters (U.S. Patent US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (U.S. Registered Patent US 10584338 B2), O2 promoter (U.S. Patent No. 10273491), tkt promoter, and yccA promoter.

[0073] 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, replacement with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon.

[0074] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) may be, but is not limited to, a mutation in the sequence of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to enhance the activity of the polypeptide, or an amino acid sequence or polynucleotide sequence improved to have stronger activity or an amino acid sequence or polynucleotide sequence improved to increase activity. The replacement may be, but is not limited to, specifically, by inserting the polynucleotide into a chromosome by homologous recombination. The vector used in this case may further include a selection marker for checking whether or not the chromosome is inserted. The selection marker is as described above.

[0075] The introduction of a foreign polynucleotide exhibiting the activity of the polypeptide 6) may be the introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide into a host cell. The foreign polynucleotide is not limited in origin or sequence as long as it exhibits the same / similar activity as the polypeptide. The method used for the introduction can be a known transformation method appropriately selected by those skilled in the art, and the introduced polynucleotide may be expressed in a host cell to produce a polypeptide, and its activity may be increased.

[0076] 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.

[0077] In addition, the above 8) analyzing the tertiary structure of a polypeptide and selecting and modifying or chemically modifying an exposed site may involve, for example, comparing sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining template protein candidates according to the degree of sequence similarity, confirming the structure based on the template protein candidates, and selecting and modifying an exposed site to be modified or chemically modified.

[0078] Such enhanced polypeptide activity may refer to, but is not limited to, an increase in the activity or concentration of the corresponding polypeptide expressed relative to the activity or concentration of the polypeptide expressed in a wild-type or unaltered microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0079] In the microorganism of the present application, modification of a part or the whole of a polynucleotide (e.g., modification to encode the above-mentioned 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 nuclease (e.g., CRISPR-Cas9) and / or (b) treatment with light and / or chemicals such as ultraviolet light and radiation. 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 may be injected into the microorganism to cause homologous recombination, thereby causing a deletion of a part or the whole of the gene. The injected nucleotide sequence or vector may contain a dominant selection marker, but is not limited to these.

[0080] 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:22 and / or a nucleotide set forth in the nucleic acid base sequence of SEQ ID NO:23.

[0081] In the microorganisms of the present application, the variants, polynucleotides, etc. are as described in other aspects above. Another aspect of the present application provides a method for producing L-valine, 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.

[0082] The method for producing L-valine of the present application may comprise the step of culturing in a medium a Corynebacterium glutamicum strain comprising a mutant of the present application, a polynucleotide of the present application, or a vector of the present application.

[0083] In the present application, the term "culture" means growing the Corynebacterium microorganism of the present application under appropriately controlled environmental conditions. The culture process of the present application can be carried out according to suitable media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culture may be, but is not limited to, a batch, continuous and / or fed-batch culture.

[0084] In the present application, the term "culture medium" refers to a substance containing 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 medium and other culture conditions used for culturing the microorganism of the genus Corynebacterium of the present application can be any medium used for culturing ordinary microorganisms without any particular limitation, and the microorganism of the genus Corynebacterium of the present application can be cultured under aerobic conditions while controlling the temperature, pH, etc. in an ordinary medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins, etc.

[0085] 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)].

[0086] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, baca, and corn steeping liquid may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and other suitable carbon sources may be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0087] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; an organic nitrogen source such as amino acids such as glutamic acid, methionine, glutamine, etc., peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more kinds, and are not limited thereto.

[0088] The phosphorus source may include potassium monophosphate, potassium diphosphate, or the corresponding sodium-containing salts. The inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and may also include amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, they are not limited thereto.

[0089] In addition, during the cultivation of the Corynebacterium glutamicum strain of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, foam generation can be suppressed using an antifoaming agent such as fatty acid polyglycol ester, etc. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas can be injected into the medium, or in order to maintain the anaerobic and microaerobic state, no gas can be injected or nitrogen, hydrogen or carbon dioxide gas can be injected, but this is not limited thereto.

[0090] In the culture of the present application, the culture temperature can be maintained at 20 to 45° C., specifically 25 to 40° C., and the culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0091] The L-valine produced by the culture of the present application is either secreted into the medium or remains intracellularly. The method for producing L-valine 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, prior to the culturing step.

