Novel variant polypeptide having acetolactate synthase activity and method for producing l-glutamic acid using same

Abstract

The present application relates to a novel variant polypeptide having acetolactate synthase activity; a polynucleotide encoding the variant polypeptide; a microorganism comprising the variant polypeptide, the polynucleotide encoding the variant polypeptide, or a vector comprising the polynucleotide; a method for producing L-glutamic acid, said method comprising the step of culturing said microorganism in a culture medium; and the use of said microorganism for the production of L-glutamic acid.

Description

Technical Field

[0001] The present disclosure relates to a novel variant polypeptide having acetolactate synthase activity; a polynucleotide encoding the variant polypeptide; a microorganism comprising the variant polypeptide, the polynucleotide encoding the variant polypeptide, or a vector comprising the polynucleotide; a method for producing L-glutamic acid, the method comprising the step of culturing the microorganism in a culture medium; and use of the microorganism for producing L-glutamic acid. Background Art

[0002] Glutamic acid is a representative amino acid produced by fermentation. It has a unique and distinctive taste and is one of the important amino acids widely used in the food field as well as in the medical field and other animal feed fields.

[0003] The common method of producing glutamic acid comprises using Brevibacterium (Brevibacterium) or Corynebacterium (Corynebacterium) microorganism and the rod-shaped bacteria comprising its mutant strain to produce (Amino Acid Fermentation, Gakkai Shuppan Center:195-215,1986) by fermentation.In addition, the method using microorganisms such as intestinal bacteria (Escherichia coli) or Bacillus (Bacillus), Streptomyces (Streptomyces), Penicillium (Penicillum), Klebsiella (Klebsiella), Erwinia (Erwinia), Pantoea (Pantoea) is known (U.S. Patent Publication No. 3220929 and U.S. Patent No. 6682912).

[0004] In addition, carried out the various research of effectively producing amino acid whose, for example, effort is developed for efficient production of amino acid whose microorganism or fermentation process.Particularly, developed target specific method, as the method for the expression of the gene of the enzyme that increases coding participation amino acid biosynthesis or the method (Korean Patent No. 10-0924065 and 10-1208480) of removing amino acid biosynthesis unwanted gene in Corynebacterium bacterial strain.In addition, also used the method for removing the gene that does not participate in amino acid production and the method for removing the gene that unknown its specific function is relevant with amino acid production.But still need to study the method for effectively producing L-amino acid with high yield. Summary of the Invention

[0005]

Technical Issues

[0006] An object of the present disclosure is to provide a microorganism capable of producing L-glutamic acid at a high yield compared to a microorganism having an existing unmodified polypeptide by culturing a microorganism comprising a variant polypeptide having acetolactate synthase activity, and a method for producing L-glutamic acid using the microorganism.

[0007]

Technical solution

[0008] One aspect of the present disclosure provides a variant polypeptide having acetolactate synthase activity, wherein the amino acid corresponding to position 298 of SEQ ID NO: 1 is substituted with a different amino acid.

[0009] In a specific embodiment, the variant polypeptide may have the amino acid corresponding to position 298 of SEQ ID NO: 1 substituted with valine.

[0010] In another specific embodiment, the variant polypeptide may consist of the amino acid sequence of SEQ ID NO:3.

[0011] Another aspect of the present disclosure provides a polynucleotide encoding the variant polypeptide.

[0012] Another aspect of the present disclosure provides a microorganism comprising the variant polypeptide, the polynucleotide encoding the variant polypeptide, or a vector comprising the polynucleotide.

[0013] In a specific embodiment, the microorganism may have increased L-glutamic acid-producing ability compared to a microorganism comprising the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same.

[0014] Regarding the microorganism according to any one of the above-mentioned specific embodiments, the microorganism may be a microorganism of the genus Corynebacterium.

[0015] Regarding the microorganism according to any one of the above-mentioned specific embodiments, the Corynebacterium microorganism may be Corynebacterium glutamicum.

[0016] Another aspect of the present disclosure provides a method for producing L-glutamic acid, the method comprising the step of culturing the microorganism in a culture medium.

[0017] In a specific embodiment, the method may further comprise the step of recovering the target substance from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium.

[0018] Another aspect of the present disclosure provides a composition for producing L-glutamic acid, which comprises the variant polypeptide; the polynucleotide encoding the variant polypeptide; a vector comprising the polynucleotide; a microorganism comprising the variant polypeptide, the polynucleotide encoding the variant polypeptide, or the vector comprising the polynucleotide; a culture of the microorganism; or a combination of two or more thereof.

[0019] Another aspect of the present disclosure provides use of the microorganism for producing L-glutamic acid.

[0020] Beneficial effects

[0021] When a microorganism comprising the variant polypeptide having acetolactate synthase activity of the present disclosure is cultured, L-glutamic acid can be produced at a high yield compared to a microorganism comprising an existing unmodified polypeptide. DETAILED DESCRIPTION

[0022] The present disclosure will be described in detail below. At the same time, each description and embodiment disclosed in the present disclosure can also be applied to other descriptions and embodiments. In other words, all combinations of the various elements disclosed in the present disclosure fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific description described below. In addition, many papers and patent documents are referenced and cited throughout the specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to further illustrate the level and scope of the subject matter to which the present disclosure belongs.

[0023] One aspect of the present disclosure provides a variant polypeptide having acetolactate synthase activity, wherein the amino acid corresponding to position 298 of SEQ ID NO: 1 is substituted with a different amino acid.

[0024] As used herein, the term "variant polypeptide having acetolactate synthase activity" refers to a variant polypeptide having acetolactate synthase activity comprising one or more amino acid substitutions in the amino acid sequence of a polypeptide having acetolactate synthase activity; or a variant polypeptide having acetolactate synthase activity comprising one or more amino acid substitutions in the parent sequence (the amino acid sequence of a polypeptide having acetolactate synthase activity).

[0025] As used herein, the term "acetolactate synthase (ilvB)" is an enzyme that produces acetolactate using pyruvate as a substrate, and may be used interchangeably with terms such as "acetohydroxyacid synthase" and "acetohydroxyacid synthase large subunit", and may be, but is not limited to, acetolactate synthase (ilvB) encoded by the ilvB gene.

[0026] The gene encoding acetolactate synthase may be derived from a microorganism of the genus Corynebacterium, specifically, it may be ilvB derived from Corynebacterium glutamicum, but is not limited thereto.

[0027] Specifically, examples of acetolactate synthase proteins may include the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence with 60% or higher homology or identity thereto, but are not limited thereto, as long as it has acetolactate synthase activity. Specifically, the amino acid sequence may include SEQ ID NO: 1 or an amino acid sequence with at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher homology or identity thereto. The sequence of SEQ ID NO: 1 can be obtained from GenBank of the known database NCBI or the Kyoto Gene and Genome Database (KEGG). For example, the sequence may be derived from Corynebacterium or Corynebacterium glutamicum, and more specifically, it may be a polypeptide / protein comprising the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. It is also obvious that accessory proteins having amino acid sequences with deletions, modifications, substitutions or additions of certain sequences also fall within the scope of the present disclosure, as long as the amino acid sequences have such homology or identity and exhibit the corresponding efficacy of the protein.

[0028] In addition, the acetolactate synthase protein having the amino acid sequence of SEQ ID NO: 1 can be encoded by a polynucleotide having or including the sequence of SEQ ID NO: 2, or a nucleotide sequence having 60% or more, 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 to the sequence of SEQ ID NO: 2, or consisting of or essentially consisting of the sequence of SEQ ID NO: 2, or a nucleotide sequence having 60% or more, 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 to the sequence of SEQ ID NO: 2, but is not limited thereto.

