LysE mutant and method for producing L-arginine using the same

Abstract

The present application provides LysE mutants.

Description

Technical Field

[0001] This application relates to LysE variants, Corynebacterium microorganisms containing the same, and a method for producing L-arginine using the same.

Background Art

[0002] L-arginine is used medicinally as a liver function promoter, brain function promoter, comprehensive amino acid preparation, etc., and has recently been in the spotlight as a food additive such as kamaboko additive, health drink additive, and salt substitute for hypertensive patients.

[0003] In order to produce industrially available high-concentration L-arginine, research on using microorganisms has been continuously conducted. Methods such as using mutant strains derived from Brevibacterium or Corynebacterium microorganisms, which are glutamate-producing strains, and using amino acid-producing strains with improved growth by cell fusion have been reported.

[0004] On the other hand, although it has been reported that lysE (lysine exporter) of Corynebacterium microorganisms, which are lysine exporters, also excretes L-arginine (Bellmann A, et al, Microbiology, 147: 1765-1774, 2001), research for further increasing L-arginine production is required.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

[0006] [Non-licensed Document 1] Bellmann A, et al, Microbiology, 147:1765-1774, 2001 [Non-licensed Document 2] Pearson et al (1988) [Proc.Natl.Acad.Sci.USA 85]:2444 [Non-licensed Document 3] Rice et al., 2000,Trends Genet.16:276-277 [Non-licensed Document 4] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed Document 5] Devereux,J.,et al,Nucleic Acids Research 12:387 (1984) [Non-licensed Document 6] Atschul,[S.] [F.,] [ET AL,J MOLEC BIOL 215]:403 (1990) [Non-licensed Document 7] Guide to Huge Computers,Martin J.Bishop,[ED.,] Academic Press,San Diego, 1994 [Non-licensed Document 8] [CARILLO ET AL.](1988) SIAM J Applied Math 48:1073 [Non-licensed Document 9] Smith and Waterman,Adv.Appl.Math (1981) 2:482

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

Non-licensed Document 17

Non - Patent Document 18

Non - Patent Document 19

Summary of the Invention

Problems to be Solved by the Invention

[0007] This application provides a LysE mutant.

Means for Solving the Problems

[0008] The object of this application is to provide a LysE mutant in which any one or more of the amino acids corresponding to the 50th, 153rd, and 215th positions of SEQ ID NO: 1 are substituted with other amino acids.

[0009] Another object of this application is to provide a polynucleotide encoding the LysE mutant of this application.

[0010] Another object of this application is to provide a microorganism containing the LysE mutant of this application or the polynucleotide encoding it.

[0011] Another object of this application is to provide a method for producing L - arginine including the step of culturing the microorganism of this application.

Effects of the Invention

[0012] The variant of this invention can be used to increase the L-arginine production yield of microorganisms. [Modes for carrying out the invention]

[0013] This can be explained in more detail as follows: On the other hand, each description and embodiment disclosed in this application can also be applied to each other different descriptions and embodiments. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the categories of this application are not considered to be limited by the specific descriptions described below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated as a whole into this specification as references, and the level of the technical field to which this application belongs and the content of this application are explained more clearly.

[0014] One aspect of this application provides a LysE variant in which one or more amino acids in the amino acids corresponding to positions 50, 153, and 215 of Sequence ID No. 1 are substituted with other amino acids.

[0015] In this application, "LysE" refers to the lysine exporter protein encoded by the lysE gene.

[0016] Specifically, the LysE may be a naturally occurring polypeptide or a wild-type polypeptide, or a mature polypeptide thereof, or may include its variants or functional fragments, but is not limited to any form that can serve as a parent for the LysE variants of this application.

[0017] The LysE sequence described in this application can be obtained from the NCBI's Genbank, a publicly known database.

[0018] As one example, LysE in this application may be derived from the genus Corynebacterium. Examples include, but are not limited to, Corynebacterium glutamicum, Corynebacterium suranareeae, Corynebacterium callunae, Corynebacterium deserti, Corynebacterium crudilactis, Corynebacterium efficiens, and Corynebacterium pacaense.

[0019] As one example, the LysE of this application may be the polypeptide of SEQ ID NO: 1. As another example, the LysE of this application may be a polypeptide having approximately 60% or more sequence identity with the polypeptide of SEQ ID NO: 1, and is included in the LysE protein without limitation as long as it has the same or equivalent activity as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0020] For example, LysE may be a protein derived from the genus Corynebacterium that has at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, or 99.5% or more identity with SEQ ID NO: 1. No.WP_003854734.1, P94633.2, WP_040967332.1, WP_074494034.1, WP_038583742.1, WP_074506107.1, WP_01543 9467.1, WP_220313759.1, WP_077311917.1, WP_172768514.1, WP_096455559.1, WP_015651045.1, WP_053544699.1 Examples of proteins include, but are not limited to, WP_066565316.1, NLZ57487.1, Q8RQM4.2, BAC18167.1, WP_006769321.1, WP_191733757.1, WP_080795286.1, WP_197088449.1, WP_047252903.1, WP_221709738.1, WP_123048290.1, and WP_042621270.1.

[0021] As an example of any of the aforementioned embodiments, LysE of this application may have, contain, or be essentially composed of the amino acid sequence described in Sequence ID No. 1, but any polypeptide / protein having the same activity may be included without limitation. Furthermore, it is obvious that polypeptides / proteins having amino acid sequences in which some sequences are deleted, modified, substituted, or added are also included within the range of polypeptides / proteins targeted for mutation in this application, as long as they have such homology or identity and exhibit L-lysine efflux activity.

[0022] In this application, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or base sequences, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0023] The sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequencing algorithms, and a default gap penalty established by the program used may be used in conjunction with them. Substantially homologous or identical sequences can generally be hybridized with whole or partial sequences under moderate to high stringent conditions. It is obvious that hybridization also includes hybridization with polynucleotides containing codons, or codons considering codon degeneracy in general, in polynucleotides.

