Mutant ribonuclease activity-regulating 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]

[0001] The present application relates to a mutant ribonuclease activity-regulating protein and a method for producing L-valine using the same. [Background technology]

[0002] L-amino acids are the basic building blocks of proteins and are used as important materials in pharmaceutical ingredients, food additives, animal feed, nutrients, insecticides, fungicides, and other products. In particular, branched chain amino acids (BCAAs) are a collective term for the essential amino acids L-valine, L-leucine, and L-isoleucine, and these branched chain amino acids have antioxidant effects and promote protein synthesis in muscle cells.

[0003] Branched-chain amino acids are mainly produced using microorganisms of the genus Corynebacterium. For example, in the case of L-valine, target substance-specific approaches are mainly used, such as increasing the expression of genes encoding enzymes involved in its biosynthesis or removing genes unnecessary for biosynthesis (Patent Documents 1 and 2). In addition, methods for producing L-valine by feedback inhibition are also being researched (Patent Document 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 8,465,962 [Patent Document 2] Korean Patent Registration No. 10-2153534 [Patent Document 3] U.S. Patent No. 1,045,7919 [Patent Document 4] Korean Patent No. 10-1947945 [Patent Document 5] U.S. Patent No. 7,662,943 [Patent Document 6] U.S. Patent No. 10584338 [Patent Document 7] U.S. Patent No. 10273491 [Patent Document 8] Korean Patent Publication No. 10-2020-0136813

Non-licensed literature

[0005]

Non-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

Non-patented document 5

Non-patent document 6

Non-patent document 7

Non-patent document 8

Non-patented document 9

[0006] The present inventors have confirmed that introducing a mutant ribonuclease activity-regulating protein into a microorganism improves L-valine production ability compared to a microorganism containing the wild-type protein, and have completed the present application. [Means for solving the problem]

[0007] The present application provides a mutant ribonuclease activity-regulating protein in which the amino acid corresponding to position 93 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid. The present application provides polynucleotides encoding the mutant ribonuclease activity-regulating proteins of the present application.

[0008] The present application provides a microorganism comprising the mutant ribonuclease activity-regulating protein of the present application or a polynucleotide encoding the same. The present application provides a method for producing L-valine, comprising the step of culturing in a medium a microorganism containing a mutant ribonuclease activity-modulating protein of the present application or a polynucleotide encoding the same. [Effects of the Invention]

[0009] When a microorganism that produces L-valine is cultured using the mutant ribonuclease activity-regulating protein of the present application, L-valine can be produced in a higher yield than when a microorganism has a conventional wild-type ribonuclease activity-regulating protein. DETAILED DESCRIPTION OF THE INVENTION

[0010] These will be described in detail below. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. Furthermore, this application is not limited to the specific descriptions below. Furthermore, many papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety, thereby more clearly explaining the state of the art to which this application pertains and the contents of this application.

[0011] One aspect of the present application provides a mutant ribonuclease activity-regulating protein in which the amino acid at position 93 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid.

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

[0013] The protein to be mutagenized in the present application may be a protein that has the activity of regulating ribonuclease activity. Specifically, the protein comprises the amino acid sequence of SEQ ID NO: 1 and has the activity of regulating ribonuclease activity, but is not limited thereto. This does not exclude meaningless additions of sequences before or after the amino acid sequence of SEQ ID NO: 1, naturally occurring mutations, or silent mutations thereof. Proteins that have the same or equivalent activity as a protein comprising the amino acid sequence of SEQ ID NO: 1 are included in the protein to be mutagenized in the present application. For example, the protein to be mutagenized in the present application may be a protein consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homologous or identical thereto. Furthermore, it goes without saying that proteins with such homology or identity and in which a portion of the amino acid sequence has been deleted, modified, substituted, or added are also included in the protein to be mutagenized in the present application, as long as the amino acid sequence has the same activity as the protein.

[0014] In the present application, the term "ribonuclease activity regulator protein" refers to a protein that regulates ribonuclease activity by binding to the endonuclease RNase E and inhibiting RNA processing, and is also used interchangeably with the term "ribonuclease E activity regulator" or "RraA." The amino acid sequence of RraA can be obtained from publicly known databases such as NCBI Genebank.

[0015] By way of example, the RraA protein of the present application is derived from a microorganism, specifically from a prokaryotic or eukaryotic microorganism, more specifically from a microorganism of the genus Corynebacterium, but is not limited thereto.

[0016] Another example is the RraA protein WP_003858525.1 derived from Corynebacterium genus microorganisms, but it goes without saying that proteins of various origins having the activity of regulating ribonuclease activity are included.

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

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

[0019] As an example, the mutant ribonuclease activity-regulating protein may be one in which the amino acid corresponding to position 93 in the amino acid sequence of SEQ ID NO: 1 is substituted 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-regulating protein may be one in which the amino acid corresponding to position 93 in the amino acid sequence of SEQ ID NO: 1 is substituted 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, tyrosine.

