Corynebacterium sp. microorganism capable of producing L-amino acids and method for producing L-amino acids using the same

Abstract

The present application relates to an L-amino acid producing Corynebacterium microorganism and a method for producing an L-amino acid using the same.

Description

[Technical field]

[0001] The present application relates to an L-amino acid producing Corynebacterium microorganism and a method for producing an L-amino acid using the same. [Background technology]

[0002] L-amino acids are used in the animal feed, pharmaceutical and cosmetic industries, and are mainly produced by fermentation using strains of the genus Corynebacterium or Escherichia. Various research projects are being conducted to develop highly efficient production strains and fermentation process technologies for the production of L-amino acids.

[0003] Specifically, approaches specific to the target substance, such as increasing the expression of genes encoding enzymes involved in L-amino acid biosynthesis or removing genes unnecessary for biosynthesis, are mainly used (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 8,048,650 [Patent Document 2] Korean Patent No. 10-2126951 [Patent Document 3] U.S. Pat. No. 1,059,046 [Patent Document 4] Korean Patent No. 10-1335789 [Patent Document 5] Korean Patent Publication No. 10-2020-0136813 [Non-patent literature]

[0005] [Non-Patent Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-Patent Document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-Patent Document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-Patent Document 4] Devereux, J., et al, Nucleic Acids Research 12: 387 (1984) [Non-Patent Document 5] Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990) [Non-Patent Document 6] Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994 [Non-Patent Document 7] [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073 [Non-Patent Document 8] Smith and Waterman, Adv. Appl. Math (1981) 2:482 [Non-Patent Document 9] Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) [Non-Patent Document 10] Gribskov et al(1986) Nucl. Acids Res. 14: 6745 [Non-Patent Document 11] J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989 [Non-Patent Document 12] FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8 [Non-Patent Document 13] Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793 [Non-Patent Document 14] Sambrook et al. Molecular Cloning 2012 [Non-Patent Document 15] Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986 [Non-Patent Document 16] "Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981) [Non-Patent Document 17] Appl. Microbiol. Biotecenol. (1999) 52:541-545 [Non-Patent Document 18] Moore, S., Stein, WH, Photometric ninhydrin method for use in the chromatography of amino acids. J. Biol. Chem.1948, 176, 367-388 Summary of the Invention [Problem to be solved by the invention]

[0006] The present applicant has confirmed that the L-amino acid producing ability of a Corynebacterium microorganism in which the activity of a protein containing the amino acid sequence of SEQ ID NO:1 is attenuated is improved, and has completed the present invention. [Means for solving the problem]

[0007] An object of the present application is to provide a Corynebacterium microorganism in which the activity of a protein comprising the amino acid sequence of SEQ ID NO:1 is attenuated.

[0008] Another object of the present application is to provide a method for producing an L-amino acid, comprising the step of culturing the microorganism in a medium.

[0009] A further object of the present application is to provide a composition for producing L-amino acids, comprising the microorganism, a medium in which it has been cultured, or a combination thereof.

[0010] Furthermore, the present application aims to provide a method for producing a Corynebacterium microorganism that produces an L-amino acid, the method comprising the step of attenuating the activity of a protein comprising the amino acid sequence of SEQ ID NO:1.

[0011] Another object of the present application is to provide a use of a Corynebacterium microorganism having attenuated activity of a protein comprising the amino acid sequence of SEQ ID NO:1 for producing L-amino acids. Effect of the Invention

[0012] By using the microorganism of the present application, L-amino acids can be produced with high efficiency. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] These will be described in detail below. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. In addition, this application is not limited to the following specific description.

[0014] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present application described herein which equivalents are intended to be encompassed by this application.

[0015] One aspect of the present application provides a microorganism of the genus Corynebacterium in which the activity of a protein comprising the amino acid sequence of SEQ ID NO:1 is attenuated.

[0016] The protein of the present application may have the amino acid sequence shown in SEQ ID NO:1, may contain the amino acid sequence, or may essentially consist of the amino acid sequence.

[0017] In the present application, the term "protein comprising the amino acid sequence of SEQ ID NO: 1" refers to a protein having ABC-Transporter permease component activity and having an NCBI number of NCgl1917.

[0018] In the present application, the protein comprising the amino acid sequence of SEQ ID NO:1 is an endogenous protein of the strain, but is not limited thereto.

[0019] The amino acid sequence of the NCgl1917 protein can be obtained from the publicly known database, NIH GenBank. For example, the amino acid sequence information of the NCgl1917 can be found in NCBI Reference Sequence WP_006284211, but is not limited thereto. For example, the NCgl1917 protein is a protein having ABC transporter permease activity derived from the genus Corynebacterium, but is not limited thereto.

[0020] In the present application, the amino acid sequence of the NCgl1917 protein may include 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 shown in SEQ ID NO: 1. It goes without saying that the present application also includes proteins having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, so long as the amino acid sequence has such homology or identity and exhibits efficacy equivalent to that of the protein of the present application.