[0092] The method for producing L-valine of the present application may further include a step of recovering L-valine from the culture medium (cultured medium) or the Corynebacterium microorganism of the present application. The recovery step may be further included after the culturing step.

[0093] Said recovery may be to collect the desired L-valine using a suitable method known in the art by the culture method of the microorganism of the present application, for example, a batch, continuous or fed-batch culture method, etc. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination of these methods can be used, and the desired L-valine can be recovered from the medium or the microorganism using a suitable method known in the art.

[0094] In addition, the L-valine production method of the present application may further include a purification step. The purification can be performed using a suitable method known in the art. As an example, when the L-valine production method 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 simultaneously or integrated into one step, but are not limited thereto.

[0095] In the methods of the present application, the variants, polynucleotides, vectors and microorganisms etc. are as described in other aspects above. Another aspect of the present application is to provide a composition for producing L-valine, comprising a Corynebacterium microorganism comprising a variant of the present application, a polynucleotide encoding the variant of the present application, or a vector comprising the polynucleotide of the present application; a medium in which the same is cultured; or a combination thereof.

[0096] The composition of the present application may further contain any suitable excipient that is commonly used in compositions for producing L-valine, and such excipients may be, for example, but are not limited to, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, or an isotonic agent.

[0097] In the composition of the present application, the variant, polynucleotide, vector, strain, medium, etc. are as described in other aspects above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0098] Hereinafter, the present application will be described in more detail 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 ordinary skilled artisans in the technical field of the present application or a similar technical field.

[0099] Example 1: Selection of mutants with increased L-valine productivity through random mutagenesis Example 1-1: Random mutagenesis through UV irradiation To select mutants with increased L-valine production, Corynebacterium glutamicum CA08-0072 (KCCM11201P, U.S. Patent No. 8,465,962), an L-valine-producing strain, was spread on a nutrient medium containing agar and cultured for 36 hours at 30° C. Several hundred colonies thus obtained were irradiated with UV light at room temperature to induce random mutations in the genome of the strain.

[0100] <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) Example 1-2: Fermentation titer experiment of mutagenized strains and strain selection To select mutants with increased L-valine production ability compared to the parent strain Corynebacterium glutamicum CA08-0072, fermentation titration experiments were performed on the randomly mutated strains. Each colony (M1-M16) was subcultured in nutrient medium, and then each strain was inoculated into a 250-ml corner baffle flask containing 25 ml 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 the analyzed L-valine concentrations are shown in Table 1 below.

[0101] <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) <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)

[0102] [Table 1]

[0103] As a result, the M5 and M12 strains were selected, which showed the greatest increase in L-valine productivity, with L-valine productivity of 178% and 181%, respectively, compared to the control strain CA08-0072.

[0104] Example 2: Confirmation of mutations through gene sequencing The M5 and M12 strains with increased L-valine production were compared with the CA08-0072 strain and wild-type Corynebacterium glutamicum strains ATCC14067, ATCC13032, and ATCC13869 by sequencing the L-valine production key genes. As a result, it was confirmed that the M5 and M12 strains contain a mutation at a specific position of citrate synthase (GltA). Specifically, it was confirmed that the 413th amino acid in GltA is replaced by glycine (G) with aspartic acid (D).

[0105] In the following Example, it was determined whether the above mutations affect the L-valine production amount of Corynebacterium microorganisms. Example 3: Construction of a vector for introducing mutations and construction of a mutant strain Example 3-1: Selection of citrate synthase mutants and vector construction A vector was constructed to replace the 413th amino acid, glycine, of GltA with aspartic acid.

[0106] To construct a vector that replaces the glycine at the 413th amino acid position with aspartic acid, PCR was performed using the primer pair of SEQ ID NOs: 25 and 26 and the primer pair of SEQ ID NOs: 27 and 28 with wild-type Corynebacterium glutamicum ATCC14067 gDNA as a template. The PCR was performed under the following conditions: denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 30 seconds were repeated 30 times, and polymerization was performed at 72°C for 5 minutes. A fragment was obtained by performing overlapping PCR again using the primer pair of SEQ ID NOs: 25 and 26 with the mixture of both fragments obtained above as a template. The 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 and 30 seconds, followed by 72°C for 5 minutes. The pDCM2 vector (Korea Publication No. 10-2020-0136813) was treated with smaI, and the PCR products obtained above were fusion cloned. Fusion cloning was performed using the In-Fusion (registered trademark) HD ​​Cloning Kit (Clontech). The plasmid obtained as a result of cloning was named pDCM2-gltA (G413D). The sequences of the primers used in this example are listed in Table 2 below.