[0024] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms such as the "FASTA" program with default parameters, for example, as in Pearson et al (1988) [Proc.Natl.Acad.Sci.USA 85]:2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), as performed 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) (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 (Including Diego, 1994, and [CARILLO ET AL.] (1988) SIAM J Applied Math 48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information Databases.

[0025] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as Needleman et al. (1970), J Mol Biol. 48:443, as is publicly known, for example, in 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 two sequences divided by the number of similarly sequenced symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program may include: (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and a weighted comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14:6745 (or EDNAFULL (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 penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0026] In this application, the term "variant" refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, resulting in a different amino acid sequence from the original polypeptide, but with maintained functions or properties. Such variants may generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the capabilities of the variant may be increased, unchanged, or decreased compared to the original polypeptide. Some variants may also include those in which one or more parts, such as the N-terminal leader sequence or transmembrane domain, are removed. Other variants may include those in which parts of the N- and / or C-terminus of a mature protein are removed. The aforementioned term "mutant" can be used interchangeably with terms such as variant, modified, mutant polypeptide, mutant protein, mutation, and divergent (in English, these may be expressed as 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 a mutated form.

[0027] Furthermore, the mutants may include the deletion or addition of amino acids that have minimal impact on the polypeptide's properties and secondary structure. For example, the N-terminus of the mutant may be conjugated with a signal (or leader) sequence involved in protein translocation co-translationally or post-translationally. The mutants may also be conjugated with other sequences or linkers for verification, purification, or synthesis.

[0028] The LysE variant provided in this application may be a polypeptide comprising an amino acid sequence in which one or more amino acids in the amino acids corresponding to positions 50, 153, and 215 of Sequence ID No. 1 are substituted with other amino acids.

[0029] As an example, the LysE variant of this application may be a variant in any polypeptide or protein having lysine efflux activity, comprising the substitution of one or more amino acids with other amino acids among the amino acids corresponding to the 50th, 153rd, and 215th positions from the N-terminus of SEQ ID NO: 1.

[0030] The aforementioned "other amino acids" are not limited to amino acids that are different from the amino acid before substitution. On the other hand, when the expression "a specific amino acid is substituted" is used in this application, it is self-evident that the substitution is made with an amino acid different from the amino acid before substitution, even without specifically stating that it is substituted with another amino acid.

[0031] As one concrete example, the variant of this application may be such that the amino acid corresponding to the 50th position in the amino acid sequence of the reference protein SEQ ID NO: 1 is an amino acid other than phenylalanine, the amino acid corresponding to the 153rd position is an amino acid other than asparagine, and the amino acid corresponding to the 215th position is an amino acid other than asparagine.

[0032] As one example of any of the aforementioned embodiment examples, the amino acid corresponding to position 50 of SEQ ID NO: 1 in the mutant may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, histidine, and arginine. As an example, the amino acid corresponding to position 50 of SEQ ID NO: 1 in the mutant may be a nonpolar amino acid selected from glycine, alanine, valine, leucine, isoleucine, methionine, tryptophan, and proline. As an example, the amino acid corresponding to position 50 of SEQ ID NO: 1 in the mutant may be a branched amino acid selected from valine, leucine, and isoleucine, but is not limited thereto.

[0033] As one example of any of the aforementioned embodiment examples, the amino acid corresponding to position 153 of SEQ ID NO: 1 in the mutant may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, glutamine, aspartic acid, glutamic acid, lysine, histidine, and arginine. As an example, the amino acid corresponding to position 153 of SEQ ID NO: 1 in the mutant may be a polar amino acid selected from serine, threonine, cysteine, tyrosine, and glutamine. As an example, the amino acid corresponding to position 153 of SEQ ID NO: 1 in the mutant may be serine, threonine, or glutamine, but is not limited to these.

[0034] As one example of any of the aforementioned embodiment examples, the amino acid corresponding to position 215 of SEQ ID NO: 1 in the mutant may be selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, glutamine, aspartic acid, glutamic acid, lysine, histidine, and arginine. As an example, the amino acid corresponding to position 215 of SEQ ID NO: 1 in the mutant may be a polar amino acid selected from serine, threonine, cysteine, tyrosine, and glutamine. As an example, the amino acid corresponding to position 215 of SEQ ID NO: 1 in the mutant 215 The amino acid corresponding to the 1-th position may be, but is not limited to, serine, threonine, or glutamine.

[0035] As an example of any of the aforementioned embodiments, the variant of this application may be one in which one or more amino acids at the 50th, 153rd, and 215th amino acid residues in the amino acid sequence of SEQ ID NO: 1 are substituted with other amino acids. As an example of any of the aforementioned embodiments, the variant of this application may be one in which one of the 50th, 153rd, and 215th amino acid residues in the amino acid sequence of SEQ ID NO: 1 are substituted with other amino acids. The amino acid after substitution of the 50th, 153rd, or 215th amino acid residue in the amino acid sequence of SEQ ID NO: 1 is the same as the amino acid after substitution of the amino acid residue corresponding to the 50th, 153rd, or 215th position in the amino acid sequence of SEQ ID NO: 1 described above.

[0036] As an example of any one of the aforementioned embodiment examples, the variant of this application has one or more amino acids at the 50th, 153rd, and 215th amino acid residues in the amino acid sequence of SEQ ID NO: 1 replaced with other amino acids, and can have at least 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more and less than 100% identity with SEQ ID NO: 1.

[0037] As an example of any of the aforementioned examples of embodiment, the variant of this application may consist of an amino acid sequence selected from Sequence IDs 27-35, or have, include, or be essentially composed of an amino acid sequence having at least 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.8% or more, or 100% homology or identity with such amino acid sequence.