[0029] As used herein, the term "variant" refers to a polypeptide in which one or more amino acids are different from the amino acids of the amino acid sequence before being conservatively replaced and / or modified, but whose function or property is maintained. Such variants can generally be identified by modifying one or more amino acids of the polypeptide or protein amino acid sequence and evaluating the properties of the modified polypeptide. In other words, the ability of the variant can be increased, unchanged or reduced compared to the polypeptide before the change. In addition, some variants can include variants in which one or more parts, such as N-terminal leader sequences or transmembrane domains have been removed. Other variants can include variants in which a portion of the N- and / or C-termini have been removed from the mature protein. The term "variant" can be used interchangeably with terms such as modification, modified polypeptide, modified protein, mutant, mutant protein and disproportionate (divergent), and is not limited thereto, as long as it is a term with variant meaning.

[0030] Furthermore, variants may include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated co-translationally or post-translationally to a protein N-terminal signal (or leader) sequence that participates in protein transfer. Furthermore, the polypeptide may be conjugated to other sequences or linkers to identify, purify, or synthesize the polypeptide.

[0031] The variant polypeptide having acetolactate synthase activity disclosed herein may be a variant polypeptide having acetolactate synthase activity in which the amino acid corresponding to position 298 of SEQ ID NO: 1 is substituted with a different amino acid, but is not limited thereto.

[0032] In one embodiment, the variant polypeptide having acetolactate synthase activity disclosed herein may have a sequence homology of 60% or more and less than 100% with the amino acid sequence of SEQ ID NO: 1, specifically, 80% or more and less than 100%, but is not limited thereto.

[0033] Specifically, the variants of the present disclosure may include substitution of the amino acid at position 298 of the N-terminus of SEQ ID NO: 1 with a different amino acid in an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or higher homology or identity with the amino acid sequence of SEQ ID NO: 1. It is also obvious that variants of amino acid sequences having deletions, modifications, substitutions, conservative substitutions or additions of some sequences also fall within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits the corresponding efficacy of the variants of the present disclosure.

[0034] There is no limitation on the “different amino acid” as long as it is different from the amino acid before substitution. Meanwhile, in the present disclosure, when it is expressed that “a specific amino acid is substituted”, it is obvious that the amino acid is substituted with an amino acid different from the amino acid before substitution, even if it is not separately indicated that the amino acid is substituted with a different amino acid.

[0035] Amino acids can generally be classified based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0036] Examples of such classifications can include positively charged (basic) amino acids such as arginine, lysine, and histidine; negatively charged (acidic) amino acids such as glutamic acid and aspartic acid; amino acids with non-polar side chains (non-polar amino acids) such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; amino acids with polar or hydrophilic side chains (polar amino acids) such as serine, threonine, cysteine, tyrosine, asparagine, and glutamine. For another example, amino acids can be divided into amino acids with charged side chains (charged amino acids) such as arginine, lysine, histidine, glutamic acid, and aspartic acid, and amino acids with uncharged side chains (uncharged amino acids; also called neutral amino acids) such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. For another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. For another example, valine, leucine, and isoleucine can be classified as branched-chain amino acids. For another example, the 20 amino acids are classified according to size. Starting from the relatively small amino acid group, amino acids can be divided into five groups: glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, and tyrosine. However, they are not necessarily limited thereto.

[0037] For example, when it is described that "the amino acid corresponding to position 298 of SEQ ID NO: 1 is replaced by a different amino acid", it means that the amino acid is replaced by valine, glycine, isoleucine, glutamic acid (glutamate), phenylalanine, arginine, aspartic acid, cysteine, asparagine, glutamine, histidine, proline, serine, tyrosine, lysine, tryptophan, methionine, threonine or leucine, excluding alanine, but is not limited thereto.

[0038] As used herein, although the expression "a protein having an amino acid sequence described by a specific sequence number" is used, it is obvious that any protein having an amino acid sequence comprising a deletion, modification, substitution, conservative substitution or addition of a certain sequence can also be used in the present disclosure, as long as the protein has the same or equivalent activity as the protein consisting of the amino acid sequence of the corresponding sequence number. Examples thereof do not exclude the addition of a sequence that does not change the function of the protein, a possible naturally occurring mutation, a silent mutation or a conservative substitution thereof upstream or downstream of the amino acid sequence, as long as it has the same or corresponding activity as the variant protein, and it is obvious that even proteins having such sequence additions or mutations fall within the scope of the present disclosure.

[0039] The "N position" disclosed herein may include the N position and the amino acid position corresponding to the N position. Specifically, the N position may include the amino acid position corresponding to any amino acid residue in the mature polypeptide disclosed in the specific amino acid sequence. The specific amino acid sequence may be the amino acid sequence of SEQ ID NO: 1.

[0040] As used herein, the term "corresponding to" refers to an amino acid residue at a listed position in a polypeptide, or an amino acid residue that is similar, identical, or homologous to a listed residue in a polypeptide. Identifying an amino acid at a corresponding position can be determining a specific amino acid in a sequence with reference to a specific sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or a reference protein.

[0041] For example, an arbitrary amino acid sequence is aligned with SEQ ID NO: 1, and on this basis, each amino acid residue in the amino acid sequence can be numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, the sequence alignment algorithms described in the present disclosure can determine the position of an amino acid or the position of a modification such as a substitution, insertion or deletion by comparing the position of the amino acid in a query sequence (also referred to as a "reference sequence").

[0042] For this alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needleman program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277), etc. can be used, but are not limited thereto, and sequence alignment programs, pairwise sequence alignment algorithms, etc. known in the art can be appropriately used.

[0043] In a specific embodiment, the variant polypeptide may have the amino acid corresponding to position 298 of SEQ ID NO: 1 replaced by an amino acid selected from the group consisting of valine, glycine, isoleucine, arginine, leucine, methionine, threonine, asparagine, glutamine, proline, serine, tryptophan, phenylalanine, histidine, cysteine, tyrosine, lysine, aspartic acid and glutamic acid, but is not limited thereto.

[0044] In one embodiment of the above embodiments, the variant polypeptide provided by the present disclosure may include the amino acid at position 298 of the N-terminus corresponding to SEQ ID NO: 1 being replaced by an amino acid selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline (which are amino acids with non-polar side chains (non-polar amino acids)).

[0045] In one embodiment of the above embodiments, the variant polypeptide provided by the present disclosure may include an amino acid corresponding to position 298 at the N-terminus of SEQ ID NO: 1 replaced by an amino acid selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine and glutamine (which are amino acids with uncharged side chains (uncharged amino acids; also known as neutral amino acids)).

[0046] In one embodiment of the above embodiments, the variant polypeptide provided by the present disclosure may include the amino acid at position 298 corresponding to the N-terminus of SEQ ID NO: 1 being replaced by an amino acid selected from valine, leucine and isoleucine (which are branched-chain amino acids).

[0047] In one embodiment of the above embodiments, the variant polypeptide provided by the present disclosure may include the amino acid at position 298 corresponding to the N-terminus of SEQ ID NO: 1 being replaced by an amino acid selected from valine, histidine, glutamic acid and glutamine.

[0048] In one embodiment of the above embodiment, the variant polypeptide having acetolactate synthase activity of the present disclosure may be a polypeptide in which the amino acid corresponding to position 298 of SEQ ID NO: 1 is substituted with valine, but is not limited thereto.

[0049] For example, the variant polypeptides of the present disclosure may include an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or higher homology or identity with SEQ ID NO: 1, provided that the amino acid valine at position 298 in the amino acid sequence corresponding to SEQ ID NO: 1 is fixed. In addition, it is obvious that variant polypeptides having amino acid sequences with deletions, modifications, substitutions, conservative substitutions or additions of some sequences also fall within the scope of the present disclosure, as long as the amino acid sequence has the corresponding efficacy of the variant polypeptides of the present disclosure and has such homology or identity.

[0050] At the same time, those skilled in the art can identify the amino acid corresponding to position 298 of the amino acid sequence of SEQ ID NO: 1 of the present disclosure in any polynucleotide sequence by sequence alignment known in the art. Unless otherwise described in the present disclosure, when describing the "amino acid at a specific position of a specific sequence number", it is obvious that the amino acid also includes the "amino acid at the corresponding position" in any amino acid sequence.

