Mutant ribonuclease activity-modulating protein and method for producing L-valine using the same

Abstract

The present application relates to a mutant ribonuclease activity-regulating protein and a method for producing L-valine using the same.

Description

Technical Field

[0004] , , , , , , ,

[0001] This application relates to a mutant ribonuclease activity regulatory protein and a method for producing L-valine using the same.

Background Art

[0002] L-amino acids are the basic building units of proteins and are used as important materials such as pharmaceutical raw materials, food additives, animal feeds, nutritional agents, insecticides, and bactericides. In particular, branched-chain amino acids (BCAAs) are a collective term for the essential amino acids L-valine, L-leucine, and L-isoleucine. The branched-chain amino acids have an antioxidant effect and an effect of promoting protein synthesis in muscle cells.

[0003] The production of branched-chain amino acids using microorganisms is mainly carried out by microorganisms of the genus Corynebacterium. For example, in the case of L-valine, an approach specific to a target substance that increases the expression of a gene encoding an enzyme involved in its biosynthesis or removes a gene unnecessary for biosynthesis is mainly used (Patent Documents 1, 2). In addition, a method for producing L-valine by feedback inhibition has been studied (Patent Document 3).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

[0005] [Non-licensed Document 1] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-licensed Document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-licensed Document 3] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed Document 4] Devereux, J., et al, Nucleic Acids Research 12: 387 (1984) [Non-licensed Document 5] Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990) [Non-licensed Document 6] Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994 [Non-licensed Document 7] [CARILLO et al / .](1988) SIAM J Applied Math 48: 1073 [Non-licensed Document 8] Smith and Waterman, Adv. Appl. Math (1981) 2:482 [Non-licensed 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] Biotechnology and Bioprocess Engineering, June 2014, Volume 19, Issue 3, pp 456-467 [Non-Patent Document 14] 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 15] Sambrook et al. Molecular Cloning 2012 [Non-Patent Document 16] Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Summary of the Invention

Problems to be Solved by the Invention

[0006] The inventors have confirmed that when a mutant ribonuclease activity regulatory protein is introduced into a microorganism, the L-valine production ability is improved compared to a microorganism containing the wild-type protein, and have thus completed this application.

Means for Solving the Problems

[0007] This application provides a mutant ribonuclease activity regulatory protein in which the amino acid corresponding to the 93rd position in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid. This application provides a polynucleotide encoding the mutant ribonuclease activity regulatory protein of this application.

[0008] <The present application provides a microorganism comprising a mutant ribonuclease activity regulatory protein of the present application or a polynucleotide encoding the same. The present application provides a method for producing L-valine, comprising culturing a microorganism comprising a mutant ribonuclease activity regulatory protein of the present application or a polynucleotide encoding the same in a medium.

Effects of the Invention

[0009] When culturing a microorganism that produces L-valine using the mutant ribonuclease activity regulatory protein of the present application, L-valine can be produced in a higher yield compared to a microorganism having a conventional wild-type ribonuclease activity regulatory protein.

Modes for Carrying Out the Invention

[0010] These will be specifically described below. Each of the descriptions and embodiments disclosed in the present application is also applicable to other descriptions and embodiments, respectively. That is, any combination of various elements disclosed in the present application is included in the present application. Further, the present application is not limited to the following specific descriptions. Furthermore, many papers and patent documents are referred to throughout this specification, and their citations are indicated. The entire disclosure content of the cited papers and patent documents is incorporated herein by reference, thereby more clearly explaining the level of the technical field to which the present application belongs and the content of the present application.

[0011] One aspect of the present application provides a mutant ribonuclease activity regulatory protein in which the amino acid corresponding to the 93rd position in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.

[0012] In this application, "mutant ribonuclease activity-regulating protein" means any polypeptide having the function of regulating ribonuclease activity, or a variant of a ribonuclease activity-regulating protein that includes a substitution of another amino acid with the amino acid corresponding to the 93rd position from the N-terminus of SEQ ID NO: 1. The aforementioned "mutant ribonuclease activity-regulating protein" is also referred to as "ribonuclease activity-regulating protein variant," "RraA variant," "mutant RraA," "rraA variant," "mutant rraA," etc.

[0013] The proteins targeted for mutation introduction in this application may be proteins that have the activity to regulate ribonuclease activity. Specifically, the protein includes the amino acid sequence of SEQ ID NO: 1 and has the activity to regulate ribonuclease activity, but is not limited to this. Meaningless sequence additions before or after the amino acid sequence of SEQ ID NO: 1, naturally occurring mutations, and silent mutations are not excluded, and any protein that has the same or equivalent activity as a protein containing the amino acid sequence of SEQ ID NO: 1 is included as a protein targeted for mutation introduction in this application. For example, the protein targeted for mutation introduction in this application may be a protein consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with it. Furthermore, it goes without saying that proteins having amino acid sequences in which some sequences are deleted, modified, substituted, or added are also included as proteins targeted for mutation in this application, as long as they have such homology or identity and exhibit the same efficacy as the aforementioned protein.

[0014] In this application, "ribonuclease activity regulator protein" refers to a protein that regulates ribonuclease activity by binding to the endonuclease RNase E and suppressing the RNA process, and is used interchangeably with "ribonuclease E activity regulator" or "RraA". The amino acid sequence of RraA is obtained from known databases such as NCBI's Genebank.

[0015] For example, the RraA protein of this application is derived from microorganisms, specifically from prokaryotic or eukaryotic microorganisms, and more specifically from microorganisms of the genus Corynebacterium, but is not limited to these.

[0016] Another example is the RraA protein, WP_003858525.1, derived from the microorganism Corynebacterium, but it goes without saying that it includes proteins of various origins that have the activity to regulate ribonuclease activity.

[0017] In this application, the original amino acid corresponding to position 93 of SEQ ID NO: 1 in the original amino acid sequence of the RraA protein to be mutated may be histidine (H).

[0018] The mutant ribonuclease activity-regulating protein of this application may be one in which the amino acid at position 93 in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid different from the original amino acid. Alternatively, the mutant ribonuclease activity-regulating protein is a mutant ribonuclease activity-regulating protein in which an amino acid different from the original amino acid is a polar or hydrophilic amino acid that has an uncharged side chain, but is not limited to this.

[0019] As an example, the mutant ribonuclease activity-modulating protein may be one in which the amino acid corresponding to the 93rd position in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid selected from the group consisting of tyrosine, arginine, lysine, aspartic acid, asparagine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, glycine, glutamic acid, and glutamine.