[0038] Furthermore, it is obvious that any amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant of this application is also included within the scope of this application, including variants in which some sequences are deleted, modified, substituted, conserved substituted, or added.

[0039] For example, the amino acid sequence may have additions or deletions of sequences that do not alter the function of the variant of this application, spontaneous mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or within it.

[0040] The term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little to no effect on the activity of a protein or polypeptide.

[0041] In this application, the term "corresponding to" refers to an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar, identical, or homologous to a residue listed in the polypeptide. Identifying the amino acid at a corresponding position may also mean determining a specific amino acid in a sequence that references a particular sequence. As used in this application, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0042] For example, any amino acid sequence can be aligned with sequence number 1, and based on this, each amino acid residue in the amino acid sequence can be numbered by referring to the position of the amino acid residue corresponding to the amino acid residue in sequence number 1. For example, a sequence alignment algorithm such as the one described in this application can verify the position of amino acids, or the position where deformations such as substitution, insertion, or deletion occur, compared to a query sequence (also called a "reference sequence").

[0043] For such sorting, one can use, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453) or the Needle program from the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16:276-277), but is not limited to these. Other sequence sorting programs and pairwise sequence comparison algorithms known in the industry can be used as appropriate.

[0044] Another aspect of this application provides a polynucleotide encoding a variant of the present application.

[0045] In this application, the term "polynucleotide" refers to a polymer of nucleotides in which nucleotide units (monomers) are covalently linked together in a long chain, and means a DNA or RNA chain of a certain length or longer.

[0046] The polynucleotides of this application can undergo various modifications to the coding region, taking into account codon degeneracy or preferred codons in organisms intended to express proteins, without altering the amino acid sequence.

[0047] As one example of the aforementioned embodiment, the polynucleotide of this application can be obtained from the lysE sequence in the NCBI Genbank, a known database.

[0048] Specifically, the polynucleotides encoding the LysE protein variant of this application may have, contain, or consist of, or be required to consist of, a nucleotide sequence that is homologous or identical to the sequence of Sequence ID No. 2 by 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%, or consist of, or be required to consist of, a nucleotide sequence that is homologous or identical to the sequence of Sequence ID No. 2 by 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%. In this case, in the homologous or identical sequences, the codon encoding the amino acid corresponding to the 50th position of Sequence ID No. 1 may be one of the codons encoding amino acids other than phenylalanine, the codon encoding the amino acid corresponding to the 153rd position may be one of the codons encoding amino acids other than asparagine, and the codon encoding the amino acid corresponding to the 215th position may be one of the codons encoding amino acids other than asparagine (for example, Sequence ID Nos. 36-38), but is not limited thereto.

[0049] Furthermore, the polynucleotides of this application are not limited to any probes that can be produced from known gene sequences, such as sequences that can hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of this application. The “stringent condition” means a condition that enables 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; FMAusubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, we can list conditions in which polynucleotides with high homology or identity hybridize with each other, with homology or identity levels 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, but do not hybridize with polynucleotides with lower homology or identity levels. Alternatively, we can list conditions in which the polynucleotides are washed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of normal Southern hybridization: 60°C, 1XSSC, 0.1% SDS, more specifically 60°C, 0.1XSSC, 0.1% SDS, or more specifically 68°C, 0.1XSSC, 0.1% SDS.

[0050] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. The term “complementary” is used to describe the relationships between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of this application may also include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the overall sequence.

[0051] Specifically, polynucleotides homologous or identical to the polynucleotide of this application can be detected using hybridization conditions that include a hybridization step at a Tm value of 55°C, and under the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0052] The appropriate stringency for hybridizing the aforementioned polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are well known in the art (e.g., J. Sambrook et al., ibid.).

[0053] Another aspect of this application provides a vector comprising a polynucleotide encoding a LysE variant. The LysE variant and the polynucleotide encoding it are as described above.

[0054] As one concrete example, the vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0055] The “vector” of this application may include a DNA product comprising a polynucleotide sequence encoding the target polypeptide, operably linked to a suitable regulatory region (or regulatory sequence) so as to enable the expression of the target polypeptide in a suitable host. The regulatory region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences regulating the termination of transcription and decoding. The vector, after being transmuted in a suitable host cell, may replicate or function independently of the host genome, or may be integrated into the genome itself.

[0056] The vectors used in this application are 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 their 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, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0057] As an example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a chromosome insertion vector within a cell. The insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited to these methods. A selection marker may further be included to confirm the presence or absence of the chromosome insertion. The selection marker is used to select cells that have been phenotyped by the vector, i.e., to confirm the presence or absence of the target nucleic acid molecule insertion, and can be a marker that confers a selectable phenotype such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of a surface polypeptide. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, thus allowing for the selection of phenotyped cells.

[0058] In this application, the term "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them, regardless of whether they are inserted into or extrachromosomal regions of the host cell, as long as they 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 into the host cell in any form that can be 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 gene structure containing all the elements necessary for its expression. The expression cassette may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to the sequences necessary for expression in the host cell, but is not limited to this.

[0059] Furthermore, the term "operably linked" in the foregoing means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the variant of this application.

[0060] Another aspect of this application provides a microorganism comprising a LysE variant or a polynucleotide encoding the variant. The LysE variant and the polynucleotide encoding it are as described above.

[0061] The microorganism of this application may include one or more of the LysE variant of this application, the polynucleotide encoding it, and a vector containing the polynucleotide.

[0062] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and naturally occurring or artificially genetically modified microorganisms in which a particular mechanism is weakened or strengthened due to causes such as the insertion of external genes or the enhancement or inactivation of the activity of endogenous genes, and may include microorganisms that undergo genetic modification for the production of a desired polypeptide, protein, or product.