[0051] In another specific embodiment, the variant polypeptide may consist of the amino acid sequence of SEQ ID NO:3.

[0052] Specifically, the variant polypeptides of the present disclosure may have or include SEQ ID NO: 3, or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more homology or identity to SEQ ID NO: 3, or may consist of or may consist essentially of the amino acid sequence.

[0053] Examples include those having sequence additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or within the amino acid sequence that do not alter the function of the disclosed variants.

[0054] "Conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitutions can generally occur based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic properties of the residues. For example, positively charged (basic) amino acids include arginine, lysine and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan and tyrosine; hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan. In addition, amino acids can also be divided into amino acids with charged side chains and amino acids with uncharged side chains. Amino acids with charged side chains include aspartic acid, glutamic acid, lysine, arginine and histidine, and amino acids with uncharged side chains can also be divided into non-polar amino acids or polar amino acids. Non-polar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, and polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Generally, conservative substitutions may have little or no effect on the activity of the resulting polypeptide. Generally, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

[0055] Another aspect of the present disclosure provides a polynucleotide encoding the variant polypeptide.

[0056] As used herein, the term "polynucleotide" refers to a DNA or RNA chain of a certain length or longer that is a polymer of nucleotides, in which nucleotide monomers are linked into the long chain by covalent bonds; more specifically, it refers to a polynucleotide fragment encoding a variant.

[0057] The polynucleotide encoding the variant polypeptide having acetolactate synthase activity of the present invention may include any polynucleotide sequence without limitation, as long as it encodes the variant polypeptide having acetolactate synthase activity of the present invention. For example, the polynucleotide encoding the variant polypeptide having acetolactate synthase activity of the present invention may be a polynucleotide sequence encoding the amino acid sequence of the variant polypeptide having acetolactate synthase activity of the present invention, but is not limited thereto.

[0058] For example, the polynucleotide may include a nucleotide sequence encoding the amino acid sequence described in SEQ ID NO: 3. For one example of the present disclosure, the polynucleotide of the present disclosure may have or include SEQ ID NO: 4. In addition, the polynucleotide of the present disclosure may consist of or consist essentially of SEQ ID NO: 4.

[0059] Taking into account the degeneracy of codons or the preferred codons in the organism in which the variants of the present invention are intended to be expressed, various modifications can be made to the coding regions of the polynucleotides of the present invention without changing the amino acid sequence of the variants of the present invention. Therefore, it is apparent that due to the degeneracy of codons, polynucleotides that are translated into polypeptides consisting of the amino acid sequence of the variants of the present invention or polypeptides having homology or identity thereto can also be included. For example, the polynucleotide of the present invention can be SEQ ID NO: 4 or a degenerate sequence thereof.

[0060] For example, the polynucleotides of the present disclosure may include, but are not limited to, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7% or 99.9% or higher homology or identity to the sequence of SEQ ID NO: 2, wherein the codon encoding alanine (which is the amino acid corresponding to position 893 of SEQ ID NO: 2) is replaced by a codon encoding an amino acid other than alanine, such as valine. It is also obvious that variants of the polynucleotide sequence having deletions, modifications, substitutions, conservative substitutions or additions of some sequences also fall within the scope of the present disclosure, as long as the polynucleotide sequence has such homology or identity and encodes the amino acid sequence of the variant polypeptide having acetolactate synthase activity of the present disclosure.

[0061] For another example, the polynucleotide of the present disclosure may have or include a nucleotide sequence having 60% or more, 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 to SEQ ID NO: 4, or may consist of or consist essentially of a nucleotide sequence having 60% or more, 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 to SEQ ID NO: 4, but is not limited thereto. Alternatively, the sequence having the above homology or identity may be a sequence in which the codon corresponding to position 298 of SEQ ID NO: 3 encoded by SEQ ID NO: 4 is fixed to a codon encoding valine.

[0062] In addition, the polynucleotides of the present disclosure may include probes prepared from known gene sequences, and may include any sequence without limitation, as long as it is a sequence that can hybridize under stringent conditions with a complementary sequence of all or part of the polynucleotide sequence of the present disclosure.

[0063] "Stringent conditions" refer to conditions that allow specific hybridization between polynucleotides. These conditions are described in detail in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). Examples include conditions in which polynucleotides with higher homology or identity, i.e., polynucleotides with homology or identity of 60% or more, 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, whereas polynucleotides with lower homology or identity do not hybridize with each other, or conditions in which washing is performed once, particularly two to three times, at a salt concentration and temperature equivalent to 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. These are general washing conditions for Southern hybridization.

[0064] Hybridization requires that the two nucleic acids have complementary sequences, although mismatches between bases are tolerated depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of the present disclosure may also include substantially similar nucleic acid sequences and isolated nucleic acid fragments that are complementary to the entire sequence.

[0065] Specifically, hybridization conditions including a hybridization step with a Tm value of 55° C. and the above conditions can be used to detect polynucleotides having homology or identity with the polynucleotides of the present disclosure. In addition, the Tm value can be 60° C., 63° C., or 65° C., but is not limited thereto, and can be appropriately adjusted by those skilled in the art according to the purpose.

[0066] The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotides and the degree of complementation, and these variables are well known in the art (eg, J. Sambrook et al., supra).

[0067] As used herein, the term "homology" or "identity" refers to the relatedness between two given amino acid sequences or nucleotide sequences and can be expressed as a percentage. The terms "homology" and "identity" are often used interchangeably.

[0068] The sequence homology or identity of conserved polynucleotides or polypeptides are determined by standard comparison algorithms and can be used together with the default gap penalty set up by the used program. Basically, homology or identical sequences can hybridize with all or part of this sequence under medium or high stringency conditions usually. Obviously, hybridization also includes polynucleotides and polynucleotide hybridizations that contain universal codons or codons that take into account codon degeneracy.

[0069] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined using known computer algorithms (such as the "FASTA" program), for example using the default parameters in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) can be used, which is implemented as follows: the Needleman program (version 5.0.0 or later) in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (including the GCG program package ((Devereux, J. et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [et al., J. Molec. Biol. 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .] (1988) SIAM J Applied Math 48:1073). For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0070] Homology, similarity or identity between polynucleotides or polypeptides can be determined by comparing sequence information using the GAP computer program, for example, as described by Needleman et al. (1970), J Mol Biol. 48: 443 and as disclosed by Smith and Waterman, Adv. Appl. Math (1981) 2: 482. Briefly, the GAP program defines homology, similarity or identity as the value obtained by dividing the number of similar aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. The default parameters of the GAP program may include: (1) a binary comparison matrix (including a value of 1 for identity and a value of 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, as disclosed in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, the terms "homology" or "identity" as used herein refer to the relatedness between sequences.

[0071] Another aspect of the present disclosure provides a vector comprising the polynucleotide of the present disclosure. The vector may be an expression vector for expressing the polynucleotide in a microorganism, but is not limited thereto.

[0072] As used herein, the term "vector" may include a DNA construct comprising a nucleotide sequence encoding a polynucleotide of a polypeptide of interest, which is operably linked to a suitable expression regulatory region (or expression control sequence) such that the polypeptide of interest can be expressed in a suitable host. The expression regulatory region may comprise a promoter capable of initiating transcription, any operator sequence controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling transcription and translation termination. The vector may be transformed into a suitable microorganism and then replicated or functioned independently of the host genome, or may be integrated into the genome itself.

[0073] The vectors used in the present disclosure are not particularly limited, but any vector known in the art can be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as phage vectors or cosmid vectors; pDZ system, pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0074] For example, the polynucleotide encoding target polypeptide can be inserted into the chromosome by the vector for the intracellular chromosome insertion. The polynucleotide insertion chromosome can be carried out by any method known in the art, for example homologous recombination, but is not limited thereto. The carrier can also comprise a selection marker for confirming that the chromosome inserts. The selection marker is used to select the cell transformed by the carrier, i.e., for confirming the insertion of the target nucleic acid molecule, and can use the mark that gives the selectable phenotype (such as drug resistance, auxotrophy, cytotoxic agent resistance or surface polypeptide expression). In the environment of processing with the selection agent, only have the cell survival of expressing the selection marker or show other phenotypic traits, and therefore can select the cell for conversion.

