Fructokine variant and method for producing L-amino acids using the same

Abstract

This application relates to a fructokinase mutant polypeptide, to a polynucleotide encoding the mutant polypeptide, to a microorganism containing the same, to a method for producing L-amino acids comprising the step of culturing the microorganism in a culture medium, and to the use of the microorganism for L-amino acid production.

Description

Technical Field

[0001] The present application relates to a fructokinase variant polypeptide, to a polynucleotide encoding said variant polypeptide, to a vector comprising the same, to a microorganism comprising said polypeptide, polynucleotide and / or vector, to a method for producing an L-amino acid comprising the step of culturing said microorganism in a medium, to a composition for producing an L-amino acid comprising at least one selected from said microorganism, a culture of said microorganism, and a fermentation product of said microorganism, and to use of said microorganism for producing an L-amino acid.

Background Art

[0002] Coryneform microorganisms are Gram-positive microorganisms mainly used for industrial production of substances having various uses such as feed including L-amino acids and various nucleic acids, pharmaceuticals, foods, and the like. Recently, diamine, keto-acid and the like have also been produced from coryneform microorganisms.

[0003] L-amino acids are the basic structural units of proteins, and are used as important raw materials for pharmaceutical raw materials, food additives, animal feed, nutrients, insecticides, fungicides, and the like. In particular, L-lysine is an essential amino acid that is not biosynthesized in vivo at all, and is known to be necessary for growth promotion, calcium metabolism, gastric juice secretion promotion, and increased resistance to diseases. Said L-lysine is variously used in feed, pharmaceuticals, foods and the like. In addition, L-tryptophan is also one of the essential amino acids, and is used as a feed additive, an infusion solution, a pharmaceutical raw material, and a health food material.

[0004] Various studies are being conducted to develop highly efficient production microorganisms and fermentation process technologies for producing the aforementioned amino acids. For example, target-specific approaches are mainly used, such as increasing the expression of genes encoding enzymes involved in amino acid biosynthesis in Corynebacterium strains, or removing genes unnecessary for amino acid biosynthesis (Patent Documents 1 and 2). In addition to these methods, methods for removing genes not involved in amino acid production, and methods for removing genes whose specific function in amino acid production is unknown, are also being utilized. However, there is still a strong need for research on methods for producing L-amino acids efficiently and in high yield. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] U.S. Patent No. 9109242 [Patent Document 2] U.S. Patent No. 8030036 [Patent Document 3] U.S. Patent No. 7662943 [Patent Document 4] U.S. Patent No. 10584338 [Patent Document 5] U.S. Patent No. 10273491 [Patent Document 6] Korean Registered Patent Publication No. 10-0620092 [Patent Document 7] Korean Registered Patent Publication No. 10-1783170 [Patent Document 8] Korean Registered Patent Publication No. 10-2363913 [Patent Document 9] Korean Registered Patent Publication No. 10-1968317 [Patent Document 10] Korean Registered Patent Publication No. 10-0159812 [Non-patent literature]

[0006] [Non-Patent Document 1] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed document 2] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-licensed document 3] J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

[0007] The problem to be solved by the present application is to provide a microorganism comprising a fructokinase variant and a method for producing an L-amino acid using the same. [Means for Solving the Problem]

[0008] One aspect of the present application provides a fructokinase mutant polypeptide in which the amino acid corresponding to position 79 or position 181 of SEQ ID NO: 1 is substituted with another amino acid.

[0009] In one embodiment, in the mutant polypeptide, the amino acid corresponding to position 79 of SEQ ID NO: 1 may be substituted with cysteine, the amino acid corresponding to position 181 of SEQ ID NO: 1 may be substituted with valine, or the mutant polypeptide may be a combination thereof.

[0010] In another embodiment, the mutant polypeptide may consist of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

[0011] In still another embodiment, the fructokinase may be encoded by the cscK gene.

[0012] Another aspect of the present application provides a polynucleotide encoding said mutant polypeptide.

[0013] Still another aspect of the present application provides a microorganism comprising at least one selected from the group consisting of a mutant polypeptide, a polynucleotide encoding said mutant polypeptide, and a vector comprising the same.

[0014] In the microorganism according to any one of the foregoing embodiments, the microorganism may be a microorganism of the genus Corynebacterium.

[0015] In the microorganism according to any one of the foregoing embodiments, the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum.

[0016] In the microorganism according to any one of the foregoing embodiments, the microorganism may have improved L-amino acid producing ability as compared to a microorganism comprising the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same.

[0017] In any of the above-mentioned specific examples of microorganisms, the microorganisms may have improved L-histidine, L-isoleucine, L-tryptophan, or L-lysine production capabilities.

[0018] A further aspect of this application provides a method for producing L-amino acids, comprising the step of culturing the microorganism in a culture medium.

[0019] As a specific example, the method may further include a step of recovering L-amino acids from the cultured microorganism, the culture product of the microorganism, the fermented product of the microorganism, or the culture medium.

[0020] As another specific example, the above method may be a method for producing L-histidine, L-isoleucine, L-tryptophan, or L-lysine.

[0021] Further embodiments of this application provide a composition for L-amino acid production comprising at least one selected from a mutant polypeptide, a polynucleotide encoding the mutant polypeptide, a vector containing the same, a microorganism containing the vector, a culture of the microorganism, and a fermentate of the microorganism.

[0022] As a specific example, the composition may be a composition for the production of L-histidine, L-isoleucine, L-tryptophan, or L-lysine. [Effects of the Invention]

[0023] When microorganisms containing the fructokinase mutant polypeptide of this application are cultured, L-amino acids can be produced in higher yield compared to microorganisms containing conventional unmodified polypeptides. [Modes for carrying out the invention]

[0024] These will be explained in detail below. Note that each description and embodiment disclosed in this application applies to other descriptions and embodiments. That is, any combination of the various elements disclosed in this application is included. Furthermore, this application is not limited to the following specific descriptions. In addition, numerous papers and patent documents are referenced throughout this specification, and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated in their entirety as references in this specification, thereby more clearly explaining the level of the art to which this application belongs and the content of this application.

[0025] One aspect of this application provides a fructokinase mutant polypeptide in which an amino acid corresponding to the 79th or 181st position of 1 is substituted with another amino acid.

[0026] In this application, "fructokinase (CscK)" refers to an enzyme that catalyzes a reaction in which a phosphate group is transferred in the presence of ATP to produce D-fructose-6-phosphate and ADP.

[0027] For the purposes of this application, the protein is also referred to as "fructose phosphorylation enzyme," "fructose kinase," or "CscK." The gene encoding the protein is, for example, the csck gene, but is not limited to this. In this application, "csck gene" is used interchangeably with "gene encoding fructokinase." Furthermore, the protein is, for example, derived from Escherichia coli, but any type of protein with activity equivalent to fructokinase is acceptable.

[0028] The fructokinase encoded by the csck gene is known in the art, and the amino acid and polynucleotide sequences of the fructokinase can be obtained from known databases, such as NCBI's GenBank, but are not limited to these.

[0029] The fructokinase protein may contain the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having 60% or more homology or identity thereto, but any protein that has fructokinase protein activity is acceptable. Specifically, meaningless sequence additions before or after the amino acid sequence represented by SEQ ID NO: 1, naturally occurring mutations, and silent mutations are not excluded, and any protein having the same or equivalent activity as a protein containing the amino acid sequence of SEQ ID NO: 1 is included in the proteins targeted for mutation introduction in this application. For example, the proteins targeted for mutation introduction in this application may consist of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. Furthermore, it goes without saying that any mutant amino acid sequence having such homology or identity and exhibiting efficacy equivalent to the fructokinase polypeptide is also included in the polypeptides targeted for mutation in this application, including those having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added.

[0030] In this application, in the original amino acid sequence of the fructokinase to be mutated, i.e., the parent sequence, the original amino acid corresponding to position 79 of SEQ ID NO: 1 is tryptophan (W), and the original amino acid corresponding to position 181 of SEQ ID NO: 1 is alanine (A).

[0031] In this application, "mutant polypeptide," "mutant protein," or "variant" refers to a polypeptide in which at least one amino acid differs from the amino acid sequence of the original polypeptide due to conservative substitution and / or modification, but the functions or properties are maintained. Such variants can generally be identified by modifying at least one amino acid in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the capabilities of the variant are improved, unchanged, or decreased compared to the original polypeptide. Some variants also include those in which at least one part, such as the N-terminal leader sequence or transmembrane domain, is removed. Other variants include those in which a portion of the N and / or C-terminus of a mature protein is removed. The term "mutant" is often used interchangeably with terms such as mutant, modified, mutant polypeptide, mutated protein, mutation, and divergent (in English, these may include modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), but any term that means mutation is acceptable.