[0020] As another example, the mutant ribonuclease activity-modulating protein may have the amino acid corresponding to position 93 in the amino acid sequence of SEQ ID NO: 1 replaced with an amino acid selected from the group consisting of tyrosine, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, asparagine, and glutamine, for example, replaced with tyrosine.

[0021] As yet another example, the mutant ribonuclease activity-modulating protein of this application may have an amino acid sequence represented by SEQ ID NO: 3 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, or 99.4% or more homology or identity thereto, may contain the above amino acid sequence, may consist of the above amino acid sequence, or may be substantially composed of the above amino acid sequence.

[0022] The mutant ribonuclease activity-modulating protein of this application may include an amino acid sequence in which the amino acid corresponding to the 93rd position relative to the amino acid sequence of Sequence ID No. 1 is an amino acid other than histidine, such as tyrosine, and which has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, or 99.4% or more homology or identity with the amino acid sequence represented by Sequence ID No. 1. Furthermore, it goes without saying that mutant ribonuclease activity-modulating proteins having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added are also included in this application, as long as they have such homology or identity and exhibit efficacy equivalent to the mutant ribonuclease activity-modulating protein of this application.

[0023] Examples include the addition or deletion of sequences that do not alter the function of the mutant ribonuclease activity-regulating protein of this application, spontaneous mutations, silent mutations, or conserved substitutions at the N-terminus, C-terminus, and / or within the amino acid sequence.

[0024] The term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can 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.

[0025] For example, among amino acids with electrically charged side chains, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; and amino acids with uncharged side chains include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0026] In this application, "mutant protein" or "variant" refers to a polypeptide in which at least one amino acid differs from the amino acid sequence of the original polypeptide due to conservative substitution and / or modification, but the functions or properties are maintained. Such variants can generally be identified by modifying at least one amino acid in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the capabilities of the variant are improved, unchanged, or decreased compared to the original polypeptide. Some variants also include those in which at least one portion, such as the N-terminal leader sequence or transmembrane domain, is removed. Other variants include those in which a portion of the N and / or C-terminus of a mature protein is removed. The term "mutant protein" is used interchangeably with terms such as mutant, modified, mutant polypeptide, mutated protein, mutation, and variant (in English, these may include modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), but any term that means mutation is acceptable. For the purposes of this application, the variant may be a polypeptide in which the amino acid corresponding to the 93rd position of the amino acid sequence of Sequence ID No. 1 is replaced with serine. For example, the variant is a polypeptide containing the amino acid sequence represented by Sequence ID No. 3, but is not limited thereto.

[0027] Furthermore, the mutants may include the deletion or addition of amino acids that have minimal effect 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 that is involved in protein translocation co-translationally or post-translationally. The mutants may also be conjugated with other sequences or linkers so that they can be identified, purified, or synthesized.

[0028] In this application, "homology" or "identity" refers to the degree to which two given amino acid sequences or base sequences are similar, and is expressed as a percentage. Homology and identity are often used interchangeably.

[0029] The sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequence algorithms, and a default gap penalty established by the program used may also be applied. Substantially, homologous or identical sequences generally hybridize with all or part of the sequence under moderate to high stringent conditions. Needless to say, hybridization includes hybridization with polynucleotides that have common codons or codons considering codon degeneracy in the polynucleotide.

[0030] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined, for example, using default parameters as described in Non-Patent Document 1 and known computer algorithms such as the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 3), as performed in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) (including the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, homology, similarity, or identity can be determined using BLAST or Clustal W from the National Center for Biotechnology Information.

[0031] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 3, as disclosed in Non-Patent Document 8, for example. In summary, the GAP program is defined as 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 (with a value of 1 for identity and 0 for non-identity) and a weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9, as in Non-Patent Document 10; (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 extended penalty of 0.5); and (3) no penalty for terminal gaps.

[0032] As an example of this application, the mutant ribonuclease activity-regulating protein of this application may have the activity to improve L-valine production capacity compared to a wild-type polypeptide that has the activity to regulate ribonuclease activity.

[0033] In this application, "corresponding to" means an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar, identical, or equivalent to a residue listed in the polypeptide. Identifying the amino acid at the corresponding position will determine the specific amino acid in the sequence referencing the particular sequence. In this application, "corresponding region" generally means a similar or corresponding position in the related protein or reference protein.

[0034] For example, by aligning any amino acid sequence with Sequence ID No. 1, each amino acid residue in the sequence can be numbered based on the number and position of amino acid residues corresponding to the amino acid residues in Sequence ID No. 1. For example, the sequence alignment algorithm in this application can be used to identify the positions of amino acids, or the positions where modifications such as substitutions, insertions, or deletions occur, by comparing it with a query sequence (also called a "reference sequence").

[0035] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Literature 3) and the Needle program from the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Literature 2) can be used, but are not limited to these. Sequence alignment programs and pairwise sequence comparison algorithms known in the art can be used as appropriate.

[0036] Another aspect of this application provides a polynucleotide encoding a mutant ribonuclease activity-regulating protein of this application. The RraA protein of this application may be encoded by the rraA gene.

[0037] For example, the rraA gene is a polynucleotide encoding WP_003858525.1 derived from a microorganism of the genus Corynebacterium, but is not limited to this. Another example is the rraA gene derived from a microorganism of the genus Corynebacterium, but is not limited to this; it goes without saying that it includes rraA genes from various sources encoding proteins with RraA protein activity.

[0038] In this application, "polynucleotide" means a polymer of nucleotides in which nucleotide monomers are covalently linked together in a long chain, and refers to a DNA or RNA chain longer than a predetermined length, and more specifically, refers to a polynucleotide fragment encoding the mutant ribonuclease activity regulating protein.

[0039] The polynucleotide encoding the mutant ribonuclease activity-modulating protein of this application may include a base sequence encoding the mutant ribonuclease activity-modulating protein in which the amino acid corresponding to position 93 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, or it may include a base sequence in which the codons corresponding to positions 277-279 in the nucleotide sequence of SEQ ID NO: 2 are substituted with codons encoding other amino acids. For example, the polynucleotide of this application may include a base sequence encoding the amino acid sequence represented by SEQ ID NO: 3. As a more specific example of this application, the polynucleotide of this application may have the sequence of SEQ ID NO: 4, or may include the said sequence. Furthermore, the polynucleotide of this application may consist of the sequence of SEQ ID NO: 4, or may be substantially composed of the said sequence.