[0107] [Table 2]

[0108] Example 3-2: Introduction of GltA mutant into L-valine producing strain and evaluation Example 3-2-1. 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: 22) was introduced into the acetolactate synthase isozyme 1 small subunit (IlvN) of wild-type Corynebacterium glutamicum ATCC14067 and ATCC13869, respectively, to create a strain with improved L-valine production ability (Korean Patent Registration No. 10-1947945).

[0109] Specifically, PCR was performed using the wild-type Corynebacterium glutamicum ATCC14067 DNA (genomic DNA) as a template and the primer pair of SEQ ID NO: 31 and 33 and the primer pair of SEQ ID NO: 32 and SEQ ID NO: 34, respectively. A mixture of both fragments obtained above was used as a template and overlapping PCR was performed again using the primer pair of SEQ ID NO: 31 and 34 to obtain a PCR fragment. PCR was performed after denaturation at 94°C for 5 minutes, 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute and 30 seconds, and then 72°C for 5 minutes. The pDCM2 vector was treated with smaI, and the PCR products obtained above were fusion cloned. Fusion cloning was performed using the In-Fusion (registered trademark) HD ​​cloning kit (Clontech). The plasmid obtained as a result of cloning was named pDCM2-ilvN (A42V). The pDCM2-ilvN(A42V) was transformed into wild-type Corynebacterium glutamicum ATCC14067 and ATCC13869 strains, respectively, to induce homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains in which the vector was inserted into the chromosome by recombination of the homologous sequence were selected in a medium containing 25 mg / L of kanamycin. The gene fragments of the selected Corynebacterium glutamicum transformants were amplified by PCR using the primer pair of SEQ ID NOs: 35 and 36, and the gene sequence was analyzed to confirm that the mutation was accurately introduced. The recombinant strains were named Corynebacterium glutamicum CJ7V and CJ8V, respectively. The sequences of the primers used in this example are shown in Table 3 below.

[0110] [Table 3]

[0111] Fermentation titer experiments were then carried out on wild-type Corynebacterium glutamicum ATCC14067 and ATCC13869 strains, and the CJ7V and CJ8V strains constructed above. Each strain subcultured in nutrient medium was inoculated into a 250-ml corner baffle flask containing 25 ml 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 4 below.

[0112] <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) <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)

[0113] [Table 4]

[0114] As can be seen from the above results, it was confirmed that the L-valine producing ability 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.

[0115] Example 3-2-2: Introduction of GltA mutant (G413D) into L-valine producing strain and evaluation The GltA mutant was introduced into the L-valine producing strain to evaluate the L-valine producing ability. The pDCM2-gltA(G413D) vector prepared in Example 3-1 was transformed into the L-valine producing strains CJ7V, CJ8V and CA08-0072 by homologous recombination on the chromosome. The strains in which the vector was inserted on the chromosome by recombination of the homologous sequence were selected on a medium containing 25 mg / L kanamycin.

[0116] Then, the gene fragment was amplified by PCR using the primer pair of SEQ ID NOs: 29 and 30 (Table 5) from the above Corynebacterium glutamicum transformant strain after the second recombination, and the mutant-inserted strain was identified by analyzing the gene sequence. The recombinant strains were named as follows, and titer evaluation was performed in the same manner as in Example 1, and the results are shown in Table 6 below.

[0117] [Table 5]

[0118] [Table 6]

[0119] As seen in Table 6 above, it was confirmed that the G413D mutant had increased L-valine productivity without a decrease in growth. Example 3-3: Evaluation of integration of GltA mutant into L-valine producing strain Example 3-3-1. Construction of GltA mutant integration vector Based on the vector prepared in Example 3-1, a vector was prepared to integrate a mutant in which the 415th amino acid of GltA, lysine (K), is replaced with histidine (H). Specifically, site-directed mutagenesis PCR (BMC Biotechnology volume 9, Article number: 61 (2009)) was performed using the pDCM2-gltA(G413D) prepared in Example 3-1 as a template and the primer pair of SEQ ID NOs: 37 and 38 (Table 7). Through this, a plasmid in which the 413th amino acid, glycine (G), is replaced with aspartic acid (D) and the 415th amino acid, lysine (K), is replaced with histidine (H) was obtained, and finally named pDCM2-gltA(G413D, K415H).