[0032] Furthermore, the variants may include the deletion or addition of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated with an N-terminal signal (or leader) sequence of a protein involved in protein transfer, either co-translationally or post-translationally. The polypeptide may also be conjugated with other sequences or linkers to enable the polypeptide to be identified, purified, or synthesized.

[0033] The fructokinase mutant polypeptide referred to in this application means a mutant polypeptide in which the amino acid corresponding to the 79th or 181st position from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid.

[0034] The aforementioned "fructokinase mutant polypeptide" is also referred to as a "fructokinase variant," "mutant CscK," or "CscK variant." The fructokinase polypeptide targeted for mutation introduction in this application is used interchangeably with "CscK," and is particularly encoded by the cscK gene, specifically cscK derived from Escherichia coli, but is not limited to these.

[0035] The fructokinase mutant polypeptide of this application may be one in which the amino acid at the position corresponding to the 79th or 181st position in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid different from the original amino acid.

[0036] In one specific example, the mutant polypeptide may be one in which the amino acid corresponding to the 79th position in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than tryptophan, which was the original amino acid; or the amino acid corresponding to the 181st position in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than alanine, which was the original amino acid; or a combination thereof.

[0037] In other specific examples, the mutant polypeptide is characterized in which the amino acid corresponding to the 79th position of SEQ ID NO: 1 is substituted with one selected from the group consisting of asparagine, valine, glycine, leucine, arginine, alanine, methionine, threonine, glutamine, proline, isoleucine, serine, phenylalanine, histidine, cysteine, tyrosine, lysine, aspartic acid, and glutamic acid, but is not limited to these.

[0038] In other specific examples, the mutant polypeptide is characterized in which the amino acid corresponding to position 181 of SEQ ID NO: 1 is substituted with one selected from the group consisting of asparagine, valine, glycine, leucine, arginine, tryptophan, methionine, threonine, glutamine, proline, isoleucine, serine, phenylalanine, histidine, cysteine, tyrosine, lysine, aspartic acid, and glutamic acid, but is not limited to these.

[0039] As an example of any of the above-described examples, the mutant polypeptide provided in this application may be one in which the amino acid corresponding to the 79th position from the N-terminus of SEQ ID NO: 1 is substituted with cysteine, or the amino acid corresponding to the 181st position from the N-terminus of SEQ ID NO: 1 is substituted with valine, or a combination thereof.

[0040] As any other example of the above-described examples, the mutant polypeptide provided in this application may contain an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% or more, or less than 100% homology or identity with SEQ ID NO: 1.

[0041] For example, the mutant polypeptide of this application may include an amino acid sequence in which the amino acid corresponding to the 79th position in the amino acid sequence represented by SEQ ID NO: 1 is fixed to cysteine, and which has at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% or more homology or identity with SEQ ID NO: 1. Alternatively, the mutant polypeptide of this application may include an amino acid sequence in which the amino acid corresponding to the 181st position in the amino acid sequence represented by SEQ ID NO: 1 is fixed to valine, and which has at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% or more homology or identity with SEQ ID NO: 1. Furthermore, the mutant polypeptide of this application may include an amino acid sequence in which the amino acid corresponding to the 79th position in the amino acid sequence represented by SEQ ID NO: 1 is fixed to cysteine, and the amino acid corresponding to the 181st position is fixed to valine, and which has at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% homology or identity with SEQ ID NO: 1. Moreover, it goes without saying that mutant polypeptides having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added are also included in this application, as long as they have such homology or identity and exhibit efficacy equivalent to the mutant polypeptide of this application.

[0042] On the other hand, a person skilled in the art can identify the amino acid corresponding to the 79th or 181st position of the amino acid sequence of Sequence ID No. 1 of this application in any given amino acid sequence using sequence alignment known in the art. It goes without saying that, without further clarification, the "amino acid at a specific position in a particular Sequence ID No. 1" in this application includes the "amino acid at a corresponding position" in any given amino acid sequence.

[0043] In further specific examples, the mutant polypeptide may consist of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.

[0044] Specifically, the mutant polypeptide of this application may have an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with the amino acid sequences of SEQ ID NO: 3, 5, or 7, may contain the said amino acid sequence, may consist of the said amino acid sequence, or may be substantially composed of the said amino acid sequence.

[0045] Examples include the addition or deletion of a sequence that does not alter the function of the variant of this application, a spontaneous mutation, a silent mutation, or a conserved substitution at the N-terminus, C-terminus, and / or within the amino acid sequence.

[0046] The aforementioned "other amino acid" may be any amino acid different from the amino acid before substitution. It goes without saying that in this application, "a specific amino acid has been substituted" means that the amino acid has been substituted with an amino acid different from the amino acid before substitution, even if it is not explicitly stated that it has been substituted with another amino acid.

[0047] Amino acids are generally classified based on the similarities of their residues in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature.

[0048] As an example of this classification, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Other examples include amino acids with electrically charged side chains (arginine, lysine, histidine, glutamic acid, and aspartic acid) and amino acids with uncharged side chains (also called neutral amino acids) (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). Further examples include phenylalanine, tryptophan, and tyrosine, which are classified as aromatic amino acids. Still further examples include valine, leucine, and isoleucine, which are classified as branched amino acids. As another example, the 20 amino acids are classified by size and, in order of increasing volume, into five groups: glycine, alanine, and serine; cysteine, proline, threonine, aspartic acid, and asparagine; valine, histidine, glutamic acid, and glutamine; isoleucine, leucine, methionine, lysine, and arginine; and phenylalanine, tryptophan, and tyrosine. However, the classification is not necessarily limited to these groups.

[0049] For example, "the amino acid corresponding to position 79 in Sequence ID No. 1 has been substituted with another amino acid" means, but is not limited to, the substitution with asparagine, valine, glycine, alanine, glutamate, phenylalanine, arginine, aspartate, cysteine, glutamine, histidine, proline, isoleucine, tyrosine, lysine, serine, methionine, threonine, or leucine.

[0050] As another example, "the amino acid corresponding to position 181 in Sequence ID No. 1 has been substituted with another amino acid" means, but is not limited to, substitution with asparagine, valine, glycine, tryptophan, glutamate, phenylalanine, arginine, aspartate, cysteine, glutamine, histidine, proline, isoleucine, tyrosine, lysine, serine, methionine, threonine, or leucine, excluding alanine.

[0051] As another example, "the amino acids corresponding to positions 79 and 181 in SEQ ID NO: 1 are substituted with other amino acids" means, but is not limited to, that the amino acid corresponding to position 79 is substituted with an amino acid other than tryptophan, and the amino acid corresponding to position 181 is substituted with an amino acid other than alanine.

[0052] Even if this application describes "a protein having an amino acid sequence represented by a specific sequence number," it goes without saying that any protein having the same or equivalent activity as the protein consisting of the amino acid sequence of said sequence number, even if it has an amino acid sequence in which some of the sequence is deleted, modified, substituted, conserved substituted, or added, can be used in this application. For example, if it has the same or equivalent activity as the mutant protein, this does not exclude the addition of sequences that do not change the function of the protein before or after the amino acid sequence, naturally occurring mutations, silent mutations, or conserved substitutions, and it goes without saying that any protein having such sequence additions or mutations is also included in this application.

[0053] In this application, "position N" includes the Nth position and the amino acid position corresponding to (correspoding with) the Nth position. Specifically, it includes the amino acid position corresponding to any amino acid residue in a mature polypeptide represented by a specific amino acid sequence. The specific amino acid sequence may be the amino acid sequence of Sequence ID No. 11.

[0054] In this application, "corresponding to" means an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar, identical, or equivalent to a residue listed in the polypeptide. Identifying the amino acid at the corresponding position will determine the specific amino acid in the sequence referencing the particular sequence. In this application, "corresponding region" generally means a similar or corresponding position in the related protein or reference protein.

[0055] For example, by aligning any amino acid sequence with Sequence ID No. 1, each amino acid residue in the sequence can be numbered based on the number and position of amino acid residues corresponding to the amino acid residues in Sequence ID No. 1. For example, the sequence alignment algorithm in this application can be used to identify the positions of amino acids, or the positions where modifications such as substitutions, insertions, or deletions occur, by comparing it with a query sequence (also called a "reference sequence").

[0056] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Literature 1) and the Needle program from the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Literature 2) can be used, but are not limited to these. Sequence alignment programs and pairwise sequence comparison algorithms known in the art can be used as appropriate.