[0063] The microorganism of this application may be, but is not limited to, a microorganism comprising one or more of the mutant of this application, a polynucleotide encoding the mutant of this application, or a vector comprising a polynucleotide encoding the mutant of this application; a microorganism modified to express the mutant of this application or a polynucleotide encoding the mutant of this application; a microorganism expressing the mutant of this application or a polynucleotide encoding the mutant of this application (e.g., a recombinant strain); or a microorganism having the mutant activity of this application (e.g., a recombinant strain).

[0064] The microorganisms of this application may have the ability to excrete and / or produce L-arginine.

[0065] The microorganisms of this application may, but are not limited to, microorganisms that naturally express the LysE protein or have arginine efflux and / or production ability; or microorganisms in which the variant of this application or the polynucleotide encoding it (or a vector containing the polynucleotide) is introduced and / or arginine efflux and / or production ability is conferred to a parent strain that lacks the LysE protein or arginine efflux and / or production ability. For example, the microorganisms of this application may, but are not limited to, microorganisms into which the LysE variant of this application has been introduced and which have increased L-arginine efflux and / or production ability.

[0066] For example, the strains of this application are cells or microorganisms that are transmuted with a vector containing the polynucleotide encoding the polynucleotide encoding the variant of this application and express the variant of this application. For the purposes of this application, the strains of this application may include all microorganisms that contain the variant of this application and are capable of effluxing or producing L-arginine. For example, the strains of this application may be recombinant strains in which a LysE variant is expressed by introducing the polynucleotide encoding the variant of this application into a naturally occurring wild-type microorganism or a microorganism that produces L-arginine, thereby increasing L-arginine efflux and / or production capacity. The recombinant strains with increased L-arginine efflux and / or production capacity may be, but are not limited to, naturally occurring wild-type microorganisms or LysE non-mutant microorganisms (i.e., microorganisms that express wild-type LysE (SEQ ID NO: 1); or microorganisms that do not express mutant LysE protein).

[0067] In this application, the term "non-myxoid microorganism" means a strain that is either wild-type or naturally occurring, or a strain before phenotypic change due to genetic mutation caused by natural or artificial factors, and does not exclude strains containing naturally occurring mutations in microorganisms. For example, the non-myxoid microorganism means a strain in which the LysE mutant described herein has not been introduced, or before its introduction. The term "non-myxoid microorganism" may be used interchangeably with "pre-deformation strain," "pre-deformation microorganism," "non-mutant strain," "non-myxoid strain," "non-mutant microorganism," or "reference microorganism."

[0068] As one concrete example, the microorganism of this application may be a microorganism of the genus Corynebacterium. For example, the microorganisms of this application include Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, and Corynebacterium ammoniagenes. It may also be Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, it may be, but is not limited to, Corynebacterium glutamicum.

[0069] The L-arginine-excreting and / or-producing microorganisms of this application may include the LysE variant disclosed herein and be characterized by increased L-arginine production and / or excretion capacity.

[0070] As an example of any of the aforementioned embodiments, a microorganism that produces and / or excretes L-arginine or a microorganism that has the ability to produce and / or excrete L-arginine may be a microorganism in which a portion of the genes in the L-arginine biosynthesis pathway is strengthened or weakened, or a portion of the genes in the L-arginine degradation pathway is strengthened or weakened.

[0071] As an example of any of the aforementioned embodiments, the microorganism may be one in which some of the genes in the L-arginine biosynthesis pathway are strengthened or weakened. Examples of L-arginine biosynthesis enzymes include N-acetylglutamyl phosphate reductase (argC), ornithine acetyltransferase (argJ), N-acetylglutamate kinase (argB), acetylornithine transaminase (argD), ornithine carbamoyltransferase (argF), argininosuccinate synthetase (argG), argininosuccinate lyase (argH), and carbamoyl phosphate synthetase (carAB). Arginine biosynthesis enzymes present on the Arg operon (argCJBDFRGH) can be regulated by the arginine repressor encoded by argR (J Bacteriol. 2002 Dec; 184(23): 6602-14). Therefore, L-arginine production capacity can be conferred or enhanced by the deficiency or weakening of the arginine repressor (argR) (US2002-0045223) and / or by overexpression of one or more of the aforementioned biosynthesis-related genes. For example, the arginine repressor may, but is not limited to, the polypeptide represented by the amino acid sequence of SEQ ID NO: 39.

[0072] In this application, the term "weakening" of polypeptide activity encompasses all cases where activity is reduced or absent compared to endogenous activity. This weakening may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decline, reduce, and attenuation.

[0073] The aforementioned weakening may include cases where the activity of the polypeptide itself is reduced or eliminated compared to the polypeptide activity originally possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide, cases where the overall polypeptide activity level and / or concentration (expression level) in the cell is lower than that of the natural strain due to inhibition of the expression of the gene encoding the polynucleotide or inhibition of translation into the polypeptide, cases where the polynucleotide is not expressed at all, and / or cases where there is no polypeptide activity despite the expression of the polynucleotide. The "intrinsic activity" refers to the activity of a specific polypeptide that was originally possessed by the parent strain, wild type, or non-mutant microorganism before the trait change due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before the change." When polypeptide activity is "inactivated, deficient, reduced, downregulated, decreased, or attenuated" compared to intrinsic activity, it means that it has decreased compared to the activity of a specific polypeptide that was originally possessed by the parent strain or non-mutant microorganism before the trait change.

[0074] The weakening of the activity of such polypeptides can be achieved by any method known in the art, but is not limited to these methods, and can be achieved by applying a variety of methods well known in the field (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.).