[0021] As yet another example, the mutant ribonuclease activity-regulating protein of the present application may have the amino acid sequence shown in 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 amino acid sequence, may consist of the amino acid sequence, or may be essentially consisting of the amino acid sequence.

[0022] The mutant ribonuclease activity-regulating protein of the present application may be one in which the amino acid corresponding to position 93 in the amino acid sequence of SEQ ID NO: 1 is an amino acid other than histidine, for example, 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% homology or identity to the amino acid sequence represented by SEQ ID NO: 1. Needless to say, the present application also includes mutant ribonuclease activity-regulating proteins having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added, so long as the amino acid sequence has such homology or identity and exhibits efficacy equivalent to that of the mutant ribonuclease activity-regulating protein of the present application.

[0023] For example, the mutant ribonuclease activity-regulating protein of the present application may have an addition or deletion of a sequence at the N-terminus, C-terminus and / or internally of the amino acid sequence, a naturally occurring mutation, a silent mutation, or a conservative substitution that does not change the function of the mutant ribonuclease activity-regulating protein of the present application.

[0024] The term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

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

[0026] As used herein, the term "mutant protein" or "variant" refers to a polypeptide that differs from the amino acid sequence of the variant by conservative substitution and / or modification of at least one amino acid, but maintains its functions or properties. 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 performance of the variant may be improved, unchanged, or decreased compared to the polypeptide of the variant. Some variants also include variants in which at least one portion, such as an N-terminal leader sequence or a transmembrane domain, has been removed. Other variants include variants in which portions have been removed from the N- and / or C-termini of the mature protein. The term "mutant protein" is often used interchangeably with "mutated," "altered," "mutant polypeptide," "mutated protein," "mutant," "mutant," "divergent," etc. (in English, "modification," "modified polypeptide," "modified protein," "mutant," "mutein," "divergent," etc.), but it may refer to any term that means mutation. For purposes of the present application, the mutant may be a polypeptide in which the amino acid at position 93 in the amino acid sequence of SEQ ID NO: 1 is substituted with serine. For example, the mutant is a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, but is not limited thereto.

[0027] The variant may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be linked to a signal (or leader) sequence involved in co- or post-translational protein translocation. The variant may also be linked to other sequences or linkers to allow identification, purification, or synthesis.

[0028] In this application, "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0029] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, optionally with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize to all or part of the sequence under moderately or highly stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides containing common codons or codons that take into account codon degeneracy in the polynucleotide.

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

[0031] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 3, as disclosed in, for example, Non-Patent Document 8. Briefly, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include: (1) a binary comparison matrix (identity takes a value of 1, non-identity a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0032] As an example of the present application, the mutant ribonuclease activity-regulating protein of the present application may have an activity that improves L-valine-producing ability compared to a wild-type polypeptide that has the activity of regulating ribonuclease activity.

[0033] "Corresponding to," as used herein, means the amino acid residue at the recited position in the polypeptide, or an amino acid residue that is similar, identical, or equivalent to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position will determine the specific amino acid of the sequence to which the particular sequence refers. "Corresponding region," as used herein, generally refers to a similar or corresponding position in a related or reference protein.

[0034] For example, when any amino acid sequence is aligned with SEQ ID NO: 1, each amino acid residue in the amino acid sequence can be numbered based on the number and position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, the sequence alignment algorithm in the present application can identify the amino acid positions or positions where modifications such as substitutions, insertions, deletions, etc. occur when compared with a query sequence (also referred to as a "reference sequence").

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

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

[0037] As an example, but not limited to, the rraA gene is a polynucleotide encoding WP_003858525.1 derived from a microorganism of the genus Corynebacterium. As another example, the rraA gene is an rraA gene derived from a microorganism of the genus Corynebacterium. However, the rraA gene is not limited to this, and it goes without saying that it includes rraA genes of various origins that encode proteins having the activity of the RraA protein.

[0038] In the present application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are covalently linked in a long chain, and refers to a DNA or RNA chain longer than a certain length, and more specifically refers to a polynucleotide fragment encoding the mutant ribonuclease activity-regulating protein.

[0039] The polynucleotide encoding the mutant ribonuclease activity-regulating protein of the present application may comprise a nucleotide sequence encoding a mutant ribonuclease activity-regulating protein in which the amino acid corresponding to position 93 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid, or may comprise a nucleotide sequence in which the codons corresponding to positions 277 to 279 in the nucleotide sequence of SEQ ID NO: 2 have been substituted with codons encoding other amino acids. For example, the polynucleotide of the present application may comprise a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 3. As a more specific example of the present application, the polynucleotide of the present application may have the sequence of SEQ ID NO: 4 or may comprise said sequence. Furthermore, the polynucleotide of the present application may consist of the sequence of SEQ ID NO: 4 or may essentially consist of said sequence.