[0040] The polynucleotides of this application can be modified in various ways in the coding region, either through codon degeneracy or by considering preferred codons in organisms that intend to express the mutant ribonuclease activity-regulating protein of this application, as long as the amino acid sequence of the mutant ribonuclease activity-regulating protein of this application does not change. Specifically, the polynucleotides of this application have 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% homology or identity with the sequence of Sequence ID No. 4, or include such a base sequence, or consist 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% homology or identity with the sequence of Sequence ID No. 4, or are substantially composed of such a base sequence, but are not limited to these. Here, in the sequence having homology or identity, the codon encoding the amino acid corresponding to the 93rd position of Sequence ID No. 1 may be one of the codons encoding an amino acid other than histidine, such as tyrosine.

[0041] Furthermore, the polynucleotide of this application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a sequence complementary 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 Non-Patent Documents 11 and 12). For example, this could involve hybridizing polynucleotides with high homology or identity, such as polynucleotides with 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 homology or identity, while not hybridizing polynucleotides with lower homology or identity. Alternatively, it could involve washing once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of a typical Southern hybridization: 60°C, 1×SSC, 0.1%SDS, more specifically 60°C, 0.1×SSC, 0.1%SDS, or more specifically 68°C, 0.1×SSC, 0.1%SDS.

[0042] Hybridization requires that the two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the 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. Therefore, the polynucleotides of this application may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0043] Specifically, polynucleotides homologous or identical to the polynucleotide of this application can be detected using hybridization conditions in which the hybridization step is performed at a Tm value of 55°C and the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited to these, and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0044] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are known in the art (e.g., Non-Patent Document 11).

[0045] Further embodiments of this application provide vectors comprising the polynucleotides of this application. The aforementioned vector is an expression vector for expressing the polynucleotide in a host cell, but is not limited to this.

[0046] The vector of this application includes a DNA product comprising a polynucleotide sequence encoding a target polypeptide, operably linked to a suitable regulatory region (or regulatory sequence) so as to enable expression of the target polypeptide in a suitable host. The regulatory region includes a promoter for initiating transcription, an optional operator sequence for regulating that transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences for regulating the termination of transcription and translation. Once transformed into a suitable host cell, the vector can replicate or function independently of the host genome and integrate into the genome itself.

[0047] 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, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used, and as plasmid vectors, pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pET series, etc. can be used. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0048] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using an intracellular chromosome introduction vector. 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 thereto. The vector may further include a selection marker to confirm whether or not the polynucleotide has been inserted into the chromosome. The selection marker is used to select cells transformed by the vector, that is, to confirm whether or not the target nucleic acid molecule has been inserted, and markers that confer selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, and expression of surface polypeptides are used. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit different phenotypes, thus allowing for the selection of transformed cells.

[0049] In this application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, thereby causing the polypeptide encoded by the polynucleotide to be expressed in the host cell. The transformed polynucleotide may be any form, regardless of whether it is inserted into or outside the host cell's chromosomes, as long as it is expressed in the host cell. The polynucleotide also contains DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the host cell in any form, as long as it is introduced and expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene structure containing all the elements necessary for its expression. Typically, the expression cassette includes 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 sequence necessary for expression in the host cell, but is not limited to this.

[0050] Furthermore, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the targeted mutant ribonuclease activity-modulating protein of this application.

[0051] Further embodiments of this application provide microorganisms comprising the mutant ribonuclease activity-modulating protein of this application or the polynucleotide of this application. The bacterial strain of this application may include the mutant ribonuclease activity-regulating protein of this application, a polynucleotide encoding the polypeptide, or a vector containing the polynucleotide of this application.

[0052] In this application, "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially, and is a microorganism in which a specific mechanism has been weakened or strengthened due to causes such as the insertion of an external gene or the strengthening or inactivation of the activity of an endogenous gene, and is a microorganism that has been genetically modified for the production of a target polypeptide, protein, or product.

[0053] The bacterial strains of this application may be strains that naturally possess L-valine production ability, or they may be microorganisms in which L-valine production ability has been conferred to strains that previously lacked it. For example, they may be microorganisms in which L-valine production ability has been improved by introducing the mutant ribonuclease activity regulating protein of this application or the polynucleotide encoding it, but are not limited to these.

[0054] The strain of this application may be a microorganism with improved L-valine production capacity compared to a parental strain that does not contain the mutant of this application, or a wild-type Corynebacterium strain. The said microorganism may have improved L-valine production capacity due to the introduction of the mutant of this application.

[0055] For example, the ribonuclease activity-regulating protein-unmodified microorganisms used as target strains for comparing whether or not the L-valine production capacity is improved include, but are not limited to, the L-valine-producing strain Corynebacterium glutamicum strain KCCM11201P (CA08-0072, Patent Document 1), the wild-type Corynebacterium glutamicum ATCC14067 (Patent Document 4) with the ilvN(A42V) mutation introduced, or the wild-type Corynebacterium glutamicum ATCC13869 (Patent Document 4).

[0056] As an example, the recombinant strain with improved production capacity is one in which the L-valine production capacity of the parent strain before mutation or the unmodified microorganism has improved by approximately 1% or more, specifically approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 6% or more, approximately 7% or more, approximately 8% or more, approximately 9% or more, approximately 10% or more, approximately 11% or more, approximately 12% or more, approximately 13% or more, or approximately 14% or more (there is no particular limit on the upper limit, for example, approximately 200% or less, approximately 150% or less, approximately 100% or less, approximately 50% or less, approximately 40% or less, approximately 30% or less, approximately 20% or less, or approximately 15% or less). However, any recombinant strain that shows a positive increase in production capacity compared to the parent strain before mutation, the unmodified microorganism, or the unmodified microorganism of ribonuclease activity-regulating protein is acceptable. As another example, recombinant strains with improved L-valine production capacity are those in which L-valine production capacity has improved by approximately 1.01 times or more, approximately 1.02 times or more, approximately 1.03 times or more, approximately 1.04 times or more, approximately 1.05 times or more, approximately 1.06 times or more, approximately 1.07 times or more, approximately 1.08 times or more, approximately 1.09 times or more, approximately 1.10 times or more, approximately 1.11 times or more, approximately 1.12 times or more, approximately 1.13 times or more, or approximately 1.14 times or more (there is no particular limit on the upper limit, for example, approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, or approximately 2 times or less), but are not limited to these.