[0075] Specifically, the weakening of the polypeptide in this application is, 1) Deletion of all or part of the gene encoding the polypeptide; 2) Modification of the gene expression regulatory region (or gene expression regulatory sequence) so that the expression of the gene encoding the polypeptide is reduced; 3) Modification of the amino acid sequence constituting the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence) so as to remove or weaken the activity of the polypeptide; 4) Modification of the gene sequence encoding the polypeptide so that the polypeptide activity is removed or weakened (e.g., deletion / substitution / addition of one or more nucleic acid bases on the nucleic acid sequence of the polypeptide gene so that it encodes a polypeptide that has been modified so that the polypeptide activity is removed or weakened); 5) Modifications of the nucleotide sequence encoding the start codon or 5'-UTR region of a polypeptide-encoding gene transcript; 6) Introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in a polypeptide-encoding gene to form a secondary structure that cannot be attached to a ribosome; 8) Addition of a promoter that is transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the polypeptide-coding gene sequence (reverse transcription engineering, RTE); or 9) A combination of two or more selected from items 1) to 8) above is also acceptable, but is not particularly limited thereto.

[0076] for example, The deletion of part or all of the gene encoding the polypeptide described in 1) above may be the removal of the entire polynucleotide encoding the endogenous target polypeptide within the chromosome, replacement with a polynucleotide in which some nucleotides are deleted, or replacement with a marker gene.

[0077] Furthermore, the modification of the regulatory region (or regulatory sequence) described in 2) above may involve deletion, insertion, non-conservative or conservative substitution, or a combination thereof, resulting in a mutation on the regulatory region (or regulatory sequence), or replacement with a sequence having weaker activity. The regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and decoding.

[0078] Furthermore, the modifications of the amino acid sequence or polynucleotide sequence described in 3) and 4) above may be, but are not limited to, deletions, insertions, non-conservative or conservative substitutions, or combinations thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, resulting in the occurrence of a sequence mutation or replacement with an amino acid sequence or polynucleotide sequence modified to have weaker activity or to have no activity, in order to weaken the activity of the polypeptide. For example, gene expression can be inhibited or weakened by introducing a mutation within the polynucleotide sequence to form a termination codon, but are not limited to this.

[0079] Furthermore, the modification of the start codon or the nucleotide sequence encoding the 5'-UTR region of the gene transcript encoding the polypeptide (5) may, for example, be replaced with a nucleotide sequence encoding another start codon with a lower polypeptide expression rate compared to the endogenous start codon, but is not limited to these.

[0080] For the introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide (6) above, see, 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].

[0081] 7) In order to form a secondary structure that ribosomes cannot attach to, the addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in the polypeptide-encoding gene may make mRNA translation impossible or reduce its rate.

[0082] The addition of a promoter that is 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 be performed to weaken the activity of the transcription of the polypeptide gene by creating complementary antisense nucleotides.

[0083] In this application, the term "enhancement" of polypeptide activity means that the activity of a polypeptide increases compared to its intrinsic activity. This enhancement may be used interchangeably with terms such as activation, upregulation, overexpression, and increase. Here, activation, enhancement, upregulation, overexpression, and increase can all include exhibiting activity that was not originally present, or exhibiting improved activity compared to the intrinsic activity or pre-mutation activity. "Intrinsic activity" means the activity of a specific polypeptide originally present in the parental strain or non-mutant microorganism before the trait change, in cases where a trait changes due to a genetic mutation caused by natural or artificial factors. This may be used interchangeably with "pre-mutation activity." "Enhancement," "upregulation," "overexpression," or "increase" of polypeptide activity compared to its intrinsic activity means that the activity and / or concentration (expression level) of a specific polypeptide originally present in the parental strain or non-mutant microorganism before the trait change is improved.

[0084] The aforementioned enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. Whether or not the polypeptide's activity has been enhanced can be confirmed by an increase in the polypeptide's activity level, expression level, or the amount of product excreted from the polypeptide.

[0085] The enhancement of the activity of the polypeptide can be achieved by applying various methods well known in the field, and is not limited as long as it can enhance the activity of the target polypeptide compared to the microorganism before deformation. Specifically, this may be, but is not limited to, methods of gene engineering and / or protein engineering that are routine methods of molecular biology and are well known to ordinary technicians in the field (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.).

[0086] Specifically, the strengthening of the polypeptide in this application is 1) Increase in the intracellular copy number of polynucleotides encoding polypeptides; 2) Replace gene expression regulatory regions on chromosomes encoding polypeptides with potent active sequences; 3) Modifications of the nucleotide sequence encoding the start codon or 5'-UTR region of a polypeptide-encoding gene transcript; 4) Modification of the amino acid sequence of the polypeptide to enhance polypeptide activity; 5) Modification of the polynucleotide sequence encoding the polypeptide so as to enhance polypeptide activity (for example, modification of the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide that has been modified to enhance polypeptide activity); 6) Introduction of a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding it; 7) Codon optimization of polynucleotides encoding polypeptides; 8) Analyze the tertiary structure of the polypeptide and select exposed sites to deform or chemically modify; or 9) A combination of two or more selected from items 1) to 8) above is also acceptable, but is not particularly limited thereto.

[0087] More specifically, The increase in the intracellular copy number of the polynucleotide encoding the polypeptide described in 1) above may be achieved by introducing into the host cell a vector that can replicate and function independently of the host, on which the polynucleotide encoding the polypeptide is operably linked. Alternatively, it may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into the chromosomes within the host cell. The introduction into the chromosomes can be performed by introducing into the host cell a vector capable of inserting the polynucleotide into the chromosomes within the host cell, but is not limited to these methods. The vector is as described above.

[0088] The replacement of a gene expression regulatory region (or regulatory sequence) on a chromosome encoding a polypeptide with a more potent sequence may, for example, involve deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the regulatory region, thereby causing a sequence mutation, or replacement with a sequence having stronger activity. The regulatory region may include, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences that regulate transcription and decoding termination. For example, the original promoter may be replaced with a potent promoter, but is not limited to these.