[0075] As used herein, the term "transformation" refers to the introduction of a vector comprising a polynucleotide encoding a target polypeptide into a microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the microorganism. The position of the converted polynucleotide can be inserted into the chromosome of the microorganism or located outside the chromosome regardless of the site, as long as it can be expressed in the microorganism. In addition, the polynucleotide comprises DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form, as long as it can be introduced into the microorganism and expressed. For example, the polynucleotide can be introduced into the microorganism in the form of an expression cassette, which is a genetic construct containing all the elements required for autonomous expression. The expression cassette can generally comprise a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette can be in the form of an expression vector capable of self-replication. In addition, the polynucleotide can be introduced into the microorganism in its own form and operably linked to the sequence required for expression in the microorganism, but is not limited thereto.

[0076] Furthermore, the term "operably linked" means that a polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the desired variant of the present disclosure.

[0077] Another aspect of the present disclosure provides a microorganism comprising the variant polypeptide, the polynucleotide encoding the variant polypeptide, or a vector comprising the polynucleotide.

[0078] In a specific embodiment, the microorganism of the present disclosure may be a microorganism having the ability to produce L-glutamic acid.

[0079] As used herein, term " microorganism (or strain) " comprises all wild-type microorganisms or natural or artificial genetically modified microorganisms, and it can be due to the insertion of exogenous genes or the activity enhancement or inactivation of endogenous genes and the microorganism that specific mechanism is weakened or strengthened, and it can be the microorganism that comprises the genetic modification for producing required polypeptide, protein or product.As used herein, " microorganism " and " strain " have identical meaning, and can be used interchangeably without restriction.

[0080] As used herein, the term "L-glutamic acid-producing microorganism" refers to a prokaryotic or eukaryotic microorganism capable of producing L-glutamic acid in a living organism, and may include microorganisms prepared by providing L-glutamic acid-producing ability to a parent strain that does not have the ability to produce L-glutamic acid, or microorganisms inherently having the ability to produce L-glutamic acid. The ability to produce L-glutamic acid can be imparted or enhanced through species improvement.

[0081] In one embodiment, the microorganism of the present invention may be a microorganism that naturally has a variant polypeptide having acetolactate synthase activity or the ability to produce L-glutamate; or a microorganism in which a variant of the present invention or a polynucleotide encoding the same (or a vector comprising the polynucleotide) is introduced into a parent strain that does not have a variant polypeptide having acetolactate synthase activity or the ability to produce L-glutamate and / or provides the parent strain with the ability to produce L-glutamate, but is not limited thereto.

[0082] In one embodiment, the microorganisms of the present invention may include all of the following: microorganisms comprising a variant polypeptide sequence having acetolactate synthase activity of the present invention due to a mutation of a gene encoding a variant polypeptide having acetolactate synthase activity on a chromosome, and / or microorganisms comprising a variant polypeptide having acetolactate synthase activity of the present invention by introducing a vector comprising a polynucleotide encoding a variant polypeptide having acetolactate synthase activity of the present invention, but are not limited thereto.

[0083] As used herein, the term "unmodified microorganism" does not exclude a strain comprising mutations that can occur naturally in the microorganism, and can be a wild-type strain or a native strain itself, or can be a strain before the change in traits due to genetic variation caused by natural or artificial factors. For example, the unmodified microorganism can be a strain into which or from which a variant polypeptide having acetolactate synthase activity described herein has not been introduced. The term "unmodified microorganism" can be used interchangeably with "strain before modification", "microorganism before modification", "unchanged strain", "unmodified strain", "unchanged microorganism", or "reference microorganism".

[0084] The microorganism having L-glutamic acid production ability of the present disclosure can be a microorganism comprising any one or more of the variant of the present disclosure, the polynucleotide of the present disclosure, and the vector comprising the polynucleotide of the present disclosure; a microorganism modified to express the variant of the present disclosure or the polynucleotide of the present disclosure; a microorganism (e.g., a recombinant strain) expressing the variant of the present disclosure or the polynucleotide of the present disclosure; or a microorganism (e.g., a recombinant strain) having the activity of the variant of the present disclosure, but is not limited thereto.

[0085] For example, the strain of the present disclosure is a cell or microorganism transformed with the polynucleotide of the present disclosure or a vector comprising a polynucleotide encoding the variant of the present disclosure to express the variant of the present disclosure, and the strain of the present disclosure can include any microorganism capable of producing L-glutamic acid by comprising the variant of the present disclosure. For example, the microorganism of the present disclosure can be a recombinant strain having increased L-glutamic acid production ability due to expression of a variant polypeptide having acetolactate synthase activity by introducing a polynucleotide encoding the variant of the present disclosure into a natural wild-type microorganism or a microorganism having L-glutamic acid production ability. The recombinant strain having increased L-glutamic acid production ability can be a microorganism in which the L-glutamic acid production ability is increased compared to a natural wild-type microorganism or an acetolactate synthase-unmodified microorganism (e.g., a microorganism expressing a wild-type acetolactate synthase or a microorganism not expressing the variant of the present disclosure), but is not limited thereto. For example, the microorganism of the present disclosure having increased L-glutamic acid production ability can be a microorganism in which the L-glutamic acid production ability is enhanced compared to a microorganism comprising a polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same, but is not limited thereto. For example, the unmodified microorganism, which is a target strain for comparison of whether the L-glutamic acid production ability is increased, can be a wild-type C. glutamicum strain, an ATCC13869 or ATCC13032 strain; or a glutamic acid-producing strain, a KFCC11074 strain (KR 10-0292299B1), but is not limited thereto.

[0086] The microorganisms disclosed herein may include any microorganisms capable of expressing the variant polypeptide having acetolactate synthase activity disclosed herein by introducing the nucleotide or vector and other various known methods.

[0087] For example, the microorganism with increased L-glutamic acid productivity can have an L-glutamic acid productivity increased by about 1% or more, specifically, about 1% or more, about 2% or more, about 3% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, or about 46% or more (the upper limit is not particularly limited, but can be, for example, about 200% or less, about 150% or less, about 100% or less, about 90% or less, about 80% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, or about 50% or less), compared to the parent strain before modification or the unmodified microorganism, but the amount of increase is not limited thereto, as long as the productivity has an increase of a positive value compared to the productivity of the parent strain before modification or the unmodified microorganism. In another example, the recombinant strain with increased L-glutamic acid-producing ability may have an L-glutamic acid-producing ability of about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, about 1.05 times or more, about 1.1 times or more, about 1.15 times or more, about 1.20 times or more, about 1.25 times or more, about 1.30 times or more, about 1.35 times or more, about 1.40 times or more, about 1.45 times or more, about 1.46 times or more (the upper limit is not particularly limited, but may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, or 1.5 times or less), as compared to the parent strain or unmodified microorganism before modification, but is not limited thereto. As used herein, the term "about" is intended to include all ranges of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values ​​equivalent to or similar to the value following the term "about", but the scope is not limited thereto.

[0088] Regarding the microorganism according to any of the above-mentioned specific embodiments, the microorganism of the present disclosure can be a microorganism of the genus Corynebacteria sp., Escherichia sp., Erwinia sp., Serratia sp., Providencia sp., Pseudomonas sp., Leptospira sp., Salmonella sp., Brevibacteria sp., Hypomononas sp., Chromobacterium sp. and Nocardia sp., fungi or yeast, in particular, a microorganism of the genus Corynebacterium, but not limited thereto.

[0089] For examples of the present disclosure, the microorganism of the present disclosure can be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris or Corynebacterium flavescens. Specifically, the microorganism of the present disclosure may be a microorganism of the genus Corynebacterium, more specifically, Corynebacterium glutamicum, but is not limited thereto.