[0057] In this application, "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but rather refers to wild-type strains or natural strains themselves, or strains before genetic mutation and changes in phenotype due to natural or artificial factors. Furthermore, in this application, "ribonuclease activity-regulating protein unmodified microorganism" refers to strains in which the ribonuclease activity-regulating protein variants described herein have not been introduced, or before they have been introduced. In this application, "ribonuclease activity-regulating protein unmodified microorganism" does not exclude strains in which other proteins or genes have been modified, other than the ribonuclease activity-regulating protein or the polynucleotide encoding it.

[0058] In this application, "unmodified microorganism" is used interchangeably with "pre-modification strain," "pre-modification microorganism," "non-mutant strain," "unmodified strain," "non-mutant microorganism," or "reference microorganism." The microorganisms of this application are, but are not limited to, microorganisms containing mutant ribonuclease activity-modulating proteins or polynucleotides encoding them, or microorganisms genetically modified to contain mutant ribonuclease activity-modulating proteins or polynucleotides encoding them (e.g., recombinant microorganisms). The term "endogenous activity" refers to the activity of a specific polypeptide that was originally present in the parent strain, wild type, or unmodified microorganism before the trait change, when a trait is altered by genetic mutation due to natural or artificial factors. This is used interchangeably with "activity before modification."

[0059] Other examples of microorganisms covered by this application include Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, and Corynebacterium striatum. These include Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, specifically Corynebacterium glutamicum, but are not limited to these.

[0060] As yet another example of this application, the recombinant microorganisms of this application may be microorganisms in which the L-valine production capacity is enhanced by further enhancement of the activity of some proteins in the L-valine biosynthesis pathway or further weakening of the activity of some proteins in the L-valine degradation pathway (Patent Documents 1, 2).

[0061] As yet another example of this application, the recombinant microorganism of this application may be a microorganism in which L-valine production ability is enhanced by further induction of feedback inhibition (Patent Document 3).

[0062] As yet another example of this application, the recombinant microorganism of this application may be a microorganism in which L-valine production capacity is enhanced by enhancing the activity of the acetolactate synthase isozyme 1 small subunit (IlvN) protein.

[0063] In any of the above-described examples, the recombinant microorganism of this application may be a microorganism in which the activity of the IlvN protein is enhanced by introducing a single mutation [ilvN(A42V); Non-Patent Literature 13] into the IlvN protein, thereby enhancing the L-valine production capacity.

[0064] The "Acetolactate synthase isozyme 1 small subunit (IlvN) protein" in this application has the activity to catalyze the conversion of two pyruvate molecules to acetolactate in the first common step of the biosynthesis pathway of branched-chain amino acids (BCAAs), including L-valine, L-leucine, and L-isoleucine. Therefore, microorganisms mutated to enhance the activity of the IlvN protein have the characteristic of having an enhanced L-valine biosynthesis pathway and are useful for L-valine production. For example, a microorganism mutated to enhance the activity of the IlvN protein may be a microorganism into which ilvN(A42V) has been introduced.

[0065] The ilvN(A42V) of this application is a protein having the amino acid sequence represented by SEQ ID NO: 13, a protein containing the amino acid sequence, a protein consisting of the amino acid sequence represented by SEQ ID NO: 13, or a protein substantially composed of the amino acid sequence, but is not limited to these. Furthermore, the ilvN(A42V) of this application may have an amino acid sequence that exhibits the activity of ilvN(A42V) and has at least 70%, 80%, 90%, 95%, or 99% homology or identity with the amino acid sequence represented by SEQ ID NO: 13, or may contain the amino acid sequence. Moreover, the ilvN(A42V) of this application may consist of an amino acid sequence that has at least 70%, 80%, 90%, 95%, or 99% homology or identity with the amino acid sequence represented by SEQ ID NO: 13, or may substantially consist of the amino acid sequence, but is not limited to these. Furthermore, the polynucleotide encoding ilvN(A42V) may have a base sequence encoding the amino acid sequence represented by Sequence ID No. 13, or may contain the said base sequence. Furthermore, the polynucleotide encoding ilvN(A42V) may consist of a base sequence encoding the amino acid sequence represented by Sequence ID No. 13, or may be substantially composed of the said base sequence. The polynucleotide encoding ilvN(A42V) of this application can be modified in various ways in the coding region, within the limits that the amino acid sequence of the ilvN(A42V) protein does not change, by codon degeneracy or by considering the preferred codon in the organism that intends to express the ilvN(A42V) protein. The polynucleotide encoding ilvN(A42V) of this application may have a base sequence that has at least 70%, 80%, 90%, 95%, or 99% or more, and less than 100% homology or identity with the base sequence of Sequence ID No. 14, or may contain the said base sequence.Furthermore, the polynucleotide encoding ilvN(A42V) of this application may consist of a base sequence that is homologous or identical to the base sequence of Sequence ID No. 14 by at least 70%, 80%, 90%, 95%, or 99%, and less than 100%, or may be substantially composed of the said base sequence, but is not limited to these.

[0066] In one embodiment, the microorganism into which ilvN(A42V) is introduced may be Corynebacterium glutamicum CJ7V or Corynebacterium glutamicum CJ8V, but is not limited to these. Regarding such L-valine-producing microorganisms, in addition to the above, the information disclosed in Patent Document 4 and others is used as reference material in this application, but is not limited to these.

[0067] In this application, "weakening" of the activity of polypeptides (including proteins identified by the names of each enzyme) is a concept that encompasses all cases where the activity is reduced compared to endogenous activity or where the activity is eliminated. The term "weakening" is used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0068] The aforementioned weakening includes at least one of the following: 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; the degree and / or concentration (expression level) of overall polypeptide activity within the cell is reduced compared to the natural strain due to inhibition of the expression of the gene encoding the polynucleotide or inhibition of translation into the polypeptide; there is no expression of the polynucleotide at all; and even if the polynucleotide is expressed, there is no polypeptide activity. "Inactivation," "deficiency," "reduction," "downregulation," "decrease," or "attenuation" of polypeptide activity compared to endogenous activity means that it is reduced compared to the activity of the specific polypeptide originally possessed by the parent strain or unmodified microorganism before the trait change.

[0069] Such weakening of polypeptide activity is not limited to these methods and can be achieved by applying various methods well known in the field (e.g., Non-Patent Documents 14, 15, etc.).