[0021] For example, the amino acid sequence may have an addition or deletion of a sequence that does not change the function of the protein of the present application, a naturally occurring mutation, a silent mutation, or a conservative substitution at the N-terminus, C-terminus, and / or internally.

[0022] In the present application, a "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structural and / or chemical properties. The protein may, for example, have at least one conservative substitution while still retaining at least one biological activity. 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. For example, among amino acids having electrically charged side chains, positively charged (basic) amino acids include arginine, lysine, and histidine, negatively charged (acidic) amino acids include glutamic acid and aspartic acid, among amino acids having uncharged side chains, nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, polar or hydrophilic amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine, and aromatic amino acids among the above amino acids include phenylalanine, tryptophan, and tyrosine.

[0023] In this application, "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0024] Sequence homology or identity of conserved polynucleotides or proteins may be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize to all or a portion of the sequence under moderate or high stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides having common codons in the polynucleotide or codons that take into account codon degeneracy.

[0025] Whether any two polynucleotide or protein sequences have homology, similarity or identity can be determined using known computer algorithms such as the "FASTA" program with default parameters as in, for example, Non-Patent Document 1. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 3), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later), which includes the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, FASTA (Non-Patent Documents 5, 6 and 7). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0026] Homology, similarity or identity of polynucleotides or proteins can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 8, pp. 1171-1175 (2002). Briefly, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include (1) a binary comparison matrix (identity takes a value of 1 and non-identity takes a value of 0) and a weighted comparison matrix of Non-Patent Document 10 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9, (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5), and (3) no penalty for terminal gaps.

[0027] In the present application, a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 1 is also referred to as an Ncgl1917 gene.

[0028] In the present application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and refers to a DNA or RNA chain longer than a certain length. Specifically, it refers to a polynucleotide fragment that codes for the above-mentioned protein.

[0029] A polynucleotide encoding a protein of the present application may include a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 1. As an example of the present application, a polynucleotide of the present application may have or include the sequence of SEQ ID NO: 2. Furthermore, a polynucleotide of the present application may consist of or essentially consist of the sequence of SEQ ID NO: 2.

[0030] The polynucleotides of the present application may have various modifications in the coding regions thereof, taking into account codon degeneracy or preferred codons in the organism in which the protein of the present application is to be expressed, as long as the amino acid sequence of the protein of the present application is not changed.

[0031] Specifically, the polynucleotide of the present application has a base sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO:2, or contains the above base sequence, or is composed of a base sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO:2, or is substantially composed of the above base sequence, but is not limited to these.

[0032] Furthermore, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a complementary sequence to all or a part of the polynucleotide sequence of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 11 and 12). For example, the conditions include conditions under which polynucleotides having high homology or identity, such as polynucleotides having a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, but polynucleotides having lower homology or identity than that do not hybridize with each other, or conditions under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to normal Southern hybridization washing conditions of 60°C, 1 x SSC, 0.1% SDS, specifically 60°C, 0.1 x SSC, 0.1% SDS, more specifically 68°C, 0.1 x SSC, 0.1% SDS.

[0033] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may include isolated nucleic acid fragments that are complementary to the entire sequence, as well as substantially similar nucleic acid sequences.

[0034] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and may be appropriately adjusted by those skilled in the art depending on the purpose.

[0035] The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables known in the art (eg, Non-Patent Document 11).

[0036] The term "microorganism (or strain)" in this application includes all wild-type microorganisms and microorganisms that have been genetically modified, either naturally or artificially, and is a microorganism in which a specific mechanism has been weakened or enhanced by, for example, inserting an exogenous gene or enhancing or inactivating the activity of an endogenous gene, and is a microorganism that has been genetically modified to produce a desired polypeptide, protein, or product.

[0037] The microorganism of the present application may be a microorganism in which the activity of a protein comprising the amino acid sequence of SEQ ID NO:1 of the present application is attenuated, a microorganism in which the expression of a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:1 of the present application is attenuated, or a microorganism (e.g., a recombinant microorganism) genetically modified by a vector so that the activity of a protein comprising the amino acid sequence of SEQ ID NO:1 of the present application is attenuated, but is not limited to these.

[0038] As an example, the attenuation of the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 is achieved by attenuating the expression of a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 or the polynucleotide of SEQ ID NO: 2. As another example, the attenuation of the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 is achieved by modification of the polynucleotide sequence, such as insertion, deletion or substitution of nucleotides, but is not limited thereto.

[0039] The microorganism of the present application may be a microorganism capable of producing an L-amino acid.

[0040] The microorganism of the present application may be a microorganism that naturally has the ability to produce L-amino acids, or a microorganism in which the ability to produce L-amino acids has been imparted to a parent strain that does not have the ability to produce L-amino acids by attenuating the activity of a protein containing the amino acid sequence of SEQ ID NO: 1 of the present application, but is not limited thereto.