[0090] Meanwhile, the microorganisms having L-glutamic acid-producing ability disclosed herein may include all of the following: natural wild-type microorganisms themselves, microorganisms having improved L-glutamic acid-producing ability by enhancing or weakening the activity of polypeptides related to the L-glutamic acid production mechanism, and microorganisms having improved L-glutamic acid-producing ability by introducing or enhancing the activity of exogenous polypeptides.

[0091] Meanwhile, it is known that Corynebacterium microorganisms can produce L-glutamic acid, but their production capacity is very low, and the genes or mechanisms responsible for this production mechanism have not yet been revealed. Therefore, the Corynebacterium microorganisms having L-glutamic acid production capacity disclosed herein may include all of the following: naturally occurring wild-type microorganisms themselves, Corynebacterium microorganisms having improved L-glutamic acid production capacity by enhancing or weakening the activity of polypeptides related to the L-glutamic acid production mechanism, and Corynebacterium microorganisms having improved L-glutamic acid production capacity by introducing or enhancing the activity of exogenous polypeptides.

[0092] As used herein, the term "increase (enhancement)" of polypeptide activity refers to an increase in the activity of a polypeptide compared to its endogenous activity. The increase can be used interchangeably with terms such as activation, upregulation, overexpression, and enhancement.

[0093] The increase may include expressing an activity that is not originally present, and expressing an activity that is improved compared to the endogenous activity or the activity before modification.

[0094] For example, "expressing an activity not originally possessed" can refer to "introducing a protein," but is not limited thereto. Protein introduction means that a gene not originally possessed by a microorganism is expressed in the microorganism, thereby expressing the activity of a specific protein or exhibiting an increased or improved activity compared to the endogenous activity or the activity of the corresponding protein before modification. For example, a polynucleotide encoding a specific protein can be introduced into the chromosome of the microorganism, or a vector containing a polynucleotide encoding a specific protein can be introduced into the microorganism, thereby expressing the activity.

[0095] "Endogenous activity" refers to the activity of a specific polypeptide originally present in the parent strain or unmodified microorganism before the trait is altered due to genetic variation caused by natural or artificial factors. This can be used interchangeably with "activity before modification."

[0096] The fact that the activity of a polypeptide is "increased" compared to the endogenous activity means that the activity of the polypeptide is improved compared to the activity and / or concentration (expression level) of the specific polypeptide originally in the parent strain or unmodified microorganism before the trait change.

[0097] For example, the increase means that the activity of the corresponding protein that was not originally present appears, or its activity or concentration is generally increased by about 1%, about 10%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%, up to about 1000% or about 2000% or more based on the activity or concentration in the wild-type protein or the initial microbial strain, but is not limited thereto.

[0098] Increased polypeptide activity can be achieved by introducing an exogenous polypeptide or increasing the activity of an endogenous polypeptide. Increased polypeptide activity can be confirmed by an increase in the activity level and expression level of the corresponding polypeptide or an increase in the amount of a product produced by the corresponding polypeptide.

[0099] In order to enhance the activity of polypeptides, various methods well known in the art can be applied. As long as the activity of the target polypeptide can be enhanced compared to the microorganism before modification, the method is not limited. Specifically, genetic engineering and / or protein engineering well known to those skilled in the art can be used. This is a conventional method of molecular biology, but the method is not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2, 1-16, Sambrook et al., Molecular Cloning 2012, etc.).

[0100] Specifically, increasing the activity of the polypeptide of the present disclosure may be:

[0101] 1) increasing the copy number of the polynucleotide encoding the polypeptide in the cell;

[0102] 2) modifying the gene expression regulatory region on the chromosome encoding the polypeptide (e.g., mutating the expression regulatory region, replacing it with a more active sequence, or inserting a more active sequence);

[0103] 3) modifying the nucleotide sequence encoding the start codon or the 5'-UTR region of the gene transcript encoding the polypeptide;

[0104] 4) Modifying the amino acid sequence of a polypeptide to enhance its activity;

[0105] 5) Modifying a polynucleotide sequence encoding a polypeptide to enhance the activity of the polypeptide (e.g., modifying the polynucleotide sequence of a polypeptide gene to encode a modified polypeptide to enhance the activity of the polypeptide);

[0106] 6) introducing an exogenous polypeptide expressing the polypeptide activity or an exogenous polynucleotide encoding the polypeptide;

[0107] 7) codon optimization of the polynucleotide encoding the polypeptide;

[0108] 8) analyzing the tertiary structure of the polypeptide to select and modify or chemically modify exposed sites; or

[0109] 9) A combination of two or more selected from 1) to 8), but not particularly limited thereto.

[0110] For example, 1) increasing the copy number of a polynucleotide encoding a polypeptide in a cell can be achieved by introducing a vector into a host cell, wherein the vector can replicate and function independently of the host, and the polynucleotide encoding the corresponding polypeptide is operably linked to the vector. Alternatively, such an increase can be achieved by introducing one copy or two or more copies of the polynucleotide encoding the corresponding polypeptide into the chromosome of the host cell. Introduction into the chromosome can be carried out by introducing a vector capable of inserting the polynucleotide into the host cell chromosome, but is not limited thereto. The vector is as described above.

[0111] 2) Replacing the gene expression regulatory region (or expression control sequence) on the chromosome encoding the polypeptide with a sequence exhibiting stronger activity can be, for example, by causing a variation in the sequence due to deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by replacing it with a sequence exhibiting stronger activity, thereby further enhancing the activity of the expression regulatory region. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence controlling transcription and translation termination, and the like. For example, the replacement may be replacing the original promoter with a stronger promoter, but is not limited thereto.

[0112] Examples of known strong promoters include cj1 to cj7 promoters (U.S. 7662943B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (U.S. 10584338B2), O2 promoter (U.S. 10273491B2), tkt promoter, yccA promoter, etc., but are not limited thereto.

[0113] 3) Modification of the nucleotide sequence of the start codon or 5'-UTR region of the gene encoding the polypeptide may be, for example, replacement with another start codon having a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0114] 4) and 5) modification of the amino acid sequence or the polynucleotide sequence can be a variation in the sequence due to deletion, insertion, non-conservative substitution or conservative substitution of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, or replacement with an amino acid sequence or a polynucleotide sequence improved to exhibit stronger activity, or replacement with an amino acid sequence or a polynucleotide sequence improved to be more active, thereby enhancing the activity of the polypeptide, but is not limited thereto. The replacement can be specifically performed by inserting the polynucleotide into the chromosome through homologous recombination, but is not limited thereto. The vector used herein can further include a selection marker for confirming the insertion of the chromosome. The selection marker is as described above.

[0115] 6) introduction of an exogenous polynucleotide exhibiting the activity of the polypeptide can be introduction of an exogenous polynucleotide encoding a polypeptide exhibiting the same or similar activity as the polypeptide into a host cell. There is no limitation on the origin or sequence thereof, as long as the exogenous polynucleotide exhibits the same or similar activity as the polypeptide. The method used in the introduction can be performed by appropriately selecting a known transformation method by those skilled in the art. As the introduced polynucleotide is expressed in the host cell, the polypeptide can be produced, and the activity thereof can be increased.

[0116] 7) codon optimization of the polynucleotide encoding the polypeptide can be codon optimization of the endogenous polynucleotide to increase transcription or translation in the host cell, or codon optimization of the exogenous polynucleotide to optimize the transcription and translation in the host cell.

[0117] 8) analysis of the tertiary structure of the polypeptide to select and modify or chemically modify the exposed site can be, for example, determining a template protein candidate from the sequence similarity by comparing the sequence information of the polypeptide to be analyzed with a database storing known protein sequence information, confirming the structure based thereon, and modifying or chemically modifying the exposed site to be modified or chemically modified.