[0070] Specifically, weakening the activity of a polypeptide in this application involves: 1) deleting all or part of the gene encoding the polypeptide; 2) modifying the gene expression regulatory region (or gene expression regulatory sequence) so that the expression of the gene encoding the polypeptide is reduced; 3) modifying the amino acid sequence constituting the polypeptide so that the activity of the polypeptide is deleted or weakened (e.g., deleting / substituting / adding one or more amino acids in the amino acid sequence); and 4) modifying the gene sequence encoding the polypeptide so that the activity of the polypeptide is deleted or weakened (e.g., modifying the polypeptide gene sequence so that it encodes a polypeptide that has been modified so that the activity of the polypeptide is deleted or weakened). 1) Deleting / substituting / adding one or more nucleic acid bases in the nucleic acid base sequence of a gene; 5) Modifying the base sequence encoding the start codon or 5'UTR region of the polypeptide-encoding gene transcript; 6) Introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementaryly to the transcript of the polypeptide-encoding gene; 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the polypeptide-encoding gene so that a secondary structure is formed that prevents ribosome attachment; 8) Adding a promoter to the 3' end of the ORF (open reading frame) of the polypeptide-encoding gene sequence to reverse transcription (Reverse transcription engineering, RTE); or 9) A combination of two or more selected from 1) to 8) above, but not limited to these.

[0071] For example, the deletion of part or all of the gene encoding the polypeptide described in 1) above may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide within the chromosome, or by substituting it with a polynucleotide or marker gene in which some nucleotides are deleted.

[0072] Furthermore, modifying the regulatory expression region (or regulatory expression sequence) described in 2) above may be carried out by causing a mutation in the regulatory expression region (or regulatory expression sequence) through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting it with a sequence having lower activity. The regulatory expression 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 translation.

[0073] Modifying the amino acid sequence or polynucleotide sequence described in 3) and 4) above is carried out by introducing a sequence mutation through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, so as to weaken the activity of the polypeptide, or by substituting it with an amino acid sequence or polynucleotide sequence modified to have lower activity, or an amino acid sequence or polynucleotide sequence modified to eliminate activity, but is not limited to these methods. For example, gene expression can be inhibited or weakened by introducing a mutation into a polynucleotide sequence to form a stop codon, but is not limited to these methods.

[0074] Modification of the start codon or 5'UTR region of the gene transcript encoding the polypeptide (as described in 5) above is performed, for example, by substituting it with a nucleotide sequence encoding another start codon that has a lower polypeptide expression rate compared to the endogenous start codon, but is not limited to this.

[0075] For example, refer to Non-Patent Document 16 for introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding the polypeptide.

[0076] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in the polypeptide-coding gene so that a secondary structure is formed that makes ribosome attachment impossible may be done by making mRNA translation impossible or slowing down the rate of mRNA translation.

[0077] Furthermore, the reverse transcription engineering (RTE) of the open reading frame (ORF) of the gene sequence encoding the polypeptide (8) may be performed by creating an antisense nucleotide complementary to the transcript of the polypeptide gene and weakening its activity.

[0078] In this application, "enhancement" of polypeptide activity means improving the polypeptide activity compared to its endogenous activity. This enhancement is used interchangeably with activation, upregulation, overexpression, and increase. Here, activation, enhancement, upregulation, overexpression, and increase all include exhibiting activity that was not originally present, or improving activity compared to endogenous activity or the activity before modification. When polypeptide activity is "enhanced," "upregulated," "overexpressed," or "improved" compared to endogenous activity, it means that it is improved compared to the activity and / or concentration (expression level) of a specific polypeptide that was originally present in the parent strain or unmodified microorganism before the trait change.

[0079] The enhancement may be carried out by introducing an exogenous polypeptide, or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether or not the activity of the polypeptide has been enhanced can be confirmed by an increase in the degree of the polypeptide's activity, its expression level, or the amount of product produced from the polypeptide.

[0080] Various methods known in the art can be applied to enhance the activity of the polypeptide, and any method that can enhance the activity of the target polypeptide compared to the microorganism before modification is acceptable. Specifically, this includes, but is not limited to, conventional methods in molecular biology, including genetic engineering and / or protein engineering known to those with ordinary skill in the art (see, for example, Non-Patent Documents 15, 17, etc.).

[0081] Specifically, the enhancement of polypeptide activity in this application is carried out by: 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the expression regulatory region of the gene on the chromosome encoding the polypeptide with a highly active sequence; 3) modifying the start codon or the base sequence encoding the 5'UTR region of the gene transcript encoding the polypeptide; 4) modifying the amino acid sequence of the polypeptide so as to enhance polypeptide activity; 5) modifying the polynucleotide sequence encoding the polypeptide so as to enhance polypeptide activity (for example, modifying the polynucleotide sequence of the polypeptide gene to encode a polypeptide modified to enhance polypeptide activity); 6) introducing an exogenous polypeptide exhibiting polypeptide activity or an exogenous polynucleotide encoding it; 7) optimizing the codon of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide and selectively modifying or chemically modifying exposed portions; or 9) a combination of two or more selected from 1) to 8) above, but is not limited to these methods.

[0082] More specifically, increasing the intracellular copy number of the polynucleotide encoding the polypeptide (as described in 1) above may be carried out by introducing into a host cell a vector into which the polynucleotide encoding the polypeptide is operably linked and which replicates and functions independently of the host. Alternatively, it may be carried out by introducing one or more copies of the polynucleotide encoding the polypeptide into the chromosomes within the host cell. The introduction into the chromosomes is carried out 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 this. The vector is as described above.

[0083] 2) The substitution of a gene expression regulatory region (or expression regulatory sequence) on a chromosome encoding a polypeptide with a more potent sequence may be carried out, for example, by generating a sequence mutation through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to further enhance the activity of the expression regulatory region, or by substituting it with a sequence having higher activity. The expression regulatory region includes, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences regulating transcription and translation termination. For example, this may be carried out by substituting the original promoter with a potent promoter, but is not limited to this.

[0084] Examples of known strong promoters include, but are not limited to, the CJ1-CJ7 promoters (Patent Document 5), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (Patent Document 6), the O2 promoter (Patent Document 7), the tkt promoter, and the yccA promoter.