[0041] As an example, the microorganism of the present application may be a strain or microorganism that has been transformed with a vector such that the activity of a protein comprising the amino acid sequence of SEQ ID NO: 1 of the present application is attenuated, and the activity of the protein comprising the amino acid sequence of SEQ ID NO: 1 of the present application is attenuated. For the purposes of the present application, the microorganism is a naturally occurring wild-type microorganism or a microorganism that produces an L-amino acid, in which the activity of the protein comprising the amino acid sequence of SEQ ID NO: 1 of the present application is attenuated, and thus the L-amino acid production ability is improved compared to an unmodified microorganism (e.g., a naturally occurring wild-type microorganism or a microorganism in which the activity of the protein comprising the amino acid sequence of SEQ ID NO: 1 has not been modified), but is not limited thereto.

[0042] For example, the unmodified microorganisms that are the subject strains for comparison as to whether the L-amino acid production ability is improved include Corynebacterium glutamicum KCCM12502P (Patent Document 2), KCCM11222P (Patent Document 3), or KCCM11248P (Patent Document 4), but are not limited to these.

[0043] For example, the strain with improved production ability has an L-amino acid production ability of about 1% or more, specifically about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 10.5% or more, about 11% or more, about 11.5% or more, about 12% or more, about 12.5% ​​or more, about 13% or more, about 13.5% or more, about 14% or more, about 14.5% or more, about 15% or more, about 15.5% or more, about 16% or more, about 16.5% or more, about 17% or more, about 17.5% or more, about 18% or more, about 18.5% or more, about 19% or more, about 19.5% or more, about 20% or more, about 20.5% or more, about 21% or more, compared to the L-amino acid production ability of the parent strain or unmodified microorganism before the mutation. or about 21.5% or more, about 22% or more, about 22.5% or more, about 23% or more, about 23.5% or more, about 24% or more, about 24.5% or more, about 25% or more, about 25.5% or more, about 26% or more, about 26.5% or more, about 27% or more, about 27.5% or more, about 28% or more, about 28.5% or more, about 29% or more, about 29.5% or more, about 30% or more, about 30.5% or more, about 31% or more, about 31.5% or more, about 32% or more, about 32.5% or more, about 33% or more, about 33.5% or more, about 34% or more, about 34.5% or more, or about 35% or more improved in productivity, but any increase in the + value compared to the productivity of the parent strain or unmodified microorganism before the mutation may be used. In another example, the recombinant strain with improved production ability has an L-amino acid production ability improved by about 1.1 times or more, about 1.12 times or more, about 1.13 times or more, 1.15 times or more, 1.16 times or more, 1.17 times or more, 1.18 times or more, 1.19 times or more, 1.2 times or more, 1.25 times or more, 1.3 times or more, or 1.35 times or more compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto. The term "about" refers to a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and may be any number that is equal to or in a similar range to the number following the term "about", but is not limited thereto.

[0044] As an example, a microorganism with improved L-amino acid production ability of the present application is one in which the activity of a protein comprising the amino acid sequence of SEQ ID NO:1 is less than 100%, for example, about 99.9% or less, about 99% or less, about 98% or less, about 97% or less, about 96% or less, about 95% or less, about 90% or less, about 80% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 5% or less, or about 0%, compared to the parent strain before mutation or an unmodified microorganism, but is not limited to these.

[0045] In the present application, the term "unmodified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type strain or a natural strain itself, or a strain before its characteristics are changed due to genetic mutation caused by natural or artificial factors. For example, the unmodified microorganism refers to a microorganism before the activity of a protein containing the amino acid sequence of SEQ ID NO: 1 in this specification is attenuated. The term "unmodified microorganism" is used interchangeably with "strain before modification", "microorganism before modification", "non-mutated strain", "non-modified strain", "non-mutated microorganism" or "reference microorganism".

[0046] As another example of the present application, the Corynebacterium 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.

[0047] In the present application, the term "attenuation" of a protein is a concept that includes all of the following: a decrease in activity compared to the endogenous activity, or the absence of activity. The term "attenuation" is also used interchangeably with other terms such as inactivation, deficiency, down-regulation, decrease, reduction, and attenuation.

[0048] The weakening includes at least one of the following: the activity of the protein itself is reduced or eliminated compared to the activity of the protein originally possessed by the microorganism due to a mutation in the polynucleotide encoding the protein, the degree and / or concentration (expression amount) of the overall protein activity in the cell is reduced compared to the natural strain due to inhibition of expression of the polynucleotide encoding it or inhibition of translation into the protein, the complete absence of expression of the polynucleotide, and the absence of protein activity even if the polynucleotide is expressed. The "endogenous activity" refers to the activity of a specific protein originally possessed by a parent strain, a wild type, or an unmodified microorganism before the trait change when the trait is changed by genetic mutation due to natural or artificial factors. This is used interchangeably with "activity before transformation." The activity of a protein is "inactivated, deficient, reduced, downregulated, decreased, attenuated" compared to the endogenous activity means that the activity of a specific protein is reduced compared to the activity of a specific protein originally possessed by a parent strain or an unmodified microorganism before the trait change.