[0118] Such an increase in the activity of the polypeptide can be an increase in the activity or concentration of the corresponding polypeptide, or an increase in the amount of a product produced by the corresponding polypeptide, based on the activity or concentration of the polypeptide expressed in the wild type or microbial strain before modification, but is not limited thereto.

[0119] As used herein, the term "attenuation" of the activity of the polypeptide has all concepts encompassing attenuation of the activity compared to the endogenous activity or lack of activity. Attenuation can be used interchangeably with terms such as lack (inactivation), deletion, disruption, down-regulation, reduction, weakening, inhibition, reduction, etc.

[0120] For example, attenuation is a state in which the protein is active even though it is not completely inactivated due to deletion, which can mean that the activity of the protein is attenuated compared to the unmodified microorganism, wild type strain, or parent strain, but is not limited thereto.

[0121] For example, the attenuation can be, but is not limited to, inactivation. The inactivation can mean that the protein is not expressed at all, or even if expressed, the activity is not present or is attenuated, compared to the parent strain or unmodified strain.

[0122] The attenuation can also include a case where the activity of the polypeptide itself is attenuated or lost compared to the original polypeptide activity of the microorganism due to a mutation in the polynucleotide encoding the polypeptide, etc., a case where the overall polypeptide activity level in the cell is lower than that of the natural strain due to inhibition of gene expression encoding the polypeptide or due to inhibition of translation into the polypeptide, a case where the gene is not expressed at all, and a case where even if the gene is expressed, the polypeptide activity is not present.

[0123] The fact that the activity of the polypeptide is attenuated compared to the endogenous activity means that the activity of the polypeptide is reduced compared to the activity of the specific polypeptide of the parent strain or unmodified microorganism before the change in traits. The attenuation of the activity of the polypeptide can be confirmed by the attenuation of the degree of activity and the expression level of the corresponding polypeptide or the reduction in the amount of product produced by the corresponding polypeptide.

[0124] For example, the attenuation means that the activity of the protein can be less than about 100%, about 90% or less, about 80% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 5% or less, or 0% of the activity of the protein of the parent strain or unmodified microorganism before the change in traits, but is not limited thereto.

[0125] For example, the inactivation can mean that the protein is not expressed at all, or even if expressed, the activity is not present or is attenuated, compared to the unmodified microorganism.

[0126] Such attenuation of the activity of the polypeptide can be performed by any method known in the art, but the method is not limited thereto, and the attenuation can be achieved by applying various methods well known in the art (for example, Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014; 15(2): 2773-2793; Sambrook et al., Molecular Cloning 2012, etc.).

[0127] Specifically, attenuating the activity of the polypeptide of the present disclosure can be:

[0128] 1) deletion of all or part of the gene encoding the polypeptide;

[0129] 2) modifying the expression regulatory region (or expression regulatory sequence) to reduce the expression of the gene encoding the polypeptide;

[0130] 3) Modifying the amino acid sequence of a polypeptide to eliminate or weaken the activity of the polypeptide (e.g., deleting / substituting / adding one or more amino acids in the amino acid sequence);

[0131] 4) Modifying a polynucleotide sequence encoding a polypeptide to eliminate or weaken the activity of the polypeptide (e.g., deleting / replacing / adding one or more nucleotide bases in the nucleotide sequence of the polypeptide gene to encode the modified polypeptide, thereby eliminating or weakening the activity of the polypeptide);

[0132] 5) modifying the nucleotide sequence of the start codon or 5'-UTR region of the gene encoding the polypeptide;

[0133] 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the gene transcript encoding the polypeptide;

[0134] 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the gene encoding the polypeptide to form a secondary structure to which ribosomes cannot attach;

[0135] 8) adding a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the polynucleotide sequence encoding the polypeptide (reverse transcription engineering, RTE); or

[0136] 9) A combination of two or more selected from 1) to 8), but not particularly limited thereto.

[0137] For example, 1) deleting part or all of a gene encoding a polypeptide can be accomplished by removing all polynucleotides encoding the endogenous polypeptide of interest from the chromosome, replacing them with polynucleotides lacking some nucleotides, or replacing them with marker genes.

[0138] This deletion of some or all of the polynucleotides can be carried out by the following: a method for deleting polynucleotides by homologous recombination using a vector for chromosome insertion in a microorganism, or inducing mutations with light (such as ultraviolet light) and / or chemicals, and then screening a method for a strain in which the target gene is deleted from the mutant obtained, but is not limited thereto. The method for deleting some or all of the genes can include a method using DNA recombination technology. For example, by injecting a nucleotide sequence or a vector containing a nucleotide sequence homologous to the target gene into a microorganism to cause homologous recombination, deleting some or all of the genes can be achieved. The nucleotide sequence or vector injected can include a dominant selection marker, but are not limited thereto.

[0139] Furthermore, 2) modification of the expression regulatory sequence can include: mutation of the expression regulatory region (or expression regulatory sequence) due to deletion, insertion, non-conservative substitution, conservative substitution, or a combination thereof, or replacement of the sequence with a sequence having weaker activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and sequences regulating transcription and translation termination.

[0140] In addition, 3) and 4) modification of the amino acid sequence or polynucleotide sequence may be: due to deletion, insertion, non-conservative substitution or conservative substitution or a combination thereof of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, the sequence is mutated, or the amino acid sequence or polynucleotide sequence is replaced with an amino acid sequence or polynucleotide sequence that is improved to have weaker activity or an amino acid sequence or polynucleotide sequence that is improved to be inactive, so that the activity of the polypeptide is weakened, but not limited thereto. For example, the expression of a gene can be inhibited or weakened by introducing a mutation into the polynucleotide sequence and forming a stop codon, but not limited thereto.

[0141] Furthermore, 5) modification of the start codon or nucleotide sequence of the 5'-UTR region of the gene encoding the polypeptide may be, for example, replacement with another start codon having a lower polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0142] In addition, 6) introduction of antisense oligonucleotides (e.g., antisense RNA) that complementarily bind to gene transcripts encoding polypeptides can be referred to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol. 1 (1) 1986].

[0143] Furthermore, 7) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure to which ribosomes cannot attach may render mRNA incapable of translation or slow down the mRNA translation rate.

[0144] 8) Adding a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the polynucleotide sequence encoding the polypeptide (reverse transcription engineering, RTE) can weaken the activity by making the antisense nucleotide complementary to the gene transcript encoding the polypeptide.

[0145] Another aspect of the present disclosure provides a method for producing L-glutamic acid, which includes the step of culturing the microorganism of the present disclosure in a culture medium.

[0146] Specifically, the method for producing L-glutamic acid disclosed herein may include the step of culturing a microorganism comprising the variant disclosed herein, the polynucleotide disclosed herein, or the vector disclosed herein in a culture medium, but is not limited thereto.

[0147] As used herein, the term "cultivation" refers to growing the microorganisms of the present disclosure under appropriately adjusted environmental conditions. The cultivation procedures of the present disclosure can be carried out according to suitable culture media or culture conditions known in the art. Such cultivation processes can be easily adjusted and adapted by those skilled in the art according to the selected strain. Specifically, the cultivation can be batch, continuous, and / or fed-batch, but is not limited thereto.

[0148] As used herein, " culture medium " refers to the mixture containing the nutrients required for cultivating the microorganism of the present disclosure as the main component, wherein the culture medium provides the nutrients comprising the necessary water, growth factors etc. for survival and growth. Particularly, for the culture medium and other culture conditions for cultivating the microorganism of the present disclosure, any culture medium that is generally used to cultivate microorganisms can be used, without particular limitation. However, the microorganism of the present disclosure can be cultivated in the general culture medium containing suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid and / or vitamin under aerobic conditions, while controlling temperature, pH value etc. For example, the culture medium for Corynebacterium strains can be found in document [" Manual of Methods for General Bacteriology " (Washington, D.C., the U.S., 1981) of the American Society of Bacteriology].