[0085] 3) Modifying the start codon or the nucleotide sequence encoding the 5'UTR region of a gene transcript encoding a polypeptide is performed, for example, by substituting it with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate compared to the endogenous start codon, but is not limited to this.

[0086] Modifying the amino acid sequence or polynucleotide sequence described in 4) and 5) above is carried out by causing a sequence mutation through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, in order to enhance the activity of the polypeptide, or by substituting it with an improved amino acid sequence or polynucleotide sequence that has higher activity, or an improved amino acid sequence or polynucleotide sequence that has improved activity. Specifically, the substitution is carried out by inserting the polynucleotide into the chromosome by homologous recombination, but is not limited to this. The vector used here may further include a selection marker to confirm whether or not it has been inserted into the chromosome.

[0087] The introduction of a foreign polynucleotide exhibiting polypeptide activity (6) above may be carried out by introducing a foreign polynucleotide encoding a polypeptide exhibiting identical or similar activity to the polypeptide into the host cell. The foreign polynucleotide may have any origin or sequence, as long as it exhibits identical or similar activity to the polypeptide. The introduction can be carried out by a person skilled in the art using a known transformation method as appropriate, and as the polynucleotide introduced as described above is expressed in the host cell, the polypeptide is produced and its activity is improved.

[0088] The optimization of codons of polynucleotides encoding polypeptides described in 7) above may be performed by optimizing endogenous polynucleotide codons so as to increase transcription or translation within the host cell, or by optimizing exogenous polynucleotide codons so as to perform optimized transcription or translation within the host cell.

[0089] The 8) analysis of the tertiary structure of the polypeptide, and the selection and modification or chemical modification of exposed portions may be carried out, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on these, and selecting and modifying or chemically modifying exposed portions.

[0090] Such enhancement of polypeptide activity is achieved by increasing the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in the wild-type or pre-modification microbial strain, or by increasing the amount of product produced from the polypeptide, but is not limited to these methods.

[0091] In the microorganisms of this application, modification of part or all of the polynucleotides can be induced by (a) homologous recombination using a chromosome introduction vector in 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 or radiation. The methods for modifying part or all of the genes include methods using DNA recombination technology. For example, deletion of part or all of the gene can be achieved by introducing a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination. The introduced nucleotide sequence or vector contains, but is not limited to, a dominant selection marker.

[0092] In the microorganism of this application, the mutant ribonuclease activity-regulating protein, polynucleotide, L-valine, etc., are as described above. A further aspect of this application provides a method for producing L-valine, comprising the step of culturing a microorganism containing the mutant ribonuclease activity-modulating protein of this application or the polynucleotide of this application in a culture medium.

[0093] The method for producing L-valine according to this application may include the step of culturing a microorganism containing the mutant ribonuclease activity-modulating protein, the polynucleotide, or the vector in a culture medium.

[0094] In this application, "cultivation" means growing the microorganisms of this application under appropriately adjusted environmental conditions. The cultivation process of this application can be carried out using suitable culture media and cultivation conditions known in the art. Such a cultivation process can be easily adjusted and used by those skilled in the art depending on the selected microorganisms. Specifically, the cultivation is batch, continuous, and / or fed-batch culture, but is not limited to these.

[0095] In this application, "culture medium" refers to a substance that is a mixture mainly composed of nutrients necessary for culturing the microorganisms of this application, and supplies 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 may be any that are normally used for culturing microorganisms, and the microorganisms of this application can be cultured in a normal culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, under aerobic conditions, with the temperature, pH, etc. adjusted.

[0096] Specifically, a culture medium for microorganisms of the genus Corynebacterium is disclosed in Non-Patent Document 18. In this application, the carbon source can be carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, and 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 liquid can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and any other carbon source in an appropriate amount can be used. These carbon sources can be used individually or in combination of two or more, but are not limited to these uses.

[0097] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate can be used, as well as organic nitrogen sources such as amino acids like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration liquid, casein hydrolysates, fish or their decomposition products, defatted soybean cake or its decomposition products. These nitrogen sources can be used individually or in combination of two or more, but are not limited to these uses.

[0098] As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or equivalent sodium-containing salts can be used. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., can be used, and in addition, amino acids, vitamins, and / or suitable precursors can be used. These components or precursors can be added to the culture medium in batches or continuously, but are not limited to these.

[0099] Furthermore, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture medium in a suitable manner during the cultivation of the microorganisms of this application. In addition, during cultivation, the formation of bubbles can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, oxygen or oxygen-containing gas may be injected into the culture medium to maintain an aerobic state, and to maintain an anaerobic and microaerobic state, it is not necessary to inject gas, but nitrogen, hydrogen, or carbon dioxide gas may be injected, but the invention is not limited to these.

[0100] In the culture described in this application, the culture temperature is maintained at 20-45°C, specifically 25-40°C, and the culture is performed for approximately 10-160 hours, but is not limited to these values. The L-valine produced by the culture described in this application is either secreted into the culture medium or remains within the cells.

[0101] The method for producing L-valine according to this application may further include, for example, the steps of preparing the microorganism of this application, preparing a culture medium for culturing the microorganism, or a combination thereof (in any order) before the culturing step.

[0102] The method for producing L-valine according to this application may further include a step of recovering L-valine from the culture medium used for the culture (the culture medium in which the culture was performed) or from the Corynebacterium glutamicum strain. The recovery step may further be included after the culture step.

[0103] The aforementioned recovery may involve collecting the target L-valine using a suitable method known in the art, depending on the microorganism culture method of this application, such as batch, continuous, or fed-batch culture. For example, various chromatography methods such as centrifugation, filtration, crystallization, treatment with protein precipitants (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, and the target L-valine can be recovered from the culture medium or microorganism using a suitable method known in the art.

[0104] Furthermore, the L-valine production method of this application may further include a purification step. The purification can be carried out by a preferred method known in the art. For example, if the L-valine production method of this application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out sequentially or discontinuously, regardless of order, simultaneously or as a single integrated step, but are not limited thereto.

[0105] In the method of this application, the mutant ribonuclease activity-regulating protein, polynucleotide, vector, bacterial strain, etc., are as described above. Further embodiments of this application provide a composition for L-valine production comprising the mutant ribonuclease activity-modulating protein of this application, a polynucleotide encoding the same, a vector containing the polynucleotide or a microorganism containing the polynucleotide of this application, a culture medium in which the microorganism is cultured, or at least two combinations thereof.