[0049] Such attenuation of protein activity can be achieved by applying various methods well known in the art, including, but not limited to, those described above (eg, Non-Patent Documents 13 and 14, etc.).

[0050] Specifically, the attenuation of a protein in the present application can be achieved by 1) deleting all or a part of a gene encoding the protein, 2) modifying an expression regulatory region (or an expression regulatory sequence) so as to reduce the expression of a gene encoding the protein, 3) modifying an amino acid sequence constituting the protein so as to delete or weaken the activity of the protein (e.g., deleting / substituting / adding one or more amino acids in the amino acid sequence), or 4) modifying a gene sequence encoding the protein so as to delete or weaken the activity of the protein (e.g., modifying the nucleus of the protein gene so as to encode a modified protein such that the activity of the protein is deleted or weakened). The modification can be carried out by, but is not limited to, one or more of the following: deleting / substituting / adding one or more nucleic acid bases in the nucleic acid sequence; 5) modifying the nucleic acid sequence encoding the start codon or 5'UTR region of the gene transcript encoding the protein; 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding the protein; 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the protein so that a secondary structure that makes ribosome attachment impossible is formed; 8) adding a promoter to the 3' end of the ORF (open reading frame) of the gene sequence encoding the protein so that it is reverse transcribed (reverse transcription engineering, RTE); or 9) a combination of two or more selected from 1) to 8).

[0051] For example, 1) deleting a part or the whole of the gene encoding a protein may be carried out by deleting the entire polynucleotide encoding an endogenous target protein in a chromosome, or by replacing it with a polynucleotide lacking some nucleotides or a marker gene.

[0052] In addition, the expression regulatory region (or expression regulatory sequence) may be modified by generating a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting a sequence having a lower activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0053] In addition, the above-mentioned 3) modification of the base sequence encoding the start codon or 5'UTR region of the gene transcription product encoding a protein can be carried out, for example, by substituting a base sequence encoding another start codon that has a lower protein expression rate compared to the endogenous start codon, but is not limited to this.

[0054] Furthermore, the modification of the amino acid sequence or polynucleotide sequence of 4) and 5) above can be performed by, but is not limited to, generating a mutation in the amino acid sequence of the protein or the polynucleotide sequence encoding the protein by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to weaken the activity of the protein, or by replacing the amino acid sequence or polynucleotide sequence with an improved amino acid sequence or polynucleotide sequence having a lower activity, or an improved amino acid sequence or polynucleotide sequence having no activity. For example, gene expression can be inhibited or weakened by introducing a mutation into a polynucleotide sequence to form a stop codon. As another example of modifying a polynucleotide sequence, the expression of the polynucleotide can be weakened by inserting a transposon into the polynucleotide sequence, and the activity of the protein encoded by the polynucleotide sequence can also be weakened. However, the modification can be performed without being limited to these.

[0055] 6) Introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcription product encoding a protein can be described, for example, in Non-Patent Document 15.

[0056] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a protein so that a secondary structure is formed that makes ribosome attachment impossible may be achieved by disabling or slowing down mRNA translation.

[0057] 8) Adding a promoter to the 3' end of the ORF (open reading frame) of a gene sequence encoding a protein so as to reverse transcribe the ORF (reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the gene transcript encoding the protein to attenuate the activity.

[0058] In this application, the term "vector" refers to a DNA product comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. The expression control region includes a promoter for initiating transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate and function independently of the host genome, and is integrated into the genome itself.

[0059] The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. may be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. may be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. may be used.

[0060] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome by a vector for intracellular chromosome introduction. The polynucleotide can be inserted into a chromosome by any method known in the art, such as, but not limited to, homologous recombination. A selection marker for confirming whether or not the target nucleic acid molecule has been introduced into the chromosome may be further included. The selection marker is for selecting cells transformed with the vector, i.e., for confirming whether or not the target nucleic acid molecule has been inserted, and a marker that confers a selectable phenotype such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide is used. In an environment treated with a selection agent, only cells expressing the selection marker survive or show a different phenotype, so that transformed cells can be selected.

[0061] In the present application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or a microorganism, thereby expressing the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide that is expressed in the host cell, regardless of whether it is inserted into the chromosome of the host cell or located extrachromosomally. The polynucleotide may include DNA and / or RNA that encodes the target polypeptide. The polynucleotide may be introduced in any form as long as it is introduced into the host cell and expressed. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic structure containing all elements necessary for its own expression. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may be introduced into the host cell in its own form and operably linked to a sequence necessary for expression in the host cell, but is not limited thereto.

[0062] Additionally, the term "operably linked" refers to a polynucleotide sequence that is functionally linked to a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target protein.

[0063] In the microorganism of the present application, modification of a part or the whole of a polynucleotide can be induced by (a) homologous recombination using a vector for chromosomal introduction in the microorganism, or genome editing using genetic scissors (engineered nuclease, e.g., CRISPR-Cas9), and / or (b) light such as ultraviolet light or radiation and / or chemical treatment, but is not limited thereto. Methods for modifying a part or the whole of the gene include methods using DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene is introduced into the microorganism to cause homologous recombination, thereby deleting a part or the whole of the gene. The introduced nucleotide sequence or vector may contain, but is not limited to, a dominant selection marker.