[0149] In the present disclosure, the carbon source may include carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc.; and glycerol, propylene glycol, etc. In addition, natural organic nutrient sources such as starch hydrolysate, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steep liquor may be used, and in particular, carbohydrates such as glucose and aseptically pretreated molasses (i.e., molasses converted to reducing sugars) may be used, and various other carbon sources may be used in appropriate amounts without limitation. These carbon sources may be used alone or in combinations of two or more, but are not limited thereto.

[0150] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; amino acids such as glutamic acid, methionine, glutamine, etc.; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition product, defatted soybean cake or its degradation product, etc. can be used. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0151] The phosphate source may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding sodium salts. Inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, and the like. Furthermore, amino acids, vitamins, and / or suitable precursors may also be included. These components or precursors may be added to the culture medium in batches or continuously. However, the present disclosure is not limited thereto.

[0152] In the process of cultivating microorganisms of the present disclosure, the pH of the culture medium can be regulated by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid in a suitable manner to the culture medium. In addition, a defoamer (such as fatty acid polyethylene glycol ester) can be added to suppress foam formation during cultivation. In addition, oxygen or oxygen-containing gas can be injected into the culture medium to maintain the aerobic state of the culture medium, or gas can not be injected, or nitrogen, hydrogen or carbon dioxide gas can be injected to maintain the anaerobic or non-aerobic state of the culture medium, but is not limited thereto.

[0153] In the culture of the present disclosure, the culture temperature may be maintained at 20° C. to 45° C., specifically, 25° C. to 40° C., and the culture may be performed for about 10 hours to 160 hours, but is not limited thereto.

[0154] L-glutamic acid produced by the culture of the present disclosure may be released into the culture medium, or may be retained in the cells.

[0155] In a specific embodiment, the method for producing L-glutamic acid disclosed herein may further include, for example, prior to the culturing step, the step of preparing the microorganism disclosed herein, preparing a culture medium for culturing the strain, or a combination of these steps (regardless of the order, in any order).

[0156] In a specific embodiment, the method for producing L-glutamic acid of the present disclosure may further include a step of recovering L-glutamic acid from the culture medium (culture medium) or the microorganism of the present disclosure. A recovery step may also be included after the culture step.

[0157] Recovery can be a method of cultivating a microorganism according to the present disclosure, such as batch, continuous or fed-batch type cultivation, by collecting L-glutamic acid using suitable methods known in the art. For example, centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, ultrasonic treatment, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography and affinity chromatography), HPLC and combinations of these methods can be used, and L-glutamic acid can be recovered from the culture medium or microorganism using suitable methods known in the art.

[0158] In addition, the method for producing L-glutamic acid of the present disclosure may further include a purification step. Purification can be performed by using a suitable method known in the art. In an exemplary embodiment, when the method for producing L-glutamic acid of the present disclosure includes a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously without regard to the order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0159] In the methods of the present disclosure, the variant polypeptides, polynucleotides, L-glutamic acid, etc. are described in other aspects.

[0160] Another aspect of the present disclosure provides a composition for producing L-glutamic acid, which comprises a variant polypeptide of the present disclosure; a polynucleotide encoding the variant polypeptide; a vector comprising the polynucleotide; or a microorganism comprising the variant polypeptide of the present disclosure, a polynucleotide encoding the variant polypeptide, or a vector comprising the polynucleotide; a culture of the microorganism; or a combination of two or more thereof.

[0161] The composition of the present disclosure may further comprise any suitable excipient commonly used in compositions for producing L-glutamic acid, examples of which may include preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonic agents, and the like, but are not limited thereto.

[0162] In a specific embodiment, the various components present in the compositions of the present disclosure may be included in microbiologically effective amounts, or in amounts that may be suitably present in the composition for manufacture.

[0163] In the composition disclosed herein, the variant polypeptide, polynucleotide, L-glutamic acid, etc. are as described in other aspects.

[0164] Another aspect of the present disclosure provides use of a microorganism comprising the variant polypeptide of the present disclosure, a polynucleotide encoding the variant polypeptide, or a vector comprising the polynucleotide for producing L-glutamic acid.

[0165] In the present disclosure, the variant polypeptides, polynucleotides, vectors, microorganisms, L-glutamic acid, etc. are described in other aspects.

[0166] Mode for Carrying Out the Invention

[0167] The present disclosure will be described in more detail below by way of exemplary embodiments. However, the following exemplary embodiments are merely intended to illustrate preferred embodiments of the present disclosure and are not intended to limit the scope of the present disclosure thereto. Furthermore, those skilled in the art or similar technical fields to which the present disclosure pertains can fully understand and readily implement technical matters not described in this specification.

[0168] Example 1. Screening of mutant strains with increased glutamate production capacity by artificial mutation method Example 1-1. Inducing artificial mutation by UV irradiation

[0169] In order to screen for mutant strains with improved glutamate (the target product of fermentation) production capacity, wild-type Corynebacterium glutamicum (ATCC13869) was first plated on a nutrient medium containing agar and cultured for 16 hours at 30° C. Hundreds of colonies thus obtained were irradiated with UV light at room temperature to induce random mutations in the strain genome.

[0170] Example 1-2. Fermentation titer experiment of mutagenized strains and screening strains

[0171] The fermentation titer test was performed on the mutant strain in which random mutations were induced in Example 1-1.

[0172] Each colony was subcultured in a nutrient medium and then cultured in a fermentation medium for 5 hours. 25% tween 40 was then added to each medium at a concentration of 0.4%, and each colony was cultured for an additional 32 hours.

[0173] <Nutrient medium>

[0174] 1% glucose, 0.5% meat extract, 1% polypeptone, 0.25% sodium chloride, 0.5% yeast extract, 2% agar, 0.2% urea, pH 7.2

[0175] <Fermentation medium>

[0176] 6% glucose, 5% calcium carbonate, 2.25% ammonium sulfate, 0.1% potassium dihydrogen phosphate, 0.04% magnesium sulfate, 10mg / L ferrous sulfate, 0.3mg / L biotin, 0.2mg / L thiamine hydrochloride

[0177] Under these conditions, cultivate each bacterium colony, screen and produce L-glutamic acid and be equal to or higher than the mutant strain of wild-type Corynebacterium glutamicum (ATCC13869).After this, use HPLC to measure the L-glutamic acid concentration of selected mutant strain.The L-glutamic acid concentration measured is shown in table 1 below.

[0178] [Table 1]

[0179] Strain name L-glutamic acid (g / L) ATCC13869 7.4 ATCC13869-al 8.1 ATCC13869-a2 7.0 ATCC13869-a3 9.0 ATCC13869-a4 7.6 ATCC13869-a5 8.7 ATCC13869-a6 7.3 ATCC13869-a7 6.9 ATCC13869-a8 8.8 ATCC13869-a9 7.4 ATCC13869-alO 6.8 ATCC13869-al l 7.9 ATCC13869-al2 8.3 ATCC13869-al3 7.2 ATCC13869-al4 7.7 ATCC13869-al5 7.5

[0180] Referring to Table 1, "ATCC13869-a3" and "ATCC13869-a8" were selected as mutant strains showing increased glutamate production compared to the wild-type strain.

[0181] Example 2. Identification of mutations by gene sequencing

[0182] In order to identify the gene mutations of the mutant strains, the genes of the ATCC13869-a3 and ATCC13869-a8 strains selected in Example 1-2 were compared with the genes of the wild-type strain.

[0183] As a result, it was confirmed that the ATCC13869-a3 and ATCC13869-a8 strains had the same mutation at a specific position of the gene ilvB (SEQ ID NO: 2) encoding acetolactate synthase (nucleotide at position 893 of the polynucleotide sequence represented by SEQ ID NO: 2 was substituted with T).

[0184] Therefore, in the following Examples 3 and 4, it was intended to determine whether the mutation affects the amount of glutamic acid produced by a microorganism of the genus Corynebacterium.