[0040] The polynucleotide of the present application can undergo various modifications in the coding region to the extent that the amino acid sequence of the mutant ribonuclease activity-regulating protein of the present application is not changed, taking into account codon degeneracy or codons preferred in the organism in which the mutant ribonuclease activity-regulating protein of the present application is to be expressed. Specifically, the polynucleotide of the present application has or contains a nucleotide sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: 4, or consists of or is substantially composed of a nucleotide sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homologous or identical to the sequence of SEQ ID NO: 4, but is not limited thereto. Here, in the homologous or identical sequence, the codon encoding the amino acid corresponding to position 93 of SEQ ID NO: 1 may be one of the codons encoding an amino acid other than histidine, for example, tyrosine.

[0041] Furthermore, the polynucleotide of the present application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 11 and 12). For example, conditions include those under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, but polynucleotides with lower homology or identity do not hybridize with each other; or conditions including washing once, specifically two to three times, at a salt concentration and temperature equivalent to those used in conventional Southern hybridization, namely, 60°C, 1×SSC, 0.1% SDS, specifically, 60°C, 0.1×SSC, 0.1% SDS, more specifically, 68°C, 0.1×SSC, 0.1% SDS.

[0042] Hybridization requires that 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 to each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0043] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and 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 of the polynucleotides and the degree of complementation, variables known in the art (eg, Non-Patent Document 11).

[0045] Yet another aspect of the present application provides a vector comprising a polynucleotide of the present application. The vector is an expression vector for expressing the polynucleotide in a host cell, but is not limited thereto.

[0046] The vector of the present application includes a DNA product comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable the target polypeptide to be expressed in a suitable host. The expression control region includes a promoter that initiates transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that regulates the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate or function independently of the host genome and is integrated into the genome itself.

[0047] The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage or cosmid vectors. Examples of plasmid vectors that may be used include pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors may be used.

[0048] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosomal introduction. The insertion of the polynucleotide into a chromosome can be achieved by any method known in the art, including, but not limited to, homologous recombination. A selection marker for determining whether or not the polynucleotide has been inserted into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to determine whether or not the target nucleic acid molecule has been inserted. Markers that confer selectable phenotypes, such as drug resistance, auxotrophy, 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 survive or exhibit a different phenotype, allowing the selection of transformed cells.

[0049] In the present application, "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, thereby expressing the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide that can be expressed in the host cell, regardless of whether it is located intrachromosomally or extrachromosomally. Furthermore, the polynucleotide may comprise DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form that can be expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to sequences necessary for expression in the host cell, but this is not limited thereto.

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

[0051] Yet another aspect of the present application provides a microorganism comprising the mutant ribonuclease activity-modulating protein of the present application or the polynucleotide of the present application. The strain of the present application may contain a mutant ribonuclease activity-regulating protein of the present application, a polynucleotide encoding said polypeptide, or a vector containing the polynucleotide of the present application.

[0052] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, and is a microorganism in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and is a microorganism that has been genetically modified to produce a desired polypeptide, protein, or product.

[0053] The strain of the present application may be a strain naturally capable of producing L-valine, or a microorganism in which L-valine-producing ability has been imparted to a strain that does not have the ability to produce L-valine, such as, but not limited to, a microorganism in which L-valine-producing ability has been improved by introducing a mutant ribonuclease activity-regulating protein of the present application or a polynucleotide encoding the same.

[0054] The strain of the present application may be a microorganism having improved L-valine-producing ability compared to a parent strain not containing the mutant of the present application or a wild-type Corynebacterium strain. The microorganism may have improved L-valine-producing ability due to the introduction of the mutant of the present application.

[0055] For example, the ribonuclease activity-regulating protein-unmodified microorganism, which is the subject strain for comparison to determine whether the L-valine-producing ability is improved, is the L-valine-producing Corynebacterium glutamicum strain KCCM11201P (CA08-0072, Patent Document 1), wild-type Corynebacterium glutamicum ATCC14067 into which the ilvN (A42V) mutation has been introduced (Patent Document 4), or wild-type Corynebacterium glutamicum ATCC13869 (Patent Document 4), but is not limited to these.

[0056] For example, the recombinant strain with improved production ability has an L-valine production ability that is improved by at least about 1%, specifically at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, or at least about 14% (there is no particular upper limit, for example, at most about 200%, at most about 150%, at most about 100%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, or at most about 15%) compared to the L-valine production ability of the parent strain or unmodified microorganism before mutation. However, any increase in + value compared to the production ability of the parent strain, unmodified microorganism, or unmodified microorganism with a ribonuclease activity-regulating protein before mutation may be used. In other examples, the recombinant strain with improved L-valine-producing ability has an L-valine-producing ability that is about 1.01-fold or more, about 1.02-fold or more, about 1.03-fold or more, about 1.04-fold or more, about 1.05-fold or more, about 1.06-fold or more, about 1.07-fold or more, about 1.08-fold or more, about 1.09-fold or more, about 1.10-fold or more, about 1.11-fold or more, about 1.12-fold or more, about 1.13-fold or more, or about 1.14-fold or more (there is no particular upper limit, for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, or about 2-fold or less) improved compared to the parent strain before mutation, an unmodified microorganism, or a microorganism unmodified with a ribonuclease activity-regulating protein, but is not limited thereto.