[0057] In this application, "conservative substitution" means that one amino acid is replaced by another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. 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; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Furthermore, amino acids are classified into those with electrically charged side chains and those with uncharged side chains. Examples of amino acids with electrically charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino acids with uncharged side chains are further classified into nonpolar amino acids and polar amino acids. Examples of nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Examples of polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conservative substitutions have little to no effect on the activity of the resulting polypeptide.

[0058] Another aspect of this application provides a polynucleotide encoding the mutant polypeptide.

[0059] In this application, "polynucleotide" means a polymer of nucleotides in which nucleotide monomers are covalently linked together in a long chain, and refers to a DNA or RNA chain longer than a predetermined length, and more specifically, refers to a polynucleotide fragment that codes for the aforementioned variant.

[0060] The polynucleotide encoding the fructokinase mutant polypeptide in this application may be any polynucleotide sequence that encodes a mutant polypeptide having fructokinase activity.

[0061] For example, the polynucleotide encoding the fructokinase mutant polypeptide of this application is a polynucleotide sequence encoding the amino acid sequence of the fructokinase mutant polypeptide of this application, but is not limited to this.

[0062] For example, it may include a nucleic acid sequence encoding an amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. For example, the polynucleotide of this application may have the sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or may include the said sequence. Alternatively, the polynucleotide of this application may consist of the sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or may be substantially composed of the said sequence.

[0063] The polynucleotides of this application can be modified in various ways in the coding region, either through codon degeneracy or by considering preferred codons in organisms that intend to express the variants of this application, as long as the amino acid sequence of the variants of this application remains unchanged. Therefore, it goes without saying that polynucleotides translated by codon degeneracy into polypeptides consisting of the amino acid sequence of the variants of this application, or polypeptides homologous or identical thereto, are also included. For example, the polynucleotides of this application may be the sequences of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or their degenerated sequences.

[0064] As an example, the polynucleotides of this application include, but are not limited to, nucleotide sequences having 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.8% or more homology or identity with the sequence of SEQ ID NO: 2, in which the codon encoding tryptophan, which corresponds to the amino acid positions 235-237 of SEQ ID NO: 2, is replaced with a codon encoding another amino acid, such as cysteine, or the codon encoding alanine, which corresponds to the amino acid positions 541-543, is replaced with a codon encoding another amino acid, such as valine.

[0065] Furthermore, it goes without saying that this application also includes variants having polynucleotide sequences in which some sequences are deleted, modified, substituted, conserved substituted, or added, as long as they have such homology or identity and encode the amino acid sequence of the fructokinase mutant polypeptide of this application.

[0066] As another example, the polynucleotides of this application may have nucleic acid sequences that are homologous or identical to SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8 by 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and less than 100%, or may contain such nucleic acid sequences, or may consist of nucleic acid sequences that are homologous or identical to SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8 by 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and less than 100%, or may be substantially composed of such nucleic acid sequences, but are not limited to these.

[0067] Furthermore, the polynucleotide of this application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of this application.

[0068] The aforementioned "stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 3 and 4). For example, this could involve hybridizing polynucleotides with high homology or identity, such as polynucleotides with 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 homology or identity, while not hybridizing polynucleotides with lower homology or identity. Alternatively, it could involve washing once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of a typical Southern hybridization: 60°C, 1×SSC, 0.1%SDS, more specifically 60°C, 0.1×SSC, 0.1%SDS, or more specifically 68°C, 0.1×SSC, 0.1%SDS.

[0069] Hybridization requires that the two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "Complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this application may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0070] Specifically, polynucleotides homologous or identical to the polynucleotide of this application can be detected using hybridization conditions in which the hybridization step is performed at a Tm value of 55°C and the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited to these, and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0071] The appropriate stringency for hybridizing the aforementioned polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are known in the art (e.g., Non-Patent Document 3).

[0072] In this application, "homology" or "identity" refers to the degree to which two given amino acid sequences or base sequences are similar, and is expressed as a percentage. Homology and identity are often used interchangeably.

[0073] The sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequence algorithms, and a default gap penalty established by the program used may also be applied. Substantially, homologous or identical sequences generally hybridize with all or part of the sequence under moderate to high stringent conditions. Needless to say, hybridization includes hybridization with polynucleotides that have common codons or codons considering codon degeneracy in the polynucleotide.

[0074] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using default parameters, such as those in Non-Patent Document 5, and known computer algorithms such as the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 1), as performed in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) (including the GCG program package (Non-Patent Document 6), BLASTP, BLASTN1, and FASTA (Non-Patent Documents 7, 8, and 9)). For example, homology, similarity, or identity can be determined using BLAST or Clustal W from the National Center for Biotechnology Information.

[0075] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 1, as disclosed in Non-Patent Document 10, for example. In summary, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) as the total number of symbols in the shorter of two sequences divided by the number of similar sequence symbols. Default parameters for the GAP program include (1) a binary comparison matrix (where identity is 1 and non-identity is 0) and a weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 11 and Non-Patent Document 12, and (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extended penalty of 0.5), and (3) no penalty for terminal gaps. Thus, "homology" or "identity" in this application indicates the relevance between sequences.

[0076] Further embodiments of this application provide a vector comprising the polynucleotide of this application, which is an expression vector for expressing the polynucleotide in a microorganism, but is not limited thereto.

[0077] In this application, “vector” means a DNA product comprising a polynucleotide sequence encoding a target polypeptide operably ligated to a suitable regulatory region (or regulatory sequence) so as to enable the expression of the target polypeptide in a suitable host. The regulatory region includes a promoter to initiate transcription, an optional operator sequence to regulate the transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences to regulate the termination of transcription and translation. When transformed into a suitable microorganism, the vector can replicate and function independently of the host genome and is integrated into the genome itself.

[0078] The vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used, and as plasmid vectors, pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pET series, etc. can be used. Specifically, pDZ, pDC, pDCM2, PDC24, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0079] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using an intracellular chromosome introduction vector. The insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. The vector may further include a selection marker to confirm whether or not the polynucleotide has been inserted into the chromosome. The selection marker is used to select cells transformed by the vector, that is, to confirm whether or not the target nucleic acid molecule has been inserted, and markers that confer selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, and expression of surface polypeptides are used. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit different phenotypes, thus allowing for the selection of transformed cells.

[0080] In this application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a microorganism or into a microorganism, thereby causing the polypeptide encoded by the polynucleotide to be expressed in the microorganism. The transformed polynucleotide may be any form that is expressed in the microorganism, regardless of whether it is inserted into or outside the chromosome of the microorganism. The polynucleotide also contains DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the microorganism in any form that is expressed upon introduction. For example, the polynucleotide may be introduced into the microorganism in the form of an expression cassette, which is a gene construct containing all the elements necessary for its expression. Typically, the expression cassette includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the microorganism in its own form and operably linked to a sequence necessary for expression in the microorganism, but is not limited to this.

[0081] Furthermore, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target variant of this application.

[0082] The method for transforming the vector of this application may be any method for introducing nucleic acids into cells, and can be carried out by selecting a standard technique suitable for the host cell, as is known in the art. Examples include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0083] A further aspect of this application provides a microorganism comprising at least one selected from the mutant polypeptide, the polynucleotide encoding the mutant polypeptide, and a vector comprising the same.

[0084] In one specific example, the microorganism of this application may be a microorganism capable of producing L-amino acids.

[0085] In this application, "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially. These are microorganisms in which a specific mechanism has been reduced or enhanced due to causes such as the insertion of external genes or the enhancement or inactivation of endogenous gene activity, and which have been genetically modified for the production of a target polypeptide, protein, or product. In this application, "microorganism" and "strain" are used interchangeably and are used together.

[0086] In this application, “recombinant microorganism” means a microorganism that has been genetically modified to exhibit a different genotype and / or phenotype compared to a naturally occurring microorganism (for example, in which the genetic modification affects the nucleic acid sequence coding of the microorganism), and includes all offspring or potential offspring of said microorganism. In this application, “recombinant microorganism,” “genetically modified microorganism,” “recombinant host cell,” “recombinant cell,” and “recombinant strain” are used interchangeably. The recombinant microorganism may express genes not found in the natural (non-recombinant) form, may not express genes expressed in the natural form, or may express natural genes in a manner different from that expressed in the natural form.

[0087] In this application, "microorganisms that produce L-amino acids" refers to prokaryotic or eukaryotic microbial strains that produce L-amino acids within their bodies, and includes all microorganisms in which L-amino acid production ability has been conferred from a parent strain that lacked L-amino acid production ability, as well as microorganisms that inherently possess L-amino acid production ability. L-amino acid production ability can be conferred or improved through selective breeding.