[0064] Another aspect of the present application provides a method for producing an L-amino acid, comprising the step of culturing in a medium a recombinant Corynebacterium microorganism in which the activity of a protein comprising the amino acid sequence of SEQ ID NO:1 is attenuated.

[0065] The protein, attenuation, microorganism, etc. containing the amino acid sequence of SEQ ID NO:1 are as described above.

[0066] The Corynebacterium microorganism is Corynebacterium glutamicum, but is not limited thereto, as described above.

[0067] The L-amino acid is at least one amino acid selected from, but not limited to, L-threonine and L-isoleucine.

[0068] In the present application, "culturing" means growing the Corynebacterium microorganism of the present application under appropriately adjusted environmental conditions. The culturing process of the present application can be carried out in a suitable medium and under suitable culture conditions known in the art. Such a culturing process can be easily adjusted and used by a person skilled in the art depending on the strain selected. Specifically, the culturing can be batch, continuous and / or fed-batch culture, but is not limited thereto.

[0069] The term "culture medium" in the present application means a mixture of nutrients necessary 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 may be any medium used for culturing ordinary microorganisms, and the microorganism of the genus Corynebacterium of the present application can be cultured in an ordinary medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, etc., under aerobic conditions by adjusting the temperature, pH, etc.

[0070] Specifically, a culture medium for Corynebacterium microorganisms is disclosed in Non-Patent Document 16.

[0071] The carbon source in the present application may be carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc., sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc., amino acids such as glutamic acid, methionine, lysine, etc. Also, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, corn steeping liquid, etc. may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and any other suitable carbon source may be used. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0072] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, or ammonium nitrate, or an organic nitrogen source such as an amino acid such as glutamic acid, methionine, or glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition product, or defatted soybean cake or its decomposition product. These nitrogen sources may be used alone or in combination of two or more kinds, but are not limited thereto.

[0073] The phosphorus source may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a sodium-containing salt equivalent thereto. The inorganic compound may be sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, or other suitable compounds, such as amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, they are not limited thereto.

[0074] In addition, during the cultivation of the Corynebacterium microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid may be added to the medium in a suitable manner to adjust the pH of the medium. Furthermore, during the cultivation, foam generation may be suppressed using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, and in order to maintain the anaerobic and microaerobic states, no gas may be injected, and nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.

[0075] In the culture of the present application, the culture temperature is maintained at 20 to 45° C., specifically 25 to 40° C., and the culture is carried out for about 10 to 160 hours, but is not limited thereto.

[0076] The L-amino acids produced by the culture of the present application are either secreted into the medium or remain intracellularly.

[0077] 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 prior to the culturing step.

[0078] 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 medium used for the culture (the medium in which the culture was carried out) or from the Corynebacterium microorganism. The recovery step may be further included after the culture step.

[0079] The recovery may involve collecting the target L-amino acid using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof may be used to recover the target L-amino acid from the medium or the microorganism using a suitable method known in the art.

[0080] In addition, the L-amino acid production method of the present application may further include a purification step. The purification can be performed by any suitable method known in the art. For example, when the L-amino acid production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0081] Yet another aspect of the present application provides a composition for producing L-amino acids, comprising a Corynebacterium microorganism in which the activity of a protein having the amino acid sequence of SEQ ID NO:1 of the present application is attenuated, a medium in which the microorganism is cultured, or a combination thereof.

[0082] The protein containing the amino acid sequence of SEQ ID NO:1, the attenuation, the microbial medium, the L-amino acids, etc. are as described above.

[0083] The composition of the present application may further contain any suitable excipient that is commonly used in compositions for producing amino acids, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0084] In the composition of the present application, the protein comprising the amino acid sequence of SEQ ID NO:1, the attenuated microorganism, the medium, the L-amino acid, etc. are as described above.

[0085] Yet another aspect of the present application provides a method for producing a Corynebacterium microorganism that produces an L-amino acid, the method comprising the step of attenuating the activity of a protein comprising the amino acid sequence of SEQ ID NO:1.

[0086] The production method may include a step of modifying a microorganism to attenuate the activity of a protein comprising the amino acid sequence of SEQ ID NO:1.

[0087] The weakening of protein activity, L-amino acids, microorganisms, etc. have been discussed above.

[0088] Yet another aspect of the present application provides use of a Corynebacterium microorganism in which the activity of a protein comprising the amino acid sequence of SEQ ID NO:1 is attenuated for production of L-amino acids.