[0089] Examples of well-known strong promoters include, but are not limited to, the CJ1-CJ7 promoters (US Registered Patent US 7662943 B2), the lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Registered Patent US 10584338 B2), O2 promoter (US Registered Patent US 10273491 B2), tkt promoter, and yccA promoter.

[0090] The modification of the start codon or the nucleotide sequence encoding the 5'-UTR region of the polypeptide-encoding gene transcript described in 3) above may, but is not limited to, substitution with, for example, a nucleotide sequence encoding another start codon with a higher polypeptide expression rate compared to the endogenous start codon.

[0091] The modifications of the amino acid sequence or polynucleotide sequence described in 4) and 5) above may be, but are not limited to, deletion, insertion, non-conservative or conservative substitution, or combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, thereby causing a sequence mutation, or replacement with an improved amino acid sequence or polynucleotide sequence that has stronger activity or an improved amino acid sequence or polynucleotide sequence that has increased activity, in order to enhance the activity of the polypeptide. Specifically, the replacement may be, but is not limited to, insertion of a polynucleotide into the chromosome by homologous recombination. The vector used in this case may further include a selection marker for confirming the presence or absence of chromosomal insertion. The selection marker is as described above.

[0092] The introduction of a foreign polynucleotide exhibiting polypeptide activity (6) above may also be the introduction of a foreign polynucleotide encoding a polypeptide exhibiting the same or similar activity as the polypeptide into the host cell. The foreign polynucleotide is not restricted in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be appropriately selected by those skilled in the art from known transformation methods, and the introduction of the polynucleotide into the host cell can generate a polypeptide and increase its activity.

[0093] The codon optimization of the polynucleotide encoding the polypeptide described in 7) above may be codon optimization of the endogenous polynucleotide so that transcription or translation increases in the host cell, or optimization of the codon of the exogenous polynucleotide so that optimized transcription or translation occurs in the host cell.

[0094] The 8) analysis of the tertiary structure of the polypeptide and the selection of exposed sites for deformation or chemical modification may, for example, involve comparing the sequence information of the polypeptide to be analyzed with a database containing sequence information of known proteins to determine candidate template proteins according to the degree of sequence similarity, confirming the structure based on that, and selecting exposed sites to be deformed or chemically modified for deformation or modification.

[0095] Such enhancement of polypeptide activity may, but is not limited to, an increase in the activity or concentration expression level of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in the wild-type or pre-deformation microbial strain, or an increase in the amount of product produced from said polypeptide.

[0096] In the microorganisms of this application, partial or complete modification of polynucleotides may be induced by (a) homologous recombination using a chromosome insertion vector within the microorganism or genome editing using an engineered nuclease (e.g., CRISPR-Cas9) and / or (b) light and / or chemical treatment such as ultraviolet light and radiation. The method for partial or complete modification of the gene may include methods using DNA recombination techniques. For example, partial or complete deletion of a gene may be achieved by injecting a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination. The injected nucleotide sequence or vector may include, but is not limited to, a dominant selection marker.

[0097] Another aspect of this application provides a method for producing L-arginine, comprising the step of culturing a microorganism containing a LysE variant or a polynucleotide encoding it.

[0098] The LysE variant, the polynucleotide encoding it, the microorganism containing it, and L-arginine are as described above.

[0099] The method for producing L-arginine according to this application may include the step of culturing a microorganism containing the LysE variant described herein or the polynucleotide encoding it or a vector containing the polynucleotide in a culture medium.

[0100] In this application, the term "culture" means growing the microorganisms of this application under appropriately controlled environmental conditions. The culture process of this application can be carried out according to suitable culture 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 batch, continuous, and / or fed-batch.

[0101] In this application, the term "culture medium" refers to a substance mixed primarily with nutrients necessary for culturing the microorganisms of this application, supplying nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganisms of this application can be any culture medium commonly used for culturing microorganisms, without any particular restrictions. However, the microorganisms of this application can be cultured under aerobic conditions in a conventional culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, while adjusting the temperature, pH, etc.

[0102] For example, culture media for Corynebacterium strains can be found in the literature ["Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington DC, USA, 1981)].

[0103] In this application, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and other appropriate amounts of carbon sources can 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.

[0104] The nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptones, NZ-amines, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their decomposition products, defatted soy cake or its decomposition products. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0105] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or their corresponding sodium-containing salts. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate, and may also include amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the culture medium in batches or continuously, but are not limited to these methods.

[0106] Furthermore, during the cultivation of the microorganisms of this application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture medium in an appropriate manner to adjust the pH of the culture medium. In addition, during cultivation, antifoaming agents such as fatty acid polyglycol esters can be used to suppress the formation of bubbles. Furthermore, in order to maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas can be injected into the culture medium, or in order to maintain an anaerobic and microaerobic state, no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limited to these methods.

[0107] In the culture described in this application, the culture temperature can be maintained at 20-45°C, specifically 25-40°C, and the culture can be performed for approximately 10-160 hours, but is not limited to this.

[0108] The L-arginine produced by the culture described in this application is either secreted into the culture medium or remains within the cells.

[0109] The method for producing L-arginine according to this application may further include, for example, a step of preparing the microorganism of this application, a step of preparing a culture medium for culturing the microorganism, or a combination thereof, prior to the culturing step (in any order).

[0110] The method for producing L-arginine according to this application may further include a step of recovering L-arginine from the culture medium (the culture medium in which the culture was performed) or from the microorganism. The recovery step may further include a step after the culture step.

[0111] The aforementioned recovery may involve collecting the desired L-arginine using appropriate methods known in the art, such as the microbial culture methods of this application, for example, batch, continuous, or fed-batch culture methods. For example, various chromatography methods such as centrifugation, filtration, treatment with a crystallizing protein precipitant (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof can be used to recover the desired L-arginine from the culture medium or microorganism using appropriate methods known in the art.