[0088] In this application, "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but rather means wild-type strains or natural strains themselves, or strains before they undergo genetic mutation and change in characteristics due to natural or artificial factors. For example, the "unmodified microorganism" refers to a strain in which the fructokinase mutant polypeptide used herein has not been introduced, or before its introduction. The "unmodified microorganism" is used interchangeably with "pre-modification strain," "pre-modification microorganism," "non-mutant strain," "unmodified strain," "non-mutant microorganism," or "reference microorganism."

[0089] The microorganisms having the L-amino acid production ability of this application include, but are not limited to, microorganisms comprising at least one of the mutant polypeptide of this application, the polynucleotide of this application, and a vector comprising the polynucleotide of this application, microorganisms modified to express the mutant polypeptide or polynucleotide of this application, microorganisms expressing the mutant polypeptide or polynucleotide of this application (e.g., recombinant strains), or microorganisms having the activity of the fructokinase mutant polypeptide of this application (e.g., recombinant strains).

[0090] For example, the strain of this application is a cell or microorganism transformed with a vector containing a polynucleotide encoding the mutant polypeptide of this application, and expressing a mutant fructokinase containing the mutant polypeptide of this application. The strain of this application may be any microorganism that contains the mutant polypeptide of this application and produces an L-amino acid.

[0091] For example, the microorganism of this application may be a recombinant strain in which a fructokinase mutant polypeptide is expressed and L-amino acid production ability is improved by introducing a polynucleotide encoding the mutant polypeptide of this application into a naturally occurring wild-type microorganism or a microorganism capable of L-amino acid production. The recombinant strain with improved L-amino acid production ability is a microorganism that has improved L-amino acid production ability compared to a naturally occurring wild-type microorganism or a microorganism that does not express the mutant polypeptide of this application (for example, a microorganism that expresses wild-type fructokinase or a microorganism that does not express the mutant polypeptide of this application), but is not limited thereto. For example, the microorganism with improved L-amino acid production ability of this application is a microorganism that has improved L-amino acid production ability compared to a microorganism containing the polypeptide of Sequence ID No. 1 or a polynucleotide encoding it, but is not limited thereto. For example, the unmodified microorganisms used as comparison targets for determining whether or not the L-amino acid production capacity has improved are, but are not limited to, Corynebacterium glutamicum strains CA14-0114, KCCM12739P, CA04-8405, or KCCM11016P.

[0092] The microorganisms of this application include all microorganisms that express the fructokinase mutant polypeptide of this application by various known methods other than the introduction of nucleic acids or vectors.

[0093] As an example, the microorganisms with improved L-amino acid production capacity show an improvement of approximately 1% or more compared to the L-amino acid production capacity of the parent strain before mutation or the unmodified microorganism. Specifically, this improvement is approximately 1% or more, approximately 2% or more, approximately 3% or more, approximately 4% or more, approximately 5% or more, approximately 6% or more, approximately 7% or more, approximately 8% or more, approximately 9% or more, approximately 10% or more, approximately 11% or more, approximately 12% or more, approximately 13% or more, approximately 14% or more, approximately 15% or more, approximately 16% or more, approximately 17% or more, approximately 18% or more, and approximately 19%. The above represents an improvement of approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, and approximately 50% or more (there are no particular restrictions on the upper limit; for example, it can be approximately 200% or less, approximately 150% or less, approximately 100% or less, approximately 90% or less, approximately 80% or less, approximately 70% or less, approximately 60% or less, approximately 55% or less, or approximately 50% or less). Any result is acceptable as long as it shows an increase in the positive value compared to the productivity of the parent strain or unmodified microorganism before mutation. As another example, the recombinant strains with improved L-amino acid production capacity showed L-amino acid production capacity of approximately 1.01 times or more, approximately 1.02 times or more, approximately 1.03 times or more, approximately 1.04 times or more, 1.05 times or more, 1.06 times or more, 1.07 times or more, 1.08 times or more, 1.09 times or more, approximately 1.1 times or more, approximately 1.11 times or more, approximately 1.12 times or more, approximately 1.13 times or more, approximately 1.14 times or more, and approximately 1.15 times or more compared to the parent strain before mutation or the unmodified microorganism. This refers to an improvement of more than double, approximately 1.16 times or more, approximately 1.17 times or more, approximately 1.18 times or more, approximately 1.19 times or more, approximately 1.20 times or more, approximately 1.25 times or more, approximately 1.30 times or more, approximately 1.35 times or more, approximately 1.40 times or more, approximately 1.45 times or more, or approximately 1.50 times or more (there is no particular limit on the upper limit; for example, it can be approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, approximately 2 times or less, or approximately 1.5 times or less), but is not limited to these. The aforementioned "about" includes a range that encompasses ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and any number within a range equivalent to or similar to the number following the term "about" is acceptable, but is not limited to these.

[0094] In the microorganisms described in any of the above-mentioned specific examples, the microorganisms of this application are, but are not limited to, microorganisms belonging to the genera Corynebacterium sp., Escherichia sp., Erwinia sp., Serratia sp., Providencia sp., Pseudomonas sp., Leptospira sp., Salmonella sp., Brevibacterium sp., Hyphomonas sp., Chromobacterium sp., and Norcardia sp., or fungi or yeast, specifically microorganisms of the genus Corynebacterium.

[0095] As an example of this application, the microorganisms of this application are Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, and Corynebacterium striatum. This may include Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganism of this application is a microorganism of the genus Corynebacterium, and more specifically, Corynebacterium glutamicum, but is not limited to these.

[0096] Specifically, the microorganisms of this application are of the genus Corynebacterium, and more specifically, Corynebacterium glutamicum, but are not limited to these.

[0097] On the other hand, the Corynebacterium microorganisms with improved L-amino acid production capacity described in this application include all Corynebacterium microorganisms with improved L-amino acid production capacity by increasing or decreasing the activity of genes related to the L-amino acid production mechanism, and Corynebacterium microorganisms with improved L-amino acid production capacity by introducing or enhancing the activity of external genes. As a specific example, this includes, but is not limited to, Corynebacterium microorganisms in which the fructokinase activity is improved and the L-amino acid production capacity is improved by introducing the mutant polypeptide described in this application.

[0098] In this application, "increase" of protein (polypeptide) activity means that the activity of the protein (polypeptide) within the host cell (microorganism) is improved compared to its endogenous activity. This increase is used interchangeably with activation, upregulation, overexpression, enhancement, etc. The host cell (microorganism) may be a prokaryotic or eukaryotic microorganism.

[0099] The aforementioned improvement in protein (polypeptide) activity includes all cases where the host cell (microorganism) exhibits protein (polypeptide) activity that it did not inherently possess, as well as an improvement in protein (polypeptide) activity compared to the endogenous activity or the activity before modification.

[0100] For example, the aforementioned "becoming able to exhibit protein (polypeptide) activity that was not inherently present" or "improvement in protein (polypeptide) activity" is due to "introduction of protein (polypeptide)," but is not limited to this.

[0101] In this application, "introduction" of a protein (polypeptide) means causing the activity of a specific protein to appear by expressing a gene in the microorganism that the microorganism did not originally possess, or causing the polypeptide activity to be enhanced, increased, or improved compared to the endogenous activity or the activity before modification of the protein. For example, this may be done by introducing the gene encoding the protein (polypeptide) into the host cell (microorganism). For example, this may be done by introducing a polynucleotide encoding the specific protein (polypeptide) into the chromosome in the host cell (microorganism), or by introducing a vector containing a polynucleotide encoding the specific protein (polypeptide) into the host cell (microorganism) to cause its activity to appear or be improved.

[0102] The aforementioned "endogenous activity" refers to the activity of a specific protein (polypeptide) that was originally present in the host cell (microorganism) before transformation or in the unmodified host cell (microorganism) when a genetic mutation occurs due to natural or artificial factors, resulting in a change in trait. This term is often confused with "activity before modification."

[0103] An increase in protein (polypeptide) activity compared to endogenous activity means that the activity and / or concentration (expression level) of the protein (polypeptide) in the host cell (microorganism) is increased compared to the original activity and / or concentration (expression level) of the said protein (polypeptide) in the host cell (microorganism) before transformation or in the unmodified host cell (microorganism).

[0104] For example, the aforementioned improvement means that activity that was not present in the corresponding protein (polypeptide) becomes apparent, or that the activity or concentration increases by approximately 1% or more, approximately 10% or more, approximately 25% or more, approximately 50% or more, approximately 75% or more, approximately 100% or more, approximately 150% or more, approximately 200% or more, approximately 300% or more, approximately 400% or more, or approximately 500% or more, and up to approximately 1000% or approximately 2000% or more, compared to the activity or concentration in the host cell (microorganism) before transformation or in the unmodified host cell (microorganism), but is not limited to these.