[0106] The composition of this application may further contain any suitable excipients commonly used in compositions for L-amino acid production. Examples of such excipients include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0107] Further embodiments of this application provide the mutant ribonuclease activity-modulating protein of this application, the polynucleotide encoding the same, a vector containing the polynucleotide, or applications for L-valine production of microorganisms containing the polynucleotide of this application.

[0108] The ribonuclease activity-modulating protein, mutant ribonuclease activity-modulating protein, polynucleotide, vector, bacterial strain, culture medium, L-valine, etc., are as described above. [Examples]

[0109] The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the application is not limited thereto. Technical matters not described herein can be fully understood and readily implemented by a skilled technician in the art of this application or a similar art. [Examples]

[0110] Creation of mutagenic bacterial strains Example 1-1. Induction of Mutation The L-valine-producing strain Corynebacterium glutamicum strain KCCM11201P (CA08-0072, Patent Document 1) was streaked onto a nutrient medium containing agar and cultured at 30°C for 36 hours. Several hundred colonies obtained in this way were irradiated with UV light at room temperature to induce random mutations in the strain's genome.

[0111] Examples 1-2. Evaluation of L-valine production capacity of mutagenic bacterial strains. In Example 1-1, fermentation titer experiments were conducted to select strains from among those that induced random mutations that showed improved L-valine production capacity compared to the parent strain KCCM11201P. Each colony was subcultured in nutrient medium, and then each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of production medium and cultured with shaking at 30°C and 200 rpm for 72 hours. Subsequently, the concentration of L-valine was evaluated using HPLC. The results are shown in Table 1.

[0112] <Nutrient medium> 10g glucose, 5g beef extract, 10g polypeptone, 2.5g sodium chloride, 5g yeast extract, 20g agar, 2g urea, pH 7.2 (per liter of distilled water) <Production culture medium> 100g glucose, 40g ammonium sulfate, 2.5g soy protein, 5g corn steep solids, 3g urea, 1g dipotassium hydrogen phosphate, 0.5g magnesium sulfate heptahydrate, 100μg biotin, 1mg thiamine HCl, 2mg calcium pantothenate, 3mg nicotinamide, 30g calcium carbonate, pH 7.0 (per liter of distilled water)

[0113] [Table 1]

[0114] As shown in Table 1, we selected strain C10, which showed the greatest improvement in L-valine production capacity compared to the parent strain KCCM11201P. [Examples]

[0115] Confirmation of mutations by gene sequencing The major genes of the C10 strains selected in Examples 1-2 were sequenced and compared with the parent strain KCCM11201P and the wild-type Corynebacterium glutamicum ATCC14067 strain. As a result, no mutations were found in the biosynthetic pathway genes directly related to L-valine production.

[0116] Therefore, when the presence or absence of mutations was confirmed by NGS (Next-Generation Sequencing) analysis, a mutation was confirmed at a specific position in the ORF (open reading frame) region of the rraA gene, which encodes the ribonuclease activity regulator protein (RraA). Specifically, compared to the parent strain KCCM11201P, strain C10 had one mutation introduced at a base located 277 bp upstream from the start codon of the rraA gene, changing the conventional base sequence from CAT (SEQ ID NO: 2) to TAT (SEQ ID NO: 4), and it was confirmed that it possessed a mutant RraA(H93Y) (SEQ ID NO: 3) in which the histidine amino acid residue at the 93rd position from the N-terminus of the RraA protein was replaced with tyrosine. [Examples]

[0117] Production of mutant expression vectors To construct a vector for introducing the H93Y mutation into the rraA gene, the genomic DNA of the C10 strain selected in Example 1-2 was extracted using the G-spin Total DNA Extraction Mini-Kit (Cat. No. 17045, Intron) according to the manufacturer's protocol. Using this genomic DNA as a template, PCR was performed using primer pairs of SEQ ID NO: 5 and SEQ ID NO: 6. The PCR conditions were denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 150 seconds, for 25 cycles, and then polymerization at 72°C for 7 minutes. As a result, a 507 bp PCR product (hereinafter referred to as "mutation fragment 1") was obtained.

[0118] As described above, the obtained mutant fragment 1 was ligated with a pDCM2 vector treated with the restriction enzyme SmaI (Patent Document 8) using an infusion cloning kit (Takara Bio Inc.), and this was transformed into E. coli DH5α by electroporation (Non-Patent Document 19) to induce homologous recombination on the chromosome (Non-Patent Document 19). Strains in which the vector was inserted on the chromosome by homologous sequence recombination were selected from LB medium containing 25 mg / L of kanamycin. From the selected E. coli transformed strains, DNA was obtained using a DNA-spin plasmid DNA purification kit (Intron) according to the manufacturer's protocol, and a pDCM2-rraA(H93Y) vector for introducing the H93Y mutation into the rraA gene, containing mutant fragment 1, was constructed.

[0119] The primer sequences used here are shown in Table 2.

[0120] [Table 2]

[0121] Example 4: Evaluation of L-valine production ability of mutant strains Example 4-1. Evaluation of L-valine production ability of the mutant Corynebacterium glutamicum KCCM11201P strain. To create an L-valine-producing strain with the H93Y mutation introduced into the rraA gene, the pDCM2-rraA(H93Y) vector prepared in Example 3 was used to transform the L-valine-producing strain KCCM11201P by homologous recombination on the chromosome. Strains in which the vector was inserted on the chromosome by homologous sequence recombination were selected from a medium containing 25 mg / L kanamycin. Subsequently, the gene fragments were amplified by PCR using primer pairs of SEQ ID NO: 5 and SEQ ID NO: 6 in the Corynebacterium glutamicum transformed strains after secondary recombination was completed, and then the strains in which the H93Y mutation was introduced into the rraA gene were confirmed by gene sequence analysis. The recombinant strain was named Corynebacterium glutamicum KCCM11201P::rraA(H93Y).

[0122] The L-valine production capacity of the parent strains KCCM11201P and KCCM11201P::rraA(H93Y) was evaluated in the same manner as in Example 1-2. The results are shown in Table 3.

[0123] [Table 3]

[0124] As a result, it was confirmed that the L-valine production capacity of the KCCM11201P::rraA(H93Y) strain was increased by 11% compared to the parent strain KCCM11201P. Example 4-2. Evaluation of L-valine production ability of the mutant Corynebacterium glutamicum CJ7V strain. To confirm whether introducing the H93Y mutation into the rraA gene improves L-valine production in other strains of Corynebacterium glutamicum that produce L-valine, a single mutation [ilvN(A42V); Non-Patent Literature 13] was introduced into the acetolactate synthase isozyme 1 small subunit (IlvN) protein of wild-type Corynebacterium glutamicum ATCC14067, and a strain with improved L-valine production was created (Patent Literature 4).