[0120] [Table 7]

[0121] Example 3-3-2. Construction and evaluation of an L-valine producing strain integrated with a GltA mutant The GltA mutation was integrated into the L-valine producing strain to evaluate the L-valine producing ability. The pDCM2-gltA (G413D, K415H) vector constructed in Example 3-3-1 was transformed into the L-valine producing strains CJ7V, CJ8V and CAO8-0072 by homologous recombination on the chromosome. The strains in which the vector was inserted on the chromosome by recombination of the homologous sequence were selected on a medium containing 25 mg / L kanamycin.

[0122] Then, the gene fragment was amplified by PCR using the primer pair of SEQ ID NOs: 29 and 30 from the above Corynebacterium glutamicum transformant strain after the second recombination, and the mutant-inserted strain was identified by analyzing the gene sequence. The recombinant strains were named as follows, and titer evaluation was performed in the same manner as in Example 1, and the results are shown in Table 8 below.

[0123] [Table 8]

[0124] As can be seen from the above results, it was confirmed that the strains into which the GltA mutant was introduced had increased L-valine productivity without any decrease in growth, compared to the parent strain into which the mutant was not introduced. The above CA08-0072:gltA(G413D) was named CA08-1687 and deposited with the Korea Microorganism Collection, a depository under the Budapest Treaty, on September 28, 2020 and was assigned the accession number KCCM12794P. The above CA08-0072:gltA(G413D, K415H) was named CA08-1689 and deposited with the Korea Microorganism Collection, a depository under the Budapest Treaty, on September 28, 2020 and was assigned the accession number KCCM12796P.

[0125] From the above description, a person skilled in the art to which the present invention pertains will understand that the present application may be implemented in other specific forms without changing the technical idea or essential features of the present application. In this regard, it should be understood that the above described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including any modified or altered forms derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

[0126] [Table 9]

[0127] [Table 10]

Claims

1. A citrate synthase mutant in which the amino acid glycine corresponding to the 413th position of the amino acid sequence of SEQ ID NO:1 is replaced with aspartic acid and which has a sequence identity of 90% or more with the amino acid sequence of SEQ ID NO:

1.

2. The variant of claim 1 , wherein the variant has a sequence identity of 98% or more with the amino acid sequence of SEQ ID NO:

1.

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

3.

4. The variant of claim 1 comprises a polypeptide having an amino acid sequence as described in 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 AADNX ４ (SEQ ID NO:24); 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, X ４ is lysine or histidine.

5. 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, 12 or 14.

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

7. A Corynebacterium microorganism comprising a citrate synthase mutant having a sequence identity of 90% or more with the amino acid sequence of SEQ ID NO:1, in which glycine, the amino acid corresponding to the 413th position in the amino acid sequence of SEQ ID NO:1, is replaced with aspartic acid, or a polynucleotide encoding said mutant.

8. The microorganism according to claim 7, which has an ability to produce L-valine.

9. The microorganism according to claim 7 , wherein the microorganism is Corynebacterium glutamicum.

10. A method for producing L-valine, comprising the step of culturing in a medium a Corynebacterium microorganism containing a citrate synthase mutant having 90% or more sequence identity with the amino acid sequence of SEQ ID NO:1, or a polynucleotide encoding said mutant, in which glycine, the amino acid corresponding to the 413th position in the amino acid sequence of SEQ ID NO:1, is replaced with aspartic acid.

11. 11. The method for producing L-valine according to claim 10, further comprising recovering L-valine from the culture medium or the microorganism.

12. A composition for producing L-valine, comprising: a Corynebacterium microorganism containing a citrate synthase mutant in which glycine, the amino acid corresponding to the 413th position in the amino acid sequence of SEQ ID NO: 1, is replaced with aspartic acid and which has a sequence identity of 90% or more with the amino acid sequence of SEQ ID NO: 1, or a polynucleotide encoding said mutant; a medium in which said microorganism is cultured; or a combination thereof.

Images