[0105] The activity of the aforementioned protein (polypeptide) can be improved by introducing an exogenous protein (polypeptide) or by improving the activity of an endogenous protein (polypeptide). Whether or not the activity of the aforementioned protein (polypeptide) has improved can be confirmed by the degree of activity, expression level, or increase in the amount of product produced by the activity of the said protein (polypeptide).

[0106] The improvement of the activity of the aforementioned protein (polypeptide) may be carried out by various methods well known in the field, and any method that improves the activity of the target protein (polypeptide) compared to that of the host cell (microorganism) before modification is acceptable. Specifically, this includes, but is not limited to, conventional methods in molecular biology, including genetic engineering and / or protein engineering that are well known to those with ordinary skill in the field (see, for example, Non-Patent Documents 13, 14, etc.).

[0107] Specifically, the improvement of the activity of the protein (polypeptide) of this application is achieved by: 1) increasing the intracellular copy number of the polynucleotide encoding the protein (polypeptide); 2) modifying the expression regulatory region of the gene on the chromosome encoding the protein (polypeptide) (for example, introducing a mutation into the expression regulatory region, substituting it with a sequence having higher activity, or inserting a sequence having higher activity); 3) modifying the base sequence encoding the start codon or 5'UTR region of the gene transcript encoding the protein (polypeptide); 4) modifying the amino acid sequence of the protein (polypeptide) to improve protein (polypeptide) activity; and 5) improving protein (polypeptide) activity. 1) Modify the polynucleotide sequence encoding the protein (polypeptide) (for example, modifying the polynucleotide sequence of the protein (polypeptide) coding gene to encode a protein (polypeptide) modified to improve protein (polypeptide) activity); 6) Introducing a foreign protein (polypeptide) exhibiting protein (polypeptide) activity or a foreign polynucleotide encoding it; 7) Optimizing the codons of the polynucleotide encoding the protein (polypeptide); 8) Analyzing the tertiary structure of the protein (polypeptide), selecting and modifying or chemically modifying exposed portions; 9) Regulating the cellular localization of the protein (polypeptide); or 10) Combining two or more of the above 1) to 9), but is not limited to these.

[0108] For example, increasing the intracellular copy number of the polynucleotide encoding the protein (polypeptide) described in 1) above may be carried out by introducing a vector containing the polynucleotide encoding the protein (polypeptide) operably linked to a suitable regulatory sequence into a host cell (microorganism). Alternatively, it may be carried out by introducing one or more copies of the polynucleotide encoding the protein (polypeptide) operably linked to a suitable regulatory sequence into the chromosomes within the host cell (microorganism). The introduction into the chromosomes is carried out by introducing a vector into the host cell (microorganism) that is capable of inserting the polynucleotide into the chromosomes within the host cell (microorganism), but is not limited to this. The vector is as described above. The regulatory sequence may be a sequence that is natural (of the same origin) as the encoding polynucleotide sequence, a foreign (derived from another gene) sequence, a variant sequence thereof, or another artificial sequence, and may also induce the expression of the polynucleotide within the host cell (microorganism).

[0109] 2) The substitution of a gene expression regulatory region (or expression regulatory sequence) on a chromosome encoding a protein (polypeptide) with a more potent sequence may be carried out, for example, by introducing a sequence mutation through deletion, insertion, substitution, or a combination thereof, or by substituting it with a sequence having higher activity, so as to further improve the activity of the expression regulatory region. The expression regulatory region includes, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, sequences regulating transcription and translation termination, etc. For example, this may be carried out by substituting the original promoter with a more potent promoter, but is not limited to this.

[0110] Examples of known strong promoters include, but are not limited to, the cj1-cj7 promoter (Patent Document 3), 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 4), the O2 promoter (Patent Document 5), the tkt promoter, and the yccA promoter.

[0111] 3) Modifying the base sequence of the start codon or 5'UTR region of a gene encoding a protein (polypeptide) can be done, for example, by modifying it to encode another start codon that has a higher protein (polypeptide) expression rate than the endogenous start codon, or by modifying it to encode an RBS (ribosome binding site) sequence that has a higher protein (polypeptide) expression rate than the endogenous RBS sequence, but is not limited to these methods.

[0112] Modifying the amino acid sequence or polynucleotide sequence of the proteins (polypeptides) described in 4) and 5) above is carried out by introducing sequence mutations through deletion, insertion, substitution, or combination thereof in the amino acid sequence or polynucleotide sequence encoding the protein (polypeptide) to improve the activity of the protein (polypeptide), or by substituting it with an amino acid sequence or polynucleotide sequence modified to improve activity, but is not limited to these methods. Such substitution is carried out, for example, by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited to this method.

[0113] The introduction of a foreign polynucleotide exhibiting the activity of a protein (polypeptide) as described in 6) above may be carried out by introducing a foreign polynucleotide encoding a protein (polypeptide) exhibiting the same or similar activity as the aforementioned protein (polypeptide) into a host cell (microorganism). The foreign polynucleotide may have any origin or sequence as long as it exhibits the same or similar activity as the aforementioned protein (polypeptide). The introduction can be carried out by a person skilled in the art who appropriately selects a known transformation method, and as described above, the introduction of the polynucleotide into the host cell results in the production of a protein (polypeptide) and improvement of its activity.

[0114] The optimization of codons of polynucleotides encoding proteins (polypeptides) described in 7) above may be carried out by optimizing endogenous polynucleotide codons so as to increase transcription or translation within the host cell (microorganism), or by optimizing exogenous polynucleotide codons so as to perform optimized transcription or translation within the host cell (microorganism).

[0115] The 8) analysis of the tertiary structure of a protein (polypeptide), and the selection and modification or chemical modification of exposed portions may be carried out, for example, by comparing the sequence information of the protein (polypeptide) to be analyzed with a database containing sequence information of known proteins, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on these, and selecting and modifying or chemically modifying exposed portions.

[0116] 9) The regulation of the intracellular location of a protein (polypeptide) may be carried out by targeting the protein (polypeptide) to a specific intracellular organelle or specific intracellular space. For example, this can be done by adding or removing a leader sequence that functions to target the protein (polypeptide), thereby targeting the periplasm or cytoplasm, but is not limited to these.

[0117] Such improvement in protein (polypeptide) activity is achieved by increasing the activity or concentration of the corresponding protein (polypeptide) compared to the activity or concentration of the protein (polypeptide) expressed in the wild-type or unmodified host cell (microorganism), or by increasing the amount of the product produced by the activity of the protein (polypeptide), but is not limited to these methods.

[0118] In the microorganisms of this application, modification of part or all of the polynucleotides can be induced by (a) homologous recombination using a chromosome introduction vector in the microorganism, or by genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) by light and / or chemical treatment such as ultraviolet light or radiation.

[0119] The microorganisms of this application may have improved L-amino acid production capacity.

[0120] In this application, "L-amino acids" include all L-amino acids produced by microorganisms through metabolic processes from various carbon sources, and specifically include, but are not limited to, L-histidine, L-isoleucine, L-tryptophan, or L-lysine.

[0121] As a specific example of this application, the Corynebacterium microorganism may have improved ability to produce at least one amino acid selected from L-histidine, L-isoleucine, L-tryptophan, and L-lysine compared to an unmodified microorganism.

[0122] Further embodiments of this application provide a method for producing L-amino acids, comprising the step of culturing a microorganism containing the mutant polypeptide of this application, a polynucleotide encoding the mutant polypeptide, or a vector containing the polynucleotide in a culture medium.

[0123] The aforementioned microorganisms are as described above.

[0124] In this application, "cultivation" means growing the microorganisms of this application under appropriately adjusted environmental conditions. The cultivation process of this application can be carried out using suitable culture media and cultivation conditions known in the art. Such a cultivation process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the cultivation is batch, continuous, and / or fed-batch culture, but is not limited to these.

[0125] The microorganisms of this application can be cultured under aerobic conditions in a conventional culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, by adjusting the temperature, pH, etc.

[0126] In this application, the carbon source can be carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration liquid can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and any other carbon source in an appropriate amount may be used. These carbon sources can be used individually or in combination of two or more, but are not limited to these uses.

[0127] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate can be used, as well as organic nitrogen sources such as amino acids like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration liquid, casein hydrolysates, fish or their decomposition products, defatted soybean cake or its decomposition products. These nitrogen sources can be used individually or in combination of two or more, but are not limited to these uses.

[0128] As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or equivalent sodium-containing salts can be used. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., can be used, and in addition, amino acids, vitamins, and / or suitable precursors can be used. These components or precursors can be added to the culture medium in batches or continuously, but are not limited to these.