[0057] In the present application, "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but refers to wild-type or naturally occurring strains themselves, or strains before their traits have changed due to genetic mutations caused by natural or artificial factors. Furthermore, in the present application, "microorganisms unmodified with a ribonuclease activity-regulating protein" refers to strains into which the ribonuclease activity-regulating protein mutants described herein have not been introduced or before their introduction. The unmodified microorganisms in the present application with a ribonuclease activity-regulating protein do not exclude strains in which modifications of proteins or genes other than modifications of the ribonuclease activity-regulating protein or the polynucleotide encoding it have been made.

[0058] In this application, the term "unmodified microorganism" is used interchangeably with "strain before modification," "microorganism before modification," "non-mutated strain," "unmodified strain," "non-mutated microorganism," or "reference microorganism." The microorganisms of the present application include, but are not limited to, microorganisms containing a mutant ribonuclease activity-regulating protein or a polynucleotide encoding the same, or microorganisms (e.g., recombinant microorganisms) that have been genetically modified to contain a mutant ribonuclease activity-regulating protein or a polynucleotide encoding the same. The term "endogenous activity" refers to the activity of a specific polypeptide that a parent strain, wild-type, or unmodified microorganism originally possessed before the trait change, when the trait is changed due to genetic mutation caused by natural or artificial factors. This term is sometimes used interchangeably with "activity before modification."

[0059] In other examples of the present application, the microorganism of the present application is Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens, specifically, but not limited to, Corynebacterium glutamicum.

[0060] As yet another example of the present application, the recombinant microorganism of the present application may be a microorganism whose L-valine-producing ability has been enhanced by further enhancing the activity of a part of the proteins in the L-valine biosynthetic pathway or by further weakening the activity of a part of the proteins in the L-valine degradation pathway (Patent Documents 1 and 2).

[0061] In yet another example of the present application, the recombinant microorganism of the present application may be a microorganism in which feedback inhibition is further induced, thereby enhancing L-valine-producing ability (Patent Document 3).

[0062] In yet another example of the present application, the recombinant microorganism of the present application may be a microorganism having enhanced L-valine-producing ability due to enhanced activity of acetolactate synthase isozyme 1 small subunit (IlvN) protein.

[0063] In any of the above-mentioned Examples, the recombinant microorganism of the present application may be a microorganism whose L-valine-producing ability has been enhanced by introducing one mutation [ilvN(A42V); Non-Patent Document 13] into the IlvN protein to enhance the activity of the IlvN protein.

[0064] The "acetolactate synthase isozyme 1 small subunit (IlvN) protein" in the present application has the activity of catalyzing the conversion of two pyruvate molecules to acetolactate in the first common step of the biosynthetic pathway of branched-chain amino acids (BCAAs) including L-valine, L-leucine, and L-isoleucine. Therefore, a microorganism mutated to enhance the activity of the IlvN protein is characterized by an enhanced L-valine biosynthetic pathway and is useful for producing L-valine. For example, the 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 the present application may be a protein having the amino acid sequence set forth in SEQ ID NO: 13, a protein containing the amino acid sequence, a protein consisting of the amino acid sequence set forth in SEQ ID NO: 13, or a protein substantially consisting of the amino acid sequence, but is not limited to these. Furthermore, the ilvN(A42V) of the present application may have an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 13, or may contain the amino acid sequence, so long as it exhibits the activity of ilvN(A42V). Furthermore, the ilvN(A42V) of the present application may be composed of an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 13, or may be substantially consisting of the amino acid sequence, but is not limited to these. Furthermore, the polynucleotide encoding ilvN(A42V) may have a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 13, or may contain said nucleotide sequence. Furthermore, the polynucleotide encoding ilvN(A42V) may consist of a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 13, or may essentially consist of said nucleotide sequence. The polynucleotide encoding ilvN(A42V) of the present application can undergo various modifications in the coding region, taking into account codon degeneracy or codons preferred in the organism in which the ilvN(A42V) protein is to be expressed, as long as the amino acid sequence of the ilvN(A42V) protein is not changed. The polynucleotide encoding ilvN(A42V) of the present application may have a nucleotide sequence that is at least 70%, 80%, 90%, 95%, or 99% homologous or identical to, and less than 100% identical to, the nucleotide sequence of SEQ ID NO: 14, or may contain said nucleotide sequence.Furthermore, the polynucleotide encoding ilvN(A42V) of the present application may consist of a base sequence that has at least 70%, 80%, 90%, 95%, or 99% or more, but less than 100%, homology or identity to the base sequence of SEQ ID NO: 14, or may be substantially composed of the base sequence, but is not limited to these.

[0066] In one embodiment, the microorganism into which ilvN(A42V) has been introduced may be, but is not limited to, Corynebacterium glutamicum CJ7V or Corynebacterium glutamicum CJ8V. Regarding such L-valine-producing microorganisms, in addition to the above-mentioned disclosures, the disclosures of Patent Document 4 and the like are used as reference materials for the present application, but are not limited thereto.