[0112] Furthermore, the L-arginine production method of this application may further include a purification step. The purification can be carried out using appropriate methods known in the art. For example, if the L-arginine production method of this application includes both a recovery step and a purification step, the recovery step and the purification step can be carried out sequentially or discontinuously, regardless of order, or simultaneously or integrated into a single step, but are not limited thereto.

[0113] In the method of this application, the LysE variant, polynucleotide, vector, microorganism / strain, etc., are as described in the other aspects described above.

[0114] Another aspect of this application is to provide a composition for L-arginine production comprising the LysE variant of this application, a polynucleotide encoding the variant, a vector containing the polynucleotide, or a microorganism containing the polynucleotide of this application; a culture medium for the same; or a combination thereof.

[0115] The composition of this application may further contain any suitable excipients commonly used in compositions for L-arginine production, such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents.

[0116] In the composition of this application, the LysE variant, polynucleotide, vector, microorganism / strain, culture medium, etc., are as described in the other aspects described above.

[0117] Another aspect of this application provides a method for increasing the L-arginine efflux and / or production capacity of a microorganism or a method for conferring L-arginine efflux and / or production capacity to a microorganism, comprising the step of modifying the microorganism to express the LysE variant of this application.

[0118] In the method for increasing the L-arginine production capacity of microorganisms according to this application, the mutants, polynucleotides, vectors, microorganisms / strains, etc., are as described in the other aspects above.

[0119] The method for modifying the microorganism may include, but is not limited to, introducing a LysE variant, a polynucleotide encoding it, or a recombinant vector containing the polynucleotide into the microorganism.

[0120] Another aspect of this application provides an application for L-arginine efflux of the LysE variant of this application.

[0121] Another aspect of this application provides an application for L-arginine production by microorganisms comprising one or more of the LysE variant of this application, the polynucleotide encoding the variant, and a vector containing the polynucleotide.

[0122] The LysE variant, polynucleotide, vector, and microorganism are as described in the other sections above.

[0123] The present application will be described in more detail below through examples and experimental examples. However, these examples and experimental examples are for illustrative purposes only, and the scope of the present application is not limited to these examples and experimental examples.

[0124] Example 1. Securing diverse mutants of LysE in which amino acid residues at positions 50, 153, and 215 are substituted. The inventors determined that amino acid residues 50, 153, and 215 of Sequence ID No. 1 are important positions for LysE activity, and created mutants in which these amino acid residues were substituted with other amino acids. Specifically, PCR was performed using the primer pairs listed in Table 1 (SEQ ID NOs. 3 and 5, SEQ ID NOs. 4 and 6, SEQ ID NOs. 3 and 7, SEQ ID NOs. 4 and 8, SEQ ID NOs. 3 and 9, SEQ ID NOs. 4 and 10, SEQ ID NOs. 3 and 11, SEQ ID NOs. 4 and 12, SEQ ID NOs. 3 and 13, SEQ ID NOs. 4 and 14, SEQ ID NOs. 3 and 15, SEQ ID NOs. 4 and 16, SEQ ID NOs. 3 and 17, SEQ ID NOs. 4 and 18, SEQ ID NOs. 3 and 19, SEQ ID NOs. 4 and 20, SEQ ID NOs. 3 and 21, SEQ ID NOs. 4 and 22) and overlapping PCR using the primer pairs of SEQ ID NOs. Homologous recombination fragments containing the respective LysE-F50L, F50I, F50V, N153S, N153T, N153Q, N215T, N215S, and N215Q mutant sequences were obtained. At that time, the PCR reaction was carried out by repeating the following steps 30 times: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and extension at 72°C for 1 minute. Subsequently, the pDCM2 vector (Republic of Korea Publication No. 10-2020-0136813), which is incapable of replicating in Corynebacterium glutamicum, and the aforementioned fragment amplified by PCR were treated with the restriction enzymes BamHI and XbaI for chromosome transduction, ligated using a DNA zygote, and then transmuted into E. coli DH5α. The resulting cells were then streaked onto LB solid medium containing kanamycin (25 mg / l).

[0125] [Table 1]

[0126] After selecting colonies that had been transformed into plasmids containing the desired gene via PCR, plasmids were obtained using plasmid extraction, and these plasmids were named pDCM2-lysE(F50L), pDCM2-lysE(F50I), pDCM2-lysE(F50V), pDCM2-lysE(N153S), pDCM2-lysE(N153T), pDCM2-lysE(N153Q), pDCM2-lysE(N215T), pDCM2-lysE(N215S), and pDCM2-lysE(N215Q).

[0127] Example 2. Preparation of a LysE mutant strain derived from Corynebacterium glutamicum KCCM10741P and evaluation of its L-arginine production capacity. To introduce the LysE mutation into the L-arginine-producing strain Corynebacterium glutamicum KCCM10741P (US 8034602 B2), it was transformed using the recombinant plasmid prepared as described above (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Subsequently, secondary recombination was performed on solid agar plates containing 4% sucrose, and PCR was performed on the transformed strains after secondary recombination using primer pairs (SEQ ID NOs: 3 and 4) to confirm that the respective mutations had been introduced into the LysE gene on the chromosome. The PCR reaction was performed by repeating the following steps 30 times: denaturation at 95°C for 30 seconds; annealing at 55°C for 30 seconds; and extension at 72°C for 1 minute. The selected LysE mutant-introduced strains were named KCCM10741P-lysE(F50L), KCCM10741P-lysE(F50I), KCCM10741P-lysE(F50V), KCCM10741P-lysE(N153S), KCCM10741P-lysE(N153T), KCCM10741P-lysE(N153Q), KCCM10741P-lysE(N215T), KCCM10741P-lysE(N215S), and KCCM10741P-lysE(N215Q).

[0128] To analyze the L-arginine production capacity of the LysE mutant strain, it was cultured together with the parent strain, Corynebacterium glutamicum KCCM10741P, using the following method.