[0129] The pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture medium in a suitable manner during the cultivation of the microorganisms of this application. Furthermore, during cultivation, the formation of bubbles can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. In addition, oxygen or oxygen-containing gas may be injected into the culture medium to maintain an aerobic state, and to maintain an anaerobic and microaerobic state, no gas injection is necessary, but nitrogen, hydrogen, or carbon dioxide gas may be injected, although the invention is not limited to these.

[0130] Furthermore, the culture medium may contain metal salts such as magnesium sulfate and iron sulfate, which are necessary for growth. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins may be used. Also, a precursor suitable for the culture medium may be used. The aforementioned raw materials are added in batch by batch or continuously during the culture process in a manner suitable for the culture, but are not limited to these.

[0131] In this application, the pH of a microbial culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture in a manner suitable for the culture. Furthermore, during cultivation, the formation of bubbles can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. In addition, oxygen or oxygen-containing gas may be injected into the culture to maintain an aerobic state, and to maintain an anaerobic and microaerobic state, no gas injection is necessary, but nitrogen, hydrogen, or carbon dioxide gas may be injected, although the invention is not limited to these.

[0132] In the culture described in this application, the culture temperature is maintained at 20-35°C, specifically 25-35°C, and the culture period is continued until a useful amount of substance is obtained, and is approximately 10-160 hours, approximately 20-130 hours, approximately 24-120 hours, approximately 36-120 hours, approximately 48-120 hours, approximately 48 hours or more, approximately 48 hours, approximately 72 hours, or approximately 120 hours, but is not limited to these.

[0133] The L-amino acids produced by the culture described in this application are either secreted into the culture medium or remain within the cells.

[0134] As a specific example of this application, the L-amino acid is at least one selected from L-histidine, L-isoleucine, L-tryptophan, and L-lysine, but is not limited to these.

[0135] In one specific example, the L-amino acid production method of the present application may further include, for example, the steps of preparing the microorganism of the present application, preparing a culture medium for culturing the strain, or a combination thereof (in any order) before the culturing step.

[0136] The method for producing L-amino acids according to this application may further include a step of recovering a target substance, specifically an L-amino acid, from the cultured microorganism, the culture product of the microorganism, the fermented product of the microorganism, or the culture medium. The recovery step may further include a step after the culture step.

[0137] The aforementioned recovery may involve collecting the target L-amino acids using a suitable method known in the art, depending on the microorganism culture method of this application, such as batch, continuous, or fed-batch culture. For example, various chromatography methods such as centrifugation, filtration, crystallization, treatment with protein precipitants (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof can be used, and the target substance, specifically L-amino acids, can be recovered from the culture medium or microorganism using a suitable method known in the art.

[0138] Furthermore, the L-amino acid production method of this application may further include a purification step. The purification can be carried out by a preferred method known in the art. For example, if the L-amino acid production method of this application includes both a recovery step and a purification step, the recovery step and the purification step may be carried out sequentially or discontinuously, regardless of order, simultaneously, or integrated as a single step, but are not limited thereto.

[0139] In the method of this application, the mutant polypeptide, introduction, L-amino acid, etc., are as described above.

[0140] Further embodiments of this application provide a composition for L-amino acid production comprising at least one selected from the mutant polypeptide of this application, a polynucleotide encoding the mutant polypeptide, a vector containing the same, a microorganism containing the vector, a culture of the microorganism, and a fermentate of the microorganism.

[0141] The composition of this application may further contain any suitable excipients commonly used in compositions for L-amino acid production. Examples of such excipients include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0142] As a specific example, each component present in the composition of this application is included in a microbiologically effective amount or in an appropriate amount for production.

[0143] In the composition of this application, the mutant polypeptide, introduced L-amino acids, etc., are as described above.

[0144] A further aspect of this application provides for the use of microorganisms with improved L-amino acid production capacity, which are introduced into the mutant polypeptide of this application and at least one selected from the polynucleotides encoding the mutant polypeptide, for L-amino acid production.

[0145] In the use of this application, the mutant polypeptide, introduction, L-amino acid, etc., are as described above.

[0146] A further aspect of this application provides a method for producing a microorganism with improved L-amino acid production ability, comprising the step of introducing the mutant polypeptide of this application and at least one selected from the polynucleotide encoding the mutant polypeptide into a Corynebacterium microorganism having L-amino acid production ability.

[0147] A further aspect of this application provides a method for improving the L-amino acid production ability of a Corynebacterium microorganism having L-amino acid production ability, comprising the step of introducing the mutant polypeptide of this application and at least one selected from the polynucleotide encoding the mutant polypeptide.

[0148] In the method of this application, the mutant polypeptide, introduction, L-amino acid, etc., are as described above. [Examples]

[0149] The present application will be described in more detail below with reference to examples and experimental cases. However, these examples and experimental cases are merely illustrative of the present application and the present application is not limited to these examples and experimental cases. [Examples]

[0150] Preparation of recombinant vectors for introducing fructokinase mutations We attempted to create a Corynebacterium glutamicum mutant with improved fructokinase activity. Specifically, to create a Tn1 deletion mutant (a gene encoding a transposon in Corynebacterium glutamicum) and a target gene insertion vector, we used the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and performed PCR using primer pairs of SEQ ID NOs. 9 and 10, and SEQ ID NOs. 11 and 12. PfuUltra™ high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction. The PCR conditions were denaturation at 95°C for 30 seconds, denaturation at 55°C for 30 seconds, and polymerization at 72°C for 1 minute. This denaturation, annealing, and polymerization reaction was repeated 28 times. As a result, DNA fragments of 511 bp and 526 bp were obtained, respectively.

[0151] [Table 1]

[0152] Next, using the genomic DNA of wild-type Escherichia coli ATCC9637 as a template, PCR was performed using primer pairs of SEQ ID NOs. 13 and 14 to obtain a wild-type csck(E.co) gene fragment. Information on the nucleotide sequence of this gene and its surrounding tissues (registration number CP002967.1) was obtained from the National Institutes of Health Gene Bank (NIH GenBank).

[0153] Furthermore, using the genomic DNA of Escherichia coli ATCC9637 as a template, PCR was performed using primer pairs of SEQ ID NOs. 13 and 15 and primer pairs of SEQ ID NOs. 16 and 14, respectively. Then, using a mixture of the resulting PCR fragments as a template, overlapping PCR was performed using primer pairs of SEQ ID NOs. 13 and 14 to obtain the "cscK(W79C,E.co)" gene fragment. Similarly, using the genomic DNA of Escherichia coli ATCC9637 as a template, PCR was performed using primer pairs of SEQ ID NOs. 13 and 17 and primer pairs of SEQ ID NOs. 18 and 14, respectively. Using a mixture of the two resulting fragments as a template, overlapping PCR was again performed using primer pairs of SEQ ID NOs. 13 and 14 to obtain the "cscK(A181V,E.co)" gene fragment. Furthermore, to generate combination mutants, the genomic DNA of Escherichia coli ATCC9637 was used as a template, and PCR was performed using primer pairs of SEQ ID NO: 13 and 15, SEQ ID NO: 16 and 17, and SEQ ID NO: 18 and 14. The resulting mixture of three fragments was then used as a template, and overlapping PCR was performed again using primer pairs of SEQ ID NO: 13 and 14 to obtain the "cscK(W79C / A181V,E.co)" gene fragment. The PCR conditions were as described above.

[0154] [Table 2]

[0155] Next, in order to use the CJ7 promoter derived from Corynebacterium ammoniagenes (SEQ ID NO: 19, Patent Document 6), a CJ7 promoter fragment was obtained by performing PCR in the same manner as described above, using the genomic DNA of Corynebacterium ammoniagenes as a template and primers SEQ ID NO: 20 and SEQ ID NO: 21.

[0156] [Table 3]

[0157] Each obtained DNA product was purified using a PCR purification kit (QUIAGEN). Recombinant plasmids were obtained by cloning the purified DNA products and the chromosome transformation vector pDC24 (SEQ ID NO: 85), which had been cleaved with SmaI restriction enzyme, using the Gibson assembly method (Non-Patent Literature 15). These plasmids were named pDC24△Tn1::cj7_cscK(E.co), pDC24△Tn1::Pcj7_cscK(W79C,E.co), pDC24△Tn1::Pcj7_cscK(A181V,E.co), and pDC24△Tn1::Pcj7_cscK(W79C / A181V,E.co), respectively. Cloning was performed by mixing the Gibson assembly reagent with each gene fragment in the calculated moles and then storing at 50°C for 1 hour. [Examples]

[0158] Evaluation of the productivity of L-amino acid-producing strains into which the cscK mutant gene has been introduced. Example 2-1. Preparation of histidine-producing strains and evaluation of histidine production capacity. Example 2-1-1. Preparation of histidine-producing bacterial strains To evaluate the L-histidine production ability of the strain into which the cscK mutant gene created in Example 1 was introduced, we first created the CA14-0114 strain, which was enhanced with genes in the histidine biosynthesis pathway, using wild-type Corynebacterium glutamicum ATCC13032 as the starting microorganism. Specifically, to eliminate the feedback inhibition of the HisG protein, the first enzyme in the L-histidine biosynthesis pathway, the codons of the hisG gene were modified so that a protein (SEQ ID NO: 22) (Non-Patent Literature 16) is expressed in which the 233rd and 235th amino acids from the N-terminus of HisG are simultaneously substituted, with glycine being replaced with histidine and with threonine with glutamine, respectively. In addition, to enhance the activity of the hisE gene, which is located in the same operon as hisG, the start codon was replaced from GTG to ATG. Furthermore, to enhance the L-histidine biosynthesis pathway, the promoters of the biosynthetic genes hisN, hisH, hisD, hisA, and hisB were replaced with stronger promoters, and the pathway was further strengthened by introducing the hisE(g1a)G(G233H / T235Q) operon and additional copies of the hisD gene.