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

[0068] The attenuation includes at least one of the following: a reduction or elimination of the activity of the polypeptide itself compared to the activity of the polypeptide originally possessed by the microorganism due to, for example, a mutation in the polynucleotide encoding the polypeptide; a reduction in the overall level and / or concentration (expression level) of polypeptide activity in the cell compared to that of a native strain due to, for example, inhibition of expression of the gene encoding the polynucleotide or inhibition of translation into the polypeptide; no expression of the polynucleotide at all; and no polypeptide activity even if the polynucleotide is expressed. "Inactivation," "deficiency," "reduction," "down-regulation," "reduction," or "attenuation" of a polypeptide activity compared to its endogenous activity means a reduction in the activity of a specific polypeptide originally possessed by the parent strain or unmodified microorganism before the transformation.

[0069] The activity of such polypeptides can be attenuated by applying various methods well known in the art, including, but not limited to, those described in Non-Patent Documents 14 and 15.

[0070] Specifically, the activity of a polypeptide of the present application can be attenuated by 1) deleting all or part of a gene encoding the polypeptide, 2) modifying an expression regulatory region (or expression regulatory sequence) so as to reduce the expression of a gene encoding the polypeptide, 3) modifying an amino acid sequence constituting the polypeptide so as to delete or attenuate the activity of the polypeptide (for example, deleting / substituting / adding one or more amino acids in the amino acid sequence), or 4) modifying a gene sequence encoding the polypeptide so as to delete or attenuate the activity of the polypeptide (for example, modifying the gene sequence encoding the polypeptide so as to encode a polypeptide modified so as to delete or attenuate the activity of the polypeptide). The modification can be carried out by, but is not limited to, any of the following: (1) deleting / substituting / adding one or more nucleic acid bases in the nucleic acid base sequence of a gene; (2) modifying the nucleic acid sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide; (3) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide; (4) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide so that a secondary structure that prevents ribosome attachment is formed; (5) adding a promoter to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide so that it can be reverse-transcribed (reverse transcription engineering, RTE); or (6) combining two or more selected from 1) to 8).

[0071] For example, 1) deleting a part or all of the gene encoding the polypeptide may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, or by substituting a polynucleotide with a partial deletion of nucleotides or a marker gene.

[0072] Furthermore, the modification of the expression regulatory region (or expression regulatory sequence) described above in 2) may be carried out by generating a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting a sequence having a lower activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0073] The modification of the amino acid sequence or polynucleotide sequence in 3) and 4) above can be carried out by, but is not limited to, generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to attenuate the activity of the polypeptide, or by substituting an amino acid sequence or polynucleotide sequence that has been improved to have lower activity or to eliminate activity. For example, gene expression can be inhibited or attenuated by, but is not limited to, introducing a mutation into a polynucleotide sequence to form a stop codon.

[0074] The base sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide (5) can be modified, for example, by substituting it with a base sequence encoding another start codon that has a lower polypeptide expression rate than the endogenous start codon, but is not limited to this.

[0075] 6) The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding the polypeptide can be described, for example, by referring to Non-Patent Document 16.

[0076] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide so that a secondary structure that prevents ribosome attachment is formed may be achieved by disabling or slowing down mRNA translation.

[0077] Furthermore, 8) adding a promoter to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide so as to induce reverse transcription (reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the transcription product of the gene encoding the polypeptide, thereby attenuating its activity.

[0078] In the present application, "enhancing" the activity of a polypeptide means improving the activity of the polypeptide compared to its endogenous activity. The term "enhancing" is used interchangeably with terms such as "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and improvement include exhibiting an activity not originally present, and improving activity compared to the endogenous activity or the activity before modification. "Enhancing," "up-regulating," "overexpressing," or "improving" the activity of a polypeptide compared to its endogenous activity means improving the activity and / or concentration (expression level) of a specific polypeptide compared to the activity and / or concentration (expression level) originally possessed by the parent strain or unmodified microorganism before the transformation.

[0079] The enhancement may be achieved by introducing a foreign polypeptide, or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide has been enhanced can be confirmed by an increase in the level of activity of the polypeptide, the expression level, or the amount of a product produced from the polypeptide.

[0080] Various methods well known in the art can be applied to enhance the activity of the polypeptide, and any method can be used as long as it can enhance the activity of the target polypeptide compared to the unmodified microorganism. Specifically, methods such as, but not limited to, conventional methods in molecular biology using genetic engineering and / or protein engineering well known to those skilled in the art (e.g., Non-Patent Documents 15 and 17).

[0081] Specifically, the activity of a polypeptide of the present application can be enhanced by, but is not limited to, 1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide, 2) replacing the expression regulatory region of a gene on a chromosome encoding the polypeptide with a sequence with stronger activity, 3) modifying the nucleotide sequence encoding the start codon or 5'UTR region of a 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 (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance polypeptide activity), 6) introducing a foreign polypeptide exhibiting polypeptide activity or a foreign polynucleotide encoding the same, 7) optimizing the codons of a 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.