[0089] The proteins, attenuations, microorganisms, L-amino acids, etc., including the amino acid sequence of SEQ ID NO:1 are as described above. EXAMPLES

[0090] The present application will be described in more detail below with reference to examples and experimental examples. However, these examples and experimental examples are merely illustrative of the present application, and the present application is not limited to these examples and experimental examples. EXAMPLES

[0091] Generation of random mutation libraries using transposons In order to obtain a strain with improved L-threonine production, a random mutation library was constructed as follows. First, Corynebacterium glutamicum KCCM12502P (Patent Document 2) was used as a parent strain, and EZ-Tn5 TM <R6Kγori / KAN-2> TnP Transposome TM The plasmid obtained using a kit (Epicentre) was transformed by the electric pulse method (Non-Patent Document 17), and approximately 20,000 colonies were obtained by smearing on a complex plate medium containing kanamycin (25 mg / l). <Complex plate medium (pH 7.0)> Glucose 10g, peptone 10g, beef extract 5g, yeast extract 5g, Brain Heart Infusion 18.5g, NaCl 2.5g, urea 2g, sorbitiol 91g, agar 20g (in 1 liter of distilled water) EXAMPLES

[0092] Screening of transposon-based random mutation libraries Approximately 20,000 colonies obtained in Example 1 were inoculated into 300 uL of the following selective medium containing kanamycin (25 mg / l), and cultured in a 96-deep well plate at 32° C. and 200 rpm for approximately 24 hours. <Selective medium (pH 8.0)> Glucose 10g, (NH4)2SO4 5.5g, MgSO4·7H2O 1.2g, KH2PO4 0.8g, K2HPO4 16.4g, biotin 100ug, thiamine HCL 1000ug, calcium pantothenate 2000ug, nicotinamide 2000ug (in 1 liter of distilled water)

[0093] The ninhydrin method was used to analyze the amount of L-threonine produced in the culture solution (Non-Patent Document 18). After the culture was completed, 10 ul of the culture supernatant and 190 ul of the ninhydrin reaction solution were reacted at 65°C for 30 minutes, and then the absorbance was measured at a wavelength of 570 nm using a spectrophotometer. Approximately 60 colonies were selected as mutant strains that showed higher absorbance than the control Corynebacterium glutamicum KCCM12502P strain. The other colonies showed absorbance equivalent to or weakened to that of the control Corynebacterium glutamicum KCCM12502P strain.

[0094] Approximately 60 strains selected as described above were cultured again in the same manner as above, and then the ninhydrin reaction was repeated. Finally, the top 10 mutant strains with improved L-threonine production ability compared to the parent strain Corynebacterium glutamicum KCCM12502P were selected and named 12502P-m1, 12502P-m2, 12502P-m3, 12502P-m4, 12502P-m5, 12502P-m6, 12502P-m7, 12502P-m8, 12502P-m9, and 12502P-m10, respectively. EXAMPLES

[0095] Analysis of L-threonine producing ability in selected random mutants In order to finally select a strain showing reproducible improvement in L-threonine production ability from the 10 mutant strains selected in Example 2, flask culture was performed using the following medium. The strains were cultured by the following method, and the L-threonine production amount was measured. First, each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of seed medium containing kanamycin (25 mg / l), and cultured with shaking at 30°C and 200 rpm for 20 hours. Next, 1 ml of the seed culture liquid was inoculated into a 250 ml corner baffle flask containing 24 ml of production medium containing kanamycin (25 mg / l), and cultured with shaking at 32°C and 200 rpm for 72 hours. The compositions of the seed medium and production medium are as follows. The culture results are shown in Table 1. <Seed medium (pH 7.0)> Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4·7H2O 0.5g, biotin 0.1mg, thiamine HCl 1mg, calcium pantothenate 22mg, nicotinamide 2mg (in 1 liter of distilled water) <Production medium (pH 7.0)> Glucose 63g, (NH4)2SO4 28g, soy protein 20g, molasses 14g, KH2PO4 1.1g, MgSO4·7H2O 1.2g, biotin 1.8mg, thiamine hydrochloride 9mg, calcium pantothenate 9mg, MnSO4 180mg, FeSO4 200mg, ZnSO4 1mg, CuSO4 1mg, CaCO3 30g (in 1L distilled water)

[0096] [Table 1]

[0097] Among the ten selected mutant strains, 12502P-m6 was finally selected as a strain with significantly improved L-threonine productivity. EXAMPLES

[0098] Elucidation of the cause of improved L-threonine productivity in the final selected strain In this example, the mutant strain finally selected in Example 3 was used to attempt to identify the gene deleted by random insertion of the transposon. Genomic DNA of 12502P-m6 was extracted, cut, ligated, transformed into E. coli DH5α, and smeared on LB solid medium containing kanamycin (25 mg / l). Twenty transformed colonies were selected, and a plasmid containing a part of the unknown gene was obtained, and then transformed into EZ-Tn5. TM <R6Kγori / KAN-2> TnP Transposome TM The nucleotide sequence was analyzed using primers SEQ ID NO: 3 and SEQ ID NO: 4 of the kit. As a result, based on the nucleotide sequence reported in the National Institutes of Health Genebank (NIH Geneback), it was confirmed that the gene containing the nucleotide sequence of SEQ ID NO: 2 (Ncgl1917) was inactivated and the activity of the protein containing the amino acid sequence of SEQ ID NO: 1 was weakened.