[0185] Example 3. Preparation of mutagenic strains and detection of glutamate production

[0186] Example 3-1. Preparation of mutagenic strains

[0187] The purpose was to prepare a mutant strain into which the mutation identified in Example 2 above was introduced. Specifically, in order to introduce a mutation (nucleotide 893 of the polynucleotide sequence shown in SEQ ID NO: 2 was replaced with T) into wild-type Corynebacterium glutamicum (ATCC 13869 and ATCC 13032), a vector for gene replacement was constructed to replace the alanine at position 298 of the acetolactate synthase shown in SEQ ID NO: 1 with valine in these strains. Using genomic DNA from ATCC 13869 as a template, a gene fragment for constructing the vector was obtained by PCR. Primers containing the polynucleotides of SEQ ID NOS: 5 to 8 were prepared based on information on the gene and nearby nucleotide sequences of Corynebacterium glutamicum (ATCC 13869) registered in the NIH GenBank.

[0188] PCR was performed by denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 20 seconds, annealing at 55°C for 20 seconds, and polymerization at 72°C for 30 seconds, followed by polymerization at 72°C for 5 minutes. Specifically, a 500-bp polynucleotide amplified using primers of SEQ ID NOS: 5 and 6 and a 500-bp polynucleotide amplified using primers of SEQ ID NOS: 7 and 8 were obtained. The two gene fragments thus obtained were ligated using a fusion enzyme to the pDZ vector (Korean Patent No. 10-0924065 and International Patent Publication No. 2008-033001) digested with the restriction enzymes BamHI and SalI to prepare a gene replacement vector designated "pDZ-ilvB(A298V)." The primer sequences used to construct the above vector are shown in Table 2 below.

[0189] [Table 2]

[0190]

[0191]

[0192] The chromosomal strain of the present invention is transformed into the wild-type strain by homologous recombination with the gene replacement vector (people such as van der Rest, Appl Microbiol Biotechnol 52:541-545,1999).In the substratum that contains 25mg / L kanamycin, screening is carried out the bacterial strain of the chromosomal strain by homologous recombination.After this, as the result of sequencing analysis, the Corynebacterium glutamicum transformant that completes secondary restructuring has confirmed that the target mutation is introduced into this bacterial strain, and this bacterial strain of introducing sudden change is named as " ATCC13869::ilvB (A298V) " and " ATCC13032::ilvB (A298V) ".

[0193] Example 3-2. Detection of glutamate production

[0194] The mutant strains ATCC13869::ilvB(A298V) and ATCC13032::ilvB(A298V) prepared by Example 3-1 and their respective wild-type Corynebacterium glutamicum (ATCC13869 and ATCC13032) were cultured in the same manner as in Example 1-2.

[0195] After the culture was completed, the L-glutamic acid concentration in each culture medium was measured. The measured L-glutamic acid concentrations are shown in Table 3 below.

[0196] [Table 3]

[0197] Strain name L-glutamic acid (g / L) ATCC13869 7.4 ATCC13869::ilvB(A298V) 9.5 ATCC13032 3.6 ATCC13032::ilvB(A298V) 4.5

[0198] As shown in Table 3, it was confirmed that the concentration of L-glutamic acid produced by the mutation-introduced Corynebacterium glutamicum ATCC13869::ilvB(A298V) strain was about 2.1 g / L (about 28%) higher than that produced by the wild-type Corynebacterium glutamicum ATCC13869.

[0199] It was also confirmed that the concentration of L-glutamic acid produced by the mutation-introduced Corynebacterium glutamicum ATCC13032::ilvB(A298V) strain was about 0.9 g / L (about 25%) higher than that produced by the wild-type Corynebacterium glutamicum ATCC13032.

[0200] In other words, it was confirmed that the variants of the present disclosure increased the L-glutamic acid-producing ability of the microorganism.

[0201] Example 4. Detection of glutamate production in KFCC11074 strain with introduced mutation

[0202] Example 4-1. Preparation of mutagenic strains

[0203] To examine whether this mutation has the same effect in strains having increased glutamate production ability in addition to the wild-type strain, this mutation was intended to be introduced into KFCC11074 strain (Korean Patent Publication No. 10-0292299), which is known as a glutamate-producing strain.

[0204] Particularly, pDZ-ilvB (A298V) carrier prepared by embodiment 3-1 is transformed into the KFCC11074 bacterial strain by the homologous recombination on the chromosome (people such as van der Rest, Appl Microbiol bio technol 52:541-545,1999).In the substratum that contains 25mg / L kanamycin, screening is carried out the bacterial strain of the chromosome by homologous recombination.After this, as the result of sequencing analysis, the Corynebacterium glutamicum transformant that completes secondary restructuring has confirmed that target mutation is introduced into this bacterial strain, and this bacterial strain of introducing sudden change is named as " KFCC11074-ilvB (A298V) ".

[0205] Example 4-2. Detection of glutamate production

[0206] Corynebacterium glutamicum KFCC11074 into which no mutation had been introduced and KFCC11074_ilvB(A298V) into which the mutation had been introduced according to Example 4-1 were cultured in the same manner as in Example 1-2.

[0207] After the culture was completed, the L-glutamic acid concentration in each culture medium was measured. The measured L-glutamic acid concentrations are shown in Table 4 below.

[0208] [Table 4]

[0209] Strain name L-glutamic acid (g / L) KFCC11074 5.7 KFCC11074 ilvB(A298V) 8.3

[0210] As shown in Table 4, it was confirmed that the concentration of L-glutamic acid produced by the mutation-introduced Corynebacterium glutamicum KFCC11074_ilvB(A298V) strain was about 2.6 g / L (about 46%) higher than that produced by Corynebacterium glutamicum KFCC11074 without the mutation.

[0211] In other words, it was confirmed that the mutation disclosed herein also increased the L-glutamic acid-producing ability of the microorganism in the strain having increased glutamic acid-producing ability.

[0212] Based on the above description, it will be understood by those skilled in the art that the present disclosure can be implemented in different specific forms without changing its technical spirit or essential features. In this regard, it should be understood that the above embodiments are not restrictive, but illustrative in all aspects. The scope of the present disclosure is defined by the appended claims rather than by the description before them, and all changes and modifications that fall within the boundaries and scope of the claims, or equivalent replacements of these boundaries and scopes, are covered by the claims.

Claims

1. A variant polypeptide having acetolactate synthase activity, wherein the amino acid corresponding to position 298 of SEQ ID NO: 1 is replaced by a different amino acid.

2. The variant polypeptide according to claim 1, wherein the amino acid corresponding to position 298 of SEQ ID NO: 1 is substituted with valine.

3. The variant polypeptide according to claim 1, wherein the variant polypeptide consists of the amino acid sequence of SEQ ID NO:

3. A polynucleotide encoding the variant polypeptide according to any one of claims 1 to 3. 5 . A microorganism comprising the variant polypeptide according to claim 1 , a polynucleotide encoding the variant polypeptide, or a vector comprising the polynucleotide. The microorganism according to claim 5 , wherein the microorganism has an increased L-glutamic acid-producing ability compared to a microorganism comprising the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same. The microorganism according to claim 5 , wherein the microorganism is a microorganism of the genus Corynebacterium . The microorganism according to claim 7 , wherein the Corynebacterium microorganism is Corynebacterium glutamicum .

9. A method for producing L-glutamic acid, comprising the step of culturing the microorganism according to claim 5 in a culture medium.

10. The method according to claim 9, further comprising the step of recovering L-glutamic acid from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium.

11. 1 kinds of compositions for the production of L-Glutamic acid, described compositions comprises the variant polypeptide according to any one in claim 1 to 3; The polynucleotide of encoding described variant polypeptide; The carrier that comprises described polynucleotide; The polynucleotide that comprises described variant polypeptide, described encoding described variant polypeptide or the described microorganism that comprises described polynucleotide; The culture of described microorganism; Or its two or more combination.

12. Use of a microorganism comprising a variant polypeptide according to any one of claims 1 to 3, a polynucleotide encoding the variant polypeptide, or a vector comprising the polynucleotide for producing L-glutamic acid.

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