[0082] More specifically, 1) increasing the intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a host cell a vector to which a polynucleotide encoding the polypeptide is operably linked, the vector replicating and functioning independently of the host. Alternatively, the polynucleotide encoding the polypeptide may be introduced into a chromosome of the host cell at one or more copies. The introduction into a chromosome may be achieved by, but is not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome of the host cell. The vector is as described above.

[0083] The expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide (2) can be replaced with a sequence with stronger activity by, for example, generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by replacing the expression regulatory region with a sequence with higher activity, so as to further enhance the activity of the expression regulatory region. Examples of the expression regulatory region include, but are not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences regulating the termination of transcription and translation. For example, this can be achieved by replacing the original promoter with a strong promoter, but is not limited to this.

[0084] Examples of known strong promoters include, but are not limited to, the CJ1 to 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] The nucleotide sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide (3) can be modified, for example, by substituting it with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon, but is not limited to this.

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

[0087] 6) Introduction of an exogenous polynucleotide exhibiting the activity of a polypeptide may be carried out by introducing into a host cell an exogenous polynucleotide encoding a polypeptide exhibiting the same or similar activity as the polypeptide. The exogenous polynucleotide may be of any origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The introduction can be carried out by a known transformation method appropriately selected by those skilled in the art, and the introduced polynucleotide is expressed in the host cell as described above, thereby producing the polypeptide and improving its activity.

[0088] 7) Optimizing the codons of a polynucleotide encoding a polypeptide may be carried out by optimizing the codons of an endogenous polynucleotide so as to increase transcription or translation within a host cell, or by optimizing the codons of an exogenous polynucleotide so as to achieve optimized transcription and translation within a host cell.

[0089] 8) Analyzing the tertiary structure of a polypeptide and selecting and altering or chemically modifying exposed portions may be carried out, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins based on the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and altering or modifying exposed portions to be altered or chemically modified.

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

[0091] In the microorganisms of the present application, partial or complete modification of a polynucleotide can be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal introduction into the microorganism, or genome editing using engineered nucleases (e.g., CRISPR-Cas9), and / or (b) light and / or chemical treatment, such as ultraviolet light or radiation. Methods for partially or completely modifying the gene include methods using DNA recombination techniques. For example, partial or complete deletion of a gene can be achieved by introducing a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism and causing homologous recombination. The introduced nucleotide sequence or vector may contain, but is not limited to, a dominant selection marker.

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

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

[0094] The term "culturing" as used herein means growing the microorganism of the present application under appropriately adjusted environmental conditions. The culturing process of the present application can be carried out using a suitable medium and culture conditions known in the art. Those skilled in the art can easily adjust such a culturing process depending on the microorganism selected. Specifically, the culturing may be batch, continuous, and / or fed-batch culture, but is not limited thereto.

[0095] The term "culture medium" as used herein refers to a mixture of nutrients necessary for culturing the microorganism of the present application as its main components, and supplies nutrients such as water essential for survival and growth, growth factors, etc. Specifically, the medium and other culture conditions used to culture the microorganism of the present application may be any medium used for culturing ordinary microorganisms, and the microorganism of the present application can be cultured in an ordinary medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins under aerobic conditions by adjusting the temperature, pH, etc.

[0096] Specifically, a culture medium for Corynebacterium microorganisms is disclosed in Non-Patent Document 18. In the present application, examples of carbon sources that can be used include carbohydrates such as glucose, saccharose, 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. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can be used. Any other carbon source can also be used in an appropriate amount. These carbon sources can be used alone or in combination of two or more, but are not limited to these.

[0097] Examples of the nitrogen source that can be used 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, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its degradation products, and defatted soybean cake or its degradation products. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0098] Examples of the phosphorus source that can be used include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and sodium-containing salts corresponding thereto. Examples of inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Other examples include amino acids, vitamins, and / or suitable precursors. These components or precursors can be added to the medium in a batch or continuous manner. However, the present invention is not limited to these.

[0099] Furthermore, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the medium in a suitable manner during cultivation of the microorganism of the present application. Furthermore, during cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, oxygen or an oxygen-containing gas may be injected into the medium to maintain an aerobic state, and nitrogen, hydrogen, or carbon dioxide gas may be injected, or no gas may be injected, to maintain anaerobic and microaerobic states, but these are not limiting.

[0100] In the culture of the present application, the culture temperature is maintained at 20 to 45°C, specifically 25 to 40°C, and the culture is carried out for about 10 to 160 hours, but is not limited thereto. The L-valine produced by the culture of the present invention is either secreted into the medium or remains intracellularly.

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

[0102] The method for producing L-valine of the present application may further include a step of recovering L-valine from the culture medium or the Corynebacterium glutamicum strain, which may be performed after the culturing step.