[0129] The parent strain Corynebacterium glutamicum KCCM10741P and the prepared LysE mutant strain were inoculated into a 250 ml corner baffle flask containing 25 ml of the seed medium described below, and cultured with shaking at 200 rpm at 30°C for 20 hours. Subsequently, 1 ml of the seed culture solution was inoculated into a 250 ml corner baffle flask containing 24 ml of production medium, and cultured with shaking at 200 rpm at 30°C for 72 hours. The compositions of the seed medium and production medium are as follows.

[0130] <Seed culture medium (pH 7.2)> Glucose 20g, Ammonium sulfate 45g, Magnesium sulfate heptahydrate 2g, Monopotassium phosphate 2g, Ammonium chloride 10g, Biotin 0.01mg, Thiamine-HCl 0.1mg, Calcium pantothenate 2mg, Nicotinamide 3mg, Ferrous sulfate 10mg, Manganese sulfate 10mg, Zinc sulfate 0.02mg, Copper sulfate 0.5mg (based on 1 liter of distilled water)

[0131] <Production medium (pH 7.2)> 60g glucose, 45g ammonium sulfate, 2g magnesium sulfate heptahydrate, 2g monopotassium phosphate, 10g ammonium chloride, 0.01mg biotin, 0.1mg thiamine-HCl, 2mg calcium pantothenate, 3mg nicotinamide, 10mg ferrous sulfate, 10mg manganese sulfate, 0.02mg zinc sulfate, 0.5mg copper sulfate, 30g calcium carbonate (based on 1 liter of distilled water)

[0132] After the culturing period, the L-arginine production capacity was measured by HPLC (Waters 2478) (Table 2).

[0133] [Table 2]

[0134] As a result, it was confirmed that all of the aforementioned LysE mutant-introduced strains showed an average increase of 20% in L-arginine production capacity compared to the parent strain.

[0135] Example 3. Production of a LysE mutant strain derived from Corynebacterium glutamicum CJ1R with L-arginine production ability and evaluation of its L-arginine production ability. We also checked whether other Corynebacterium glutamicum strains that produce L-arginine exhibited similar effects to those in Example 2, as described below.

[0136] A strain of Corynebacterium glutamicum capable of producing L-arginine was created by introducing a single mutation (ΔargR) into the wild-type strain (ATCC13869). Specifically, a recombinant vector for the deletion of the argR gene (SEQ ID NO: 40) was prepared in the same manner as in Example 1, and the primers used for vector preparation are shown in Table 3.

[0137] [Table 3]

[0138] After selecting plasmids containing the target fragment via PCR, these plasmids were named pDCM2-ΔargR. Corynebacterium glutamicum wild-type strains were transformed using pDCM2-ΔargR in the same manner as in Example 2. The transformed strains were then identified as lacking argR on the chromosome via PCR using primer pairs (SEQ ID NOs. 23 and 26), and these were named CJ1R.

[0139] Mutant strains were created by introducing nine different LysE variants into CJ1R using the same method as in Example 2, and were named CJ1R-lysE(F50L), CJ1R-lysE(F50I), CJ1R-lysE(F50V), CJ1R-lysE(N153S), CJ1R-lysE(N153T), CJ1R-lysE(N153Q), CJ1R-lysE(N215T), CJ1R-lysE(N215S), and CJ1R-lysE(N215Q).

[0140] To analyze the L-arginine production capacity of the LysE mutant strain derived from CJ1R, it was cultured in the same manner as in Example 2, and after the completion of the culture, the L-arginine production capacity was measured by HPLC (Waters 2478) (Table 4).

[0141] [Table 4]

[0142] As a result, we confirmed that all strains introduced with the mutant LysE showed an average increase of 19% in L-arginine production capacity compared to the parent strain.

[0143] From the above description, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. In this regard, it should be understood that the embodiments described above are merely illustrative and not limiting. The scope of this application should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims, as described below, and their equivalent concepts, rather than from the above detailed description.

[0144] [Table 5] JPEG0007854500000006.jpg196133 JPEG0007854500000007.jpg196134 JPEG0007854500000008.jpg196134 JPEG0007854500000009.jpg196135 JPEG0007854500000010.jpg196140

Claims

1. A LysE variant having 90% or more sequence identity with SEQ ID NO: 1, wherein one or more amino acids in the amino acids corresponding to the 50th, 153rd, and 215th positions of SEQ ID NO: 1 are substituted with other amino acids. The amino acid corresponding to the 50th position is substituted with valine, leucine, or isoleucine, and / or The amino acid corresponding to position 153 is substituted with serine, threonine or glutamine, and / or The amino acid corresponding to position 215 is substituted with serine, threonine, or glutamine. A LysE mutant with improved L-arginine efflux compared to LysE having the amino acid sequence of SEQ ID NO:

1.

2. The LysE mutant according to claim 1, wherein the LysE mutant is derived from the genus Corynebacterium.

3. A LysE variant according to claim 1, wherein one of the amino acids at the 50th, 153rd, and 215th amino acid residues in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, The amino acid at the 50th amino acid residue is substituted with valine, leucine, or isoleucine, or The amino acid at amino acid residue 153 is substituted with serine, threonine, or glutamine, or The LysE mutant is characterized by the substitution of the 215th amino acid residue with serine, threonine, or glutamine.

4. A polynucleotide encoding a variant according to any one of claims 1 to 3.

5. A microorganism of the genus Corynebacterium comprising the LysE variant described in any one of claims 1 to 3 or a polynucleotide encoding the LysE variant.

6. The microorganism according to claim 5, wherein the microorganism is Corynebacterium glutamicum.

7. A method for producing L-arginine, comprising the step of culturing a microorganism of the genus Corynebacterium containing the LysE mutant described in any one of claims 1 to 3 or a polynucleotide encoding the LysE mutant.

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