[0099] [Table 2] EXAMPLES

[0100] Preparation of recombinant vectors and strains for attenuating the activity of a protein containing the amino acid sequence of SEQ ID NO:1 A recombinant vector for deleting a gene containing the nucleotide sequence of SEQ ID NO: 2 confirmed in Example 4 on the chromosome of a Corynebacterium strain was prepared by the following method. First, using genomic DNA extracted from the WT strain as a template, primers of SEQ ID NO: 5 and 6 were synthesized in which a restriction enzyme SmaI recognition site was inserted into the 5' fragment located 249 bp upstream of the Ncgl1917 gene. Primers of SEQ ID NO: 7 and 8 were synthesized in which a restriction enzyme SmaI recognition site was inserted into the 3' fragment located 410 bp downstream of the Ncgl1917 gene (Table 3).

[0101] [Table 3]

[0102] Specifically, PCR [Non-Patent Document 11] was performed using the chromosomal DNA of wild-type Corynebacterium glutamicum ATCC13032 as a template. In this way, a DNA fragment of 249 bp upstream and 410 bp downstream of the portion encoding the protein encoded by the nucleotide sequence of SEQ ID NO:2 was obtained. Here, the PCR conditions were denaturation at 94°C for 2 minutes, followed by 30 cycles of denaturation at 94°C for 1 minute, annealing at 56°C for 1 minute, and polymerization at 72°C for 40 seconds, followed by polymerization reaction at 72°C for 10 minutes. On the other hand, the pDCM2 vector (Patent Document 5) treated with the restriction enzyme SmaI and then heat-treated at 65°C for 20 minutes was ligated to the insert DNA fragment amplified by the PCR using the Infusion Cloning Kit, and then transformed into Escherichia coli DH5α. The above strain was smeared on LB solid medium containing kanamycin (25 mg / l). Colonies transformed with the vector into which the target gene was inserted were selected by PCR using primers SEQ ID NO:5 and SEQ ID NO:8, and then a plasmid was obtained by a conventional plasmid extraction method. The plasmid was named pDCM2-ΔNcgl1917. The prepared vector was transformed into the threonine-producing Corynebacterium glutamicum KCCM12502P strain by electric pulse method. The strain in which the Ncgl1917 gene was inactivated in the KCCM12502P strain was named KCCM12502P::Δ1917. EXAMPLES

[0103] Evaluation of L-threonine production ability of a strain in which the activity of the protein containing the amino acid sequence of SEQ ID NO: 1, derived from Corynebacterium glutamicum KCCM12502P, was attenuated In order to analyze the L-threonine producing ability of the Corynebacterium glutamicum KCCM12502P::Δ1917 strain prepared as described above, flask culture was performed using the following medium. As a control, the parent strain Corynebacterium glutamicum KCCM12502P was used and cultured in the following manner to measure the L-threonine production amount. Each strain was inoculated into a 250-ml corner baffle flask containing 25 ml of seed medium and cultured at 30°C and 200 rpm for 20 hours with shaking. Next, 1 ml of the seed culture was inoculated into a 250-ml corner baffle flask containing 24 ml of production medium and cultured at 32°C and 200 rpm for 72 hours with shaking. The compositions of the seed medium and production medium are as follows. The culture results are shown in Table 4. <Seed medium (pH 7.0)> Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4·7H2O 0.5g, biotin 0.1mg, thiamine HCl 1mg, calcium pantothenate 22mg, nicotinamide 2mg (in 1 liter of distilled water) <Production medium (pH 7.0)> Glucose 63g, (NH4)2SO4 28g, soy protein 20g, molasses 14g, KH2PO4 1.1g, MgSO4·7H2O 1.2g, biotin 1.8mg, thiamine hydrochloride 9mg, calcium pantothenate 9mg, MnSO4 180mg, FeSO4 200mg, ZnSO4 1mg, CuSO4 1mg, CaCO3 30g (in 1L distilled water)

[0104] [Table 4]

[0105] These results confirmed that deleting the gene containing the nucleotide sequence of SEQ ID NO:2 from the L-threonine producing strain Corynebacterium glutamicum KCCM12502P improved the L-threonine production ability by an average of 35% compared to the parent strain.

[0106] Therefore, it was confirmed that the L-threonine producing ability of Corynebacterium microorganisms can be improved by deleting the gene containing the nucleotide sequence of SEQ ID NO: 2.