[0103] The target L-valine may be collected using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof can be used. The target L-valine can be collected from the medium or the microorganism using a suitable method known in the art.

[0104] The L-valine production method of the present application may further include a purification step. The purification can be performed by any suitable method known in the art. For example, when the L-valine production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, simultaneously, or integrated into a single step, but is not limited thereto.

[0105] In the method of the present application, the mutant ribonuclease activity-regulating protein, polynucleotide, vector, strain, etc. are as described above. Yet another aspect of the present application provides a composition for producing L-valine, comprising a mutant ribonuclease activity-regulating protein of the present application, a polynucleotide encoding the same, a vector containing the polynucleotide or a microorganism containing the polynucleotide of the present application, a culture medium in which the same has been cultured, or a combination of at least two of them.

[0106] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing L-amino acids, including, but not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, isotonicity agents, etc.

[0107] Yet another aspect of the present application provides use of the mutant ribonuclease activity-regulating protein of the present application, a polynucleotide encoding the same, a vector comprising the polynucleotide, or a microorganism comprising the polynucleotide of the present application in the production of L-valine.

[0108] The ribonuclease activity-regulating protein, mutant ribonuclease activity-regulating protein, polynucleotide, vector, strain, medium, L-valine, etc. are as described above. [Example]

[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 present application is not limited thereto. Note that technical matters not described in this specification are well understood and easily implemented by skilled artisans in the technical field of the present application or a similar technical field. [Example]

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

[0111] Example 1-2. Evaluation of L-valine production ability of mutagenized strains A fermentation titer experiment was conducted to select strains with improved L-valine production ability compared to the parent strain KCCM11201P from the strains randomly mutated in Example 1-1. Each colony was subcultured in nutrient medium, and then each strain was inoculated into a 250 ml corner-baffled flask containing 25 ml of production medium and cultured at 30°C and 200 rpm for 72 hours with shaking. The L-valine concentration was then evaluated using HPLC. The results are shown in Table 1.

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

[0113] [Table 1]

[0114] As shown in Table 1, the C10 strain was selected, which showed the greatest improvement in L-valine productivity compared to the parent strain KCCM11201P. [Example]

[0115] Confirmation of mutations by gene sequencing The major genes of the C10 strain selected in Example 1-2 were sequenced and compared with the parent strain KCCM11201P and the wild-type Corynebacterium glutamicum ATCC 14067. As a result, no mutations were found in the biosynthetic pathway genes directly related to L-valine production.

[0116] Next-generation sequencing (NGS) analysis confirmed the presence or absence of mutations at a specific position in the open reading frame (ORF) region of the rraA gene, which encodes the ribonuclease activity regulator protein (RraA). Specifically, compared with the parent strain KCCM11201P, the C10 strain contained a single mutation at a base located 277 bp upstream from the start codon of the rraA gene, resulting in a mutation from the original base sequence CAT (SEQ ID NO: 2) to TAT (SEQ ID NO: 4), and a substitution of the histidine at the 93rd amino acid residue from the N-terminus of the RraA protein with tyrosine, resulting in RraA(H93Y) (SEQ ID NO: 3). [Example]

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

[0118] The mutagenized fragment 1 obtained as described above was ligated to the pDCM2 vector (Patent Document 8) treated with the restriction enzyme SmaI using an infusion cloning kit (Takara Bio Inc.), and the resulting vector was transformed into Escherichia coli DH5α by electroporation (Non-Patent Document 19), inducing homologous recombination on the chromosome (Non-Patent Document 19). Strains in which the vector had been integrated into the chromosome through recombination of the homologous sequences were selected from LB medium containing 25 mg / L of kanamycin. DNA was obtained from the selected E. coli transformants using a DNA-spin plasmid DNA purification kit (Intron) according to the manufacturer's protocol, and the pDCM2-rraA(H93Y) vector containing the mutagenized fragment 1 was prepared for the introduction of the H93Y mutation into the rraA gene.

[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-expressing strains Example 4-1. Evaluation of L-valine production ability of mutant-expressing Corynebacterium glutamicum KCCM11201P strain To prepare an L-valine-producing strain carrying the H93Y mutation in the rraA gene, the pDCM2-rraA(H93Y) vector prepared in Example 3 was transformed into the L-valine-producing strain KCCM11201P by homologous recombination on the chromosome. Strains in which the vector had been integrated into the chromosome through recombination of the homologous sequences were selected from medium containing 25 mg / L kanamycin. After the secondary recombination, PCR was performed on the transformed Corynebacterium glutamicum strain using the primer pair of SEQ ID NO:5 and SEQ ID NO:6 to amplify the gene fragment. Subsequently, the strain carrying the H93Y mutation in the rraA gene was identified by gene sequence analysis. The recombinant strain was designated Corynebacterium glutamicum KCCM11201P::rraA(H93Y).