[0125] First, to prepare a vector for introducing the A42V mutation into the ilvN gene, genomic DNA from the wild-type Corynebacterium glutamicum ATCC14067 strain was extracted using a G-spin Total DNA Extraction Mini-Kit according to the manufacturer's protocol. Using this genomic DNA as a template, PCR was performed using primer pairs of SEQ ID NOs. 7 and 8, and SEQ ID NOs. 9 and 10, respectively, to obtain gene fragments A and B. The PCR conditions were denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 60 seconds, followed by polymerization at 72°C for 7 minutes, for 25 cycles. As a result, gene fragment A of 528 bp and gene fragment B of 509 bp were obtained. Using the obtained gene fragments A and B as described above as templates, overlapping PCR was performed using primer pairs of SEQ ID NOs. 6 and 9. As a result, a PCR product of 1010 bp (hereinafter referred to as "mutation-introduced fragment 2") was obtained.

[0126] As described above, the obtained mutant fragment 2 was treated with the restriction enzyme SmaI, and then ligated with a pDCM2 vector treated with the same restriction enzyme. E. coli DH5α was transformed by electroporation to induce homologous recombination on the chromosome. Strains in which the vector was inserted on the chromosome by homologous sequence recombination were selected from kanamycin-containing LB medium. From the selected E. coli transformants, DNA was obtained from a DNA-spin plasmid DNA purification kit according to the manufacturer's protocol, and a pDCM2-ilvN(A42V) vector for introducing the A42V mutation into the ilvN gene, containing mutant fragment 2, was constructed.

[0127] The primer sequences used here are shown in Table 4.

[0128] [Table 4]

[0129] As described above, the prepared pDCM2-ilvN(A42V) vector was used to transform wild-type Corynebacterium glutamicum ATCC14067 via homologous recombination on the chromosome. Strains in which the vector was inserted on the chromosome by homologous sequence recombination were selected from a culture medium containing 25 mg / L kanamycin. Subsequently, the transformed Corynebacterium glutamicum strains, after secondary recombination had been completed, were subjected to PCR using primer pairs of SEQ ID NOs. 7 and SEQ ID NOs. 10 to amplify the gene fragments, and then gene sequence analysis confirmed the presence of the A42V mutation in the ilvN gene. The recombinant strain was named Corynebacterium glutamicum CJ7V.

[0130] Finally, similar to Example 4-1, Corynebacterium glutamicum CJ7V was transformed with the pDCM2-rraA(H93Y) vector. The recombinant strain was named Corynebacterium glutamicum CJ7V::rraA(H93Y).

[0131] The L-valine production capacity of the parent strains CJ7V and CJ7V::rraA(H93Y) was evaluated in the same manner as in Example 1-2. The results are shown in Table 5.

[0132] [Table 5]

[0133] As a result, it was confirmed that the L-valine production capacity of the CJ7V::rraA(H93Y) strain was increased by 14% compared to the parent strain CJ7V. Example 4-3. Evaluation of L-valine production ability of the mutant Corynebacterium glutamicum CJ8V strain. A strain with improved L-valine production capacity was created by introducing a single mutation [ilvN(A42V)] into the IlvN protein of wild-type Corynebacterium glutamicum ATCC13869 (Patent Document 4).

[0134] The pDCM2-ilvN(A42V) vector prepared in Example 4-2 was used to transform wild-type Corynebacterium glutamicum ATCC13869 strain. Strains in which the vector was inserted into the chromosome by homologous sequence recombination were selected from a medium containing kanamycin 25 mg / L. Subsequently, the transformed Corynebacterium glutamicum strains, after secondary recombination had been completed, were subjected to PCR using primer pairs of SEQ ID NO: 11 and SEQ ID NO: 12 to amplify the gene fragments, and then the strains in which the A42V mutation had been introduced into the ilvN gene were confirmed by gene sequence analysis. The recombinant strain was named Corynebacterium glutamicum CJ8V.

[0135] The primer sequences used here are shown in Table 6.

[0136] [Table 6]

[0137] Finally, similar to Example 4-1, Corynebacterium glutamicum CJ8V was transformed with the pDCM2-rraA(H93Y) vector. The recombinant strain was named Corynebacterium glutamicum CJ8V::rraA(H93Y).

[0138] The L-valine production capacity of the parent strains CJ8V and CJ8V::rraA(H93Y) was evaluated in the same manner as in Example 1-2. The results are shown in Table 7.

[0139] [Table 7]

[0140] As a result, it was confirmed that the L-valine production capacity of the CJ8V::rraA(H93Y) strain was increased by 10% compared to the parent CJ8V strain. From the above explanation, 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. It should be understood that the above embodiments are merely illustrative and not limiting. This application should be interpreted as including all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. A mutant ribonuclease activity-regulating protein in which the amino acid corresponding to the 93rd position in the amino acid sequence of Sequence ID No. 1 is substituted with tyrosine, and which has the amino acid sequence of Sequence ID No. 3 or an amino acid sequence that is 90% or more identical thereto.

2. A polynucleotide encoding a mutant ribonuclease activity-regulating protein as described in claim 1.

3. A microorganism comprising a mutant ribonuclease activity-regulating protein or a polynucleotide encoding the same, wherein the amino acid corresponding to the 93rd position in the amino acid sequence of Sequence ID No. 1 is substituted with tyrosine, and the mutant ribonuclease activity-regulating protein has the amino acid sequence of Sequence ID No. 3 or an amino acid sequence having 90% or more identity thereto.

4. The microorganism according to claim 3, wherein the microorganism has improved L-valine production ability compared to a Corynebacterium microorganism containing a wild-type ribonuclease activity-regulating protein having the amino acid sequence of Sequence ID No. 1 or a polynucleotide encoding it.

5. The microorganism according to claim 3, wherein the microorganism is a microorganism of the genus Corynebacterium.

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

7. A method for producing L-valine, comprising the step of culturing a microorganism containing a mutant ribonuclease activity-modulating protein or a polynucleotide encoding the same in a culture medium, wherein the mutant ribonuclease activity-modulating protein has the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having 90% or more identity thereto.

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