[0107] These results confirmed that inactivating the protein containing the amino acid sequence of SEQ ID NO: 1 in an L-threonine-producing strain is effective in improving L-threonine production ability. Therefore, the strain KCCM12502P::Δ1917 was named CA09-0907 and internationally deposited at the Korea Center for Microorganisms (KCCM) on February 1, 2021 under the deposit number KCCM12946P. EXAMPLES

[0108] Construction of a strain with reduced activity of the protein containing the amino acid sequence of SEQ ID NO: 1 derived from Corynebacterium glutamicum KCCM11222P and evaluation of L-threonine production ability In order to confirm whether other strains belonging to Corynebacterium glutamicum that produce L-threonine have the same effect as above, a strain was prepared by deleting a gene containing the nucleotide sequence of SEQ ID NO: 2 from Corynebacterium glutamicum KCCM11222P (Patent Document 3), which is an L-threonine producing strain, in the same manner as in Example 5, and named KCCM11222P::Δ1917. In order to analyze the L-threonine producing ability of the prepared Corynebacterium glutamicum KCCM11222P::Δ1917 strain, it was cultured in the same manner as in Example 6. As a control group, the parent strain Corynebacterium glutamicum KCCM11222P strain was used. The culture results are shown in Table 5.

[0109] [Table 5]

[0110] From these results, it was confirmed that the L-threonine producing ability of Corynebacterium glutamicum KCCM11222P, which is an L-threonine producing strain, was improved by an average of 12% when the gene containing the nucleotide sequence of SEQ ID NO: 2 was deleted. From the results of Examples 5 to 7, it was confirmed that the L-threonine producing ability was improved in Corynebacterium microorganisms by weakening the activity of the protein containing the amino acid sequence of SEQ ID NO: 1. EXAMPLES

[0111] Construction of a strain with reduced activity of the protein containing the amino acid sequence of SEQ ID NO:1 derived from Corynebacterium glutamicum KCCM11248P and evaluation of L-isoleucine production ability In order to confirm whether or not the strains belonging to Corynebacterium glutamicum that produce L-isoleucine have the same effect of increasing L-amino acid production as described above, a strain was prepared by deleting a gene containing the nucleotide sequence of SEQ ID NO:2 from Corynebacterium glutamicum KCCM11248P (Patent Document 4), an L-isoleucine producing strain, as in Example 5, and named KCCM11248P::Δ1917. In order to analyze the L-isoleucine producing ability of the prepared Corynebacterium glutamicum KCCM11248P::Δ1917 strain, flask culture was performed using the following medium. As a control group, the parent strain Corynebacterium glutamicum KCCM11248P was used, and the amount of L-isoleucine produced was measured by culturing it in the following manner. First, each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of seed medium, and cultured with shaking at 30°C and 200 rpm for 20 hours. Next, 1 ml of the seed culture was inoculated into a 250 ml corner baffle flask containing 24 ml of production medium, and cultured with shaking at 32°C and 200 rpm for 72 hours. The compositions of the seed medium and production medium are as follows. The culture results are shown in Table 6. <Seed medium (pH 7.0)> Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4·7H2O 0.5g, biotin 0.1mg, thiamine HCl 1mg, calcium pantothenate 22mg, nicotinamide 2mg (in 1 liter of distilled water) <Production medium (pH 7.2)> Glucose 100g, yeast extract 5g, (NH4)2SO4 16g, KH2PO4 1g, MgSO4·7H2O 1g, FeSO4·7H2O 10mg, MnSO4·H2O 10mg, biotin 200μg (in 1L distilled water)

[0112] [Table 6]

[0113] From these results, it was confirmed that the L-isoleucine producing ability of Corynebacterium glutamicum KCCM11248P, which is an L-isoleucine producing strain, is improved by an average of 27% when the gene containing the nucleotide sequence of SEQ ID NO: 2 is deleted. Therefore, it was confirmed that the L-isoleucine producing ability is improved in Corynebacterium microorganisms by weakening the activity of the protein containing the amino acid sequence of SEQ ID NO: 1.

[0114] From the above description, a person skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present application should be interpreted as including all modifications and variations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

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Claims

1. A Corynebacterium microorganism in which the gene encoding the protein comprising the amino acid sequence of SEQ ID NO:1 is deleted.

2. The microorganism of the genus Corynebacterium according to claim 1, which produces an L-amino acid.

3. The Corynebacterium microorganism according to claim 1, wherein the Corynebacterium microorganism is Corynebacterium glutamicum.

4. The Corynebacterium microorganism according to claim 2, wherein the L-amino acid is at least one selected from the group consisting of L-threonine and L-isoleucine.

5. The microorganism of claim 1, wherein the microorganism has improved L-amino acid production ability compared to a microorganism that is not deficient in a gene encoding a protein comprising the amino acid sequence of SEQ ID NO:1, and the L-amino acid is at least one selected from L-threonine and L-isoleucine.

6. 6. A method for producing an L-amino acid, comprising the step of culturing the Corynebacterium microorganism according to claim 1 in a medium, wherein the L-amino acid is at least one selected from the group consisting of L-threonine and L-isoleucine.

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

8. 7. The method according to claim 6, further comprising the step of recovering an L-amino acid from the microorganism or the medium, wherein the L-amino acid is at least one selected from L-threonine and L-isoleucine.

9. 6. A composition for producing an L-amino acid, comprising the microorganism according to claim 1, wherein the L-amino acid is at least one selected from the group consisting of L-threonine and L-isoleucine.

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