[0122] The L-valine-producing abilities of the parent strains KCCM11201P and KCCM11201P::rraA(H93Y) were 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-producing ability 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 mutant-expressing Corynebacterium glutamicum CJ7V strain To confirm whether the introduction of the H93Y mutation in the rraA gene also improves L-valine production in other strains of Corynebacterium glutamicum that produce L-valine, a single mutation [ilvN(A42V); Non-Patent Document 13] was introduced into the acetolactate synthase isozyme 1 small subunit (IlvN) protein of wild-type Corynebacterium glutamicum ATCC14067 to generate a strain with improved L-valine production (Patent Document 4).

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

[0126] The resulting mutagenized fragment 2 was digested with the restriction enzyme SmaI and then ligated with the pDCM2 vector digested with the same restriction enzyme. This vector was then transformed into E. coli DH5α by electroporation to induce homologous recombination. Strains in which the vector had been integrated into the chromosome via homologous recombination were selected from the selected E. coli transformants using a DNA-spin plasmid DNA purification kit according to the manufacturer's protocol. DNA was then isolated from the selected E. coli transformants using the DNA-spin plasmid DNA purification kit according to the manufacturer's protocol, creating the pDCM2-ilvN(A42V) vector containing mutagenized fragment 2 for the introduction of the A42V mutation into the ilvN gene.

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

[0128] [Table 4]

[0129] The pDCM2-ilvN(A42V) vector constructed as described above was transformed into wild-type Corynebacterium glutamicum ATCC14067 by homologous recombination on the chromosome. Strains in which the vector had been integrated into the chromosome via homologous recombination were selected from medium containing 25 mg / L kanamycin. After the secondary recombination, the Corynebacterium glutamicum transformed strains were subjected to PCR using the primer pair of SEQ ID NO: 7 and SEQ ID NO: 10 to amplify the gene fragment. Subsequently, the strains in which the A42V mutation had been introduced into the ilvN gene were identified by gene sequence analysis. The recombinant strain was designated Corynebacterium glutamicum CJ7V.

[0130] Finally, the pDCM2-rraA(H93Y) vector was transformed into the Corynebacterium glutamicum CJ7V in the same manner as in Example 4-1, and the recombinant strain was named Corynebacterium glutamicum CJ7V::rraA(H93Y).

[0131] The L-valine-producing abilities of the parent strains CJ7V and CJ7V::rraA(H93Y) were 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-producing ability 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 mutant-expressing Corynebacterium glutamicum CJ8V strain A single mutation [ilvN(A42V)] was introduced into the IlvN protein of wild-type Corynebacterium glutamicum ATCC13869 to create a strain with improved L-valine production ability (Patent Document 4).

[0134] The wild-type Corynebacterium glutamicum ATCC13869 strain was transformed with the pDCM2-ilvN(A42V) vector prepared in Example 4-2. Strains in which the vector had been integrated into the chromosome via homologous sequence recombination were selected from medium containing 25 mg / L kanamycin. After the secondary recombination, PCR was performed on the Corynebacterium glutamicum transformed strains using the primer pair of SEQ ID NOs: 11 and 12 to amplify the gene fragment. Subsequently, gene sequence analysis was performed to confirm that the A42V mutation had been introduced into the ilvN gene. The recombinant strain was designated Corynebacterium glutamicum CJ8V.

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

[0136] [Table 6]

[0137] Finally, the pDCM2-rraA(H93Y) vector was transformed into the Corynebacterium glutamicum CJ8V in the same manner as in Example 4-1, and the recombinant strain was named Corynebacterium glutamicum CJ8V::rraA(H93Y).

[0138] The L-valine-producing abilities of the parent strains CJ8V and CJ8V::rraA(H93Y) were 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-producing ability of the CJ8V::rraA(H93Y) strain was increased by 10% compared to the parent strain, CJ8V. From the above description, those skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing the technical idea or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present application should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalent concepts.

Claims

1. A mutant ribonuclease activity regulating protein in which the amino acid corresponding to position 93 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid.

2. The mutant ribonuclease activity-regulating protein according to claim 1, wherein the other amino acid is an amino acid selected from the group consisting of tyrosine, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

3. The mutant ribonuclease activity-regulating protein according to claim 1 , wherein the other amino acid is tyrosine.

4. The mutant ribonuclease activity-regulating protein according to claim 1, wherein the mutant ribonuclease activity-regulating protein has the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having 70% or more homology thereto.

5. A polynucleotide encoding the mutant ribonuclease activity-regulating protein according to any one of claims 1 to 4.

6. A microorganism comprising a mutant ribonuclease activity-regulating protein in which the amino acid corresponding to position 93 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid, or a polynucleotide encoding the same.

7. The microorganism according to claim 6, wherein the microorganism has improved L-valine-producing ability compared to a Corynebacterium microorganism containing a wild-type ribonuclease activity-regulating protein having the amino acid sequence of SEQ ID NO: 1 or a polynucleotide encoding the same.

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

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

10. A method for producing L-valine, comprising the step of culturing in a medium a microorganism containing a mutant ribonuclease activity-regulating protein in which the amino acid corresponding to position 93 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid, or a polynucleotide encoding the same.

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