[0159] Example 2-1-1-1. Preparation of a histidine-producing strain with eliminated feedback inhibition. To create a histidine-producing strain with the feedback inhibition eliminated, PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, with primer pairs of SEQ ID NOs. 23 and 24, and primer pairs of SEQ ID NOs. 25 and 26. The PCR reaction was performed in the same manner as in Example 1. Using the two amplified DNA fragments as described above as templates, PCR was performed in the same manner as in Example 1 using primers of SEQ ID NOs. 23 and 26 to obtain the "hisE(g1a)G(G233H / T235Q)" gene fragment. Furthermore, the upstream region of the hisE gene was obtained by performing PCR using the chromosomal DNA of ATCC13032 as a template, with primers of SEQ ID NOs. 27 and 28.

[0160] [Table 4]

[0161] To replace the promoter with a stronger one, PCR was performed in the same manner as in Example 1, using the synthetic promoter Pspl13 promoter (SEQ ID NO: 29, Patent Document 7) as a template and primers SEQ ID NOs: 30 and 31.

[0162] The pDC24 vector was treated with the restriction enzyme Sma1, and then the upstream DNA fragment of the amplified hisE gene, the Pspl13 promoter, and the "hisE(g1a)G(G233H / T235Q)" gene fragment were cloned by Gibson assembly to obtain a recombinant plasmid. This plasmid was named pDC24△Pn_hisEG::Pspl13_hisE(g1a)G(G233H / T235Q). Gibson cloning was performed in the same manner as in Example 1.

[0163] The prepared pDC24△Pn_hisEG::Pspl13_hisE(g1a)G(G233H / T235Q) vector was used to transform Corynebacterium glutamicum ATCC13032 via electroporation. Subsequently, a secondary crossover process was followed, and a mutation was introduced into the conventional hisG gene to eliminate feedback inhibition. By substituting the start codon of the hisE gene, a strain with enhanced hisE activity was obtained. The genetic manipulation was confirmed by PCR using primers SEQ ID NOs. 32 and 33, and by genome sequencing, which amplified the external regions of the upstream and downstream regions of the homologous recombination in which the gene was inserted, respectively. The strain obtained in this way was named CJ-HIS1.

[0164] Example 2-1-1-2. Preparation of a histidine-producing strain with an enhanced biosynthetic pathway by promoter substitution. Next, plasmids were constructed to replace the wild-type promoters of the biosynthetic genes hisN, hisH, hisD, hisA, and hisB with strong promoters in order to enhance their activity.

[0165] Specifically, using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template, PCR was performed in the same manner as in Example 1 using the primers SEQ ID NOs. 34 and 35, SEQ ID NOs. 36 and 37, SEQ ID NOs. 38 and 39, SEQ ID NOs. 40 and 41, and SEQ ID NOs. 42 and 43 to obtain the upstream regions of the hisN, hisH, hisD, hisA, and hisB genes. Furthermore, using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template, the downstream regions of the hisN, hisH, hisD, hisA, and hisB genes were obtained, respectively, using the primers SEQ ID NOs. 44 and 45, SEQ ID NOs. 46 and 47, SEQ ID NOs. 48 and 49, SEQ ID NOs. 50 and 51, and SEQ ID NOs. 52 and 53.

[0166] To replace the endogenous promoters of the hisN, hisH, and hisD genes with the strong promoter Pcj7 promoter, PCR was performed using Corynebacterium ammoniagenes genomic DNA as a template and primers SEQ ID NOs. 54 and 55, SEQ ID NOs. 56 and 57, and SEQ ID NOs. 58 and 59, in the same manner as in Example 1.

[0167] Furthermore, in order to replace the endogenous promoters of the hisA and hisB genes with the strong promoter Pspl13 promoter, PCR was performed in the same manner as in Example 1, using the Pspl13 promoter as a template and primers SEQ ID NO: 60 and SEQ ID NO: 61, and SEQ ID NO: 62 and SEQ ID NO: 63.

[0168] Recombinant plasmids were obtained by treating the pDC24 vector with the restriction enzyme Sma1, and then cloning the upstream DNA fragments of the amplified hisN, hisH, and hisD genes, the Pcj7 promoter fragment, and the downstream DNA fragments of the hisN, hisH, and hisD genes using Gibson assembly. These were named pDC24△Pn::cj7_hisN, pDC24△Pn::cj7_hisH, and pDC24△Pn::cj7_hisD. Furthermore, recombinant plasmids were obtained by treating the pDC24 vector with the restriction enzyme Sma1, and then cloning the upstream DNA fragments of the amplified hisA and hisB genes, the Pspl13 promoter fragment, and the downstream DNA fragments of hisA and hisB genes using Gibson assembly. These were named pDC24△Pn::Pspl13_hisA and pDC24△Pn::Pspl13_hisB. Gibson cloning was performed in the same manner as in Example 1.

[0169] The prepared pDC24△Pn::cj7_hisN vector was used to transform CJ-HIS1 prepared in Example 2-1-1-1 by electroporation. Subsequently, a strain with enhanced hisN gene was obtained by substituting the promoter of the original hisN gene through a secondary crossover process. The genetic manipulation was confirmed by PCR using primers SEQ ID NOs. 64 and 65, and by genome sequencing, which amplified the external regions of the upstream and downstream regions of the homologous recombination in which the enhanced gene was inserted, respectively. The strain obtained in this way was named CJ-HIS2.

[0170] Next, the prepared pDC24△Pn::cj7_hisH vector was used to transform the CJ-HIS2 strain prepared as described above by electroporation. Subsequently, a secondary crossover process was carried out, and a strain with enhanced hisH gene was obtained by replacing the promoter in the conventional hisH gene. The genetic manipulation was confirmed by PCR using primers SEQ ID NO: 66 and SEQ ID NO: 67, and by genome sequencing, which amplified the external regions of the upstream and downstream regions of the homologous recombination in which the enhanced gene was inserted, respectively. The strain obtained in this way was named CJ-HIS3.

[0171] Next, the prepared pDC24△Pn::cj7_hisD vector was used to transform the CJ-HIS3 strain prepared as described above by electroporation. Subsequently, a secondary crossover process was carried out, and the promoter of the original hisD gene was replaced to obtain a strain in which the gene was strengthened. This genetic manipulation was confirmed by PCR using primers SEQ ID NO: 68 and SEQ ID NO: 69, and by genome sequencing. The strain obtained in this way was named CJ-HIS4.

[0172] Next, the prepared pDC24△Pn::Pspl13_hisA vector was used to transform the CJ-HIS4 strain prepared as described above by electroporation. Subsequently, a secondary crossover process was carried out, and the promoter of the original hisA gene was replaced to obtain a strain in which the gene was strengthened. This genetic manipulation was confirmed by PCR using primers SEQ ID NO: 70 and SEQ ID NO: 71, and by genome sequencing. The strain obtained in this way was named CJ-HIS5.

[0173] Next, the prepared pDC24△Pn::Pspl13_hisB vector was used to transform the CJ-HIS5 strain prepared as described above by electroporation. Subsequently, a secondary crossover process was carried out, and the promoter of the original hisB gene was replaced to obtain a strain in which the gene was strengthened. This genetic manipulation was confirmed by PCR using primers SEQ ID NO: 72 and SEQ ID NO: 73, and by genome sequencing. The strain obtained in this way was named CJ-HIS6.

[0174] [Table 5] JPEG2026531073000006.jpg139149

[0175] Example 2-1-1-3. Preparation of a histidine-producing strain with an enhanced biosynthetic pathway through gene insertion. Next, using NCgl1021, a gene known to encode a transposon in Corynebacterium glutamicum, as an insertion site, the hisE(g1a)G(G233H / T235Q) operon and the hisD gene were additionally inserted. Specifically, in order to create NCgl1021 (SEQ ID NO: 74) deletion and target gene insertion vectors, the chromosome of ATCC13032 was used as a template, and PCR was performed in the same manner as in Example 1 using primer pairs of SEQ ID NO: 75 and SEQ ID NO: 76, and SEQ ID NO: 77 and SEQ ID NO: 78, thereby amplifying the left homologous arm region and the right homologous arm region of NCgl1021. Using the vector pDC24△Pn::Pspl13_hisE(g1a)G(G233H / T235Q) prepared in Example 2-1-1-1 as a template, PCR was performed in the same manner as in Example 1 using primers of Sequence ID No. 79 and Sequence ID No. 80 to obtain the "Pspl13_hisE(g1a)G(G233H / T235Q)" gene fragment.

[0176] Furthermore, the "Pcj7_hisD" gene fragment was obtained by performing PCR in the same manner as in Example 1, using the vector pDC24△Pn::cj7_hisD prepared in Example 2-1-1-2 as a template and primers SEQ ID NO: 81 and SEQ ID NO: 82.

[0177] The pDC24 vector was treated with the restriction enzyme Sma1, and then the amplified left homologous arm region of NCgl1021, the right homologous arm region of NCgl1021, and the gene fragments "Pspl13_hisE(g1a)G(G233H / T235Q)" and "Pcj7_hisD" were cloned by Gibson assembly to obtain a recombinant plasmid. This plasmid was named "pDC24△NCgl1021::Pspl13_hisE(g1a)G(G233H / T235Q)-Pcj7_hisD". Gibson cloning was performed in the same manner as in Example 1. The prepared vector "pDC24△NCgl1021::Pspl13_hisE(g1a)G(G233H / T235Q)-Pcj7_hisD" was used to transform CJ-HIS6 prepared in Example 2-1-1-2 by electroporation. Subsequently, a strain was obtained in which the histidine biosynthesis pathway was enhanced by the insertion of an additional gene through a secondary cross-reaction process. The genetic manipulation was confirmed by PCR using primers SEQ ID NOs. 83 and 84, respectively, to amplify the external regions of the upstream and downstream regions of the homologous recombination in which the gene was inserted, and by genome sequencing. The strain obtained in this way was named CA14-0114.

[0178] [Table 6]

[0179] Example 2-1-2. Preparation of a transformed strain by introducing the csck(E.co) mutant gene into a histidine-producing strain. The recombinant vectors pDC24△Tn1::Pcj7_cscK(E.co), pDC24△Tn1::Pcj7_cscK(W79C,E.co), pDC24△Tn1::Pcj7_cscK(A181V,E.co), and pDC24△Tn1::Pcj7_cscK(W79C / A181V,E.co) prepared in Example 1 were used to transform the histidine-producing strain CA14-0114 prepared in Example 2-1-1 using the electropulse method, and then transformed strains were obtained from a selective medium containing 25 mg / L of kanamycin. These were named CA14-0114△Tn1::Pcj7_cscK(E.co), CA14-0114△Tn1::Pcj7_cscK(W79C,E.co), CA14-0114△Tn1::Pcj7_cscK(A181V,E.co), and CA14-0114△Tn1::Pcj7_cscK(W79C / A181V,E.co), respectively.

[0180] Example 2-1-3. Evaluation of histidine production capacity To confirm the L-histidine production ability of the strains prepared in Example 2-1-2, they were cultured and evaluated using the following method. Each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of the following seed medium and cultured with shaking at 30°C and 200 rpm for 20 hours. Next, 1 ml of seed culture solution was inoculated into a 250 ml corner baffle flask containing 25 ml of production medium and cultured with shaking at 30°C and 200 rpm for 48 hours. <Seed culture medium (pH 7.0)> Glucose 5%, bactopeptone 1%, sodium chloride 0.25%, yeast extract 1%, urea 0.4% (per liter of distilled water) <Production medium (pH 7.0)> Raw sugar 6%, ammonium sulfate 2%, potassium dihydrogen phosphate 0.1%, magnesium sulfate heptahydrate 0.05%, CSL (corn maceration solution) 2.0%, biotin 200 μg / L, calcium carbonate 30 g / L (per liter of distilled water)

[0181] [Table 7]

[0182] As shown in the table above, introducing the csck wild-type gene from Escherichia coli resulted in an approximately 4.6% increase in histidine production compared to the parent strain CA14-0114. Furthermore, introducing the cscK mutant gene resulted in an approximately 2.2% to 8.8% increase in histidine production compared to CA14-0114△Tn1::Pcj7-cscK(WT,E.co) which had the wild-type cscK gene introduced. Thus, it was found that csck gene mutations improve fructokinase activity, thereby enhancing the L-histidine production capacity of the strain.

[0183] Example 2-2. Preparation of isoleucine-producing bacterial strains with introduced CscK mutation and evaluation of isoleucine production capacity. Example 2-2-1. Preparation of a transformed strain by introducing the csck(E.co) mutant gene into an isoleucine-producing bacterial strain. To confirm whether introducing a fructokinase mutant gene derived from Escherichia coli improves the isoleucine production ability of Corynebacterium glutamicum strains capable of producing L-isoleucine, the recombinant vectors pDC24△Tn1::Pcj7_cscK(E.co), pDC24△Tn1::Pcj7_cscK(W79C,E.co), pDC24△Tn1::Pcj7_cscK(A181V,E.co), and pDC24△Tn1::Pcj7_cscK(W79C / A181V,E.co) prepared in Example 1 were used to transform the isoleucine-producing strain KCCM12739P (Patent Document 8) using the electropulse method. Subsequently, transformed strains were obtained from a selective medium containing 25 mg / L of kanamycin. These were named KCCM12739P△Tn1::Pcj7_cscK(E.co), KCCM12739P△Tn1::Pcj7_cscK(W79C,E.co), KCCM12739P△Tn1::Pcj7_cscK(A181V,E.co), and KCCM12739P△Tn1::Pcj7_cscK(W79C / A181V,E.co), respectively.

[0184] Example 2-2-2. Evaluation of isoleucine production capacity To confirm the L-isoleucine production ability of the bacterial strains prepared in Example 2-2-1, they were cultured and evaluated using the following method. The parent strain and the mutant strain were inoculated into a 250 ml corner baffle flask containing 25 ml of isoleucine production medium, and then cultured with shaking at 32°C and 200 rpm for 60 hours. <Production medium (pH 7.2)> Glucose 10%, Yeast extract 0.2%, Ammonium sulfate 1.6%, Potassium dihydrogen phosphate 0.1%, Magnesium sulfate heptahydrate 0.1%, Iron sulfate heptahydrate 10 mg / l, Manganese sulfate monohydrate 10 mg / l, Biotin 200 μg / l (per 1 L of distilled water)

[0185] [Table 8]

[0186] As shown in the table above, introducing the csck wild-type gene from Escherichia coli resulted in an approximately 9.5% increase in isoleucine production compared to the parent strain KCCM12739P. Furthermore, introducing the cscK mutant gene resulted in an approximately 8.6% to 30.4% increase in isoleucine production compared to KCCM12739P△Tn1::Pcj7-cscK(WT,E.co) which had the wild-type cscK gene introduced. Thus, it was found that csck gene mutations improve fructokinase activity, thereby enhancing the L-isoleucine production capacity of the strain.

[0187] Examples 2-3. Preparation of tryptophan-producing bacterial strains with introduced CscK mutations and evaluation of tryptophan production capacity. Example 2-3-1. Preparation of a transformed strain by introducing the csck(E.co) mutant gene into a tryptophan-producing strain. To confirm whether introducing a fructokinase mutant gene derived from Escherichia coli improves tryptophan production in Corynebacterium glutamicum strains capable of producing L-tryptophan, the recombinant vectors pDC24△Tn1::Pcj7_cscK(E.co), pDC24△Tn1::Pcj7_cscK(W79C,E.co), pDC24△Tn1::Pcj7_cscK(A181V,E.co), and pDC24△Tn1::Pcj7_cscK(W79C / A181V,E.co) prepared in Example 1 were used to transform the tryptophan-producing strain CA04-8405 (Patent Document 9) using the electropulse method. Subsequently, transformed strains were obtained from a selective medium containing 25 mg / L of kanamycin. These were named CA04-8405△Tn1::Pcj7_cscK(E.co), CA04-8405△Tn1::Pcj7_cscK(W79C,E.co), CA04-8405△Tn1::Pcj7_cscK(A181V,E.co), and CA04-8405△Tn1::Pcj7_cscK(W79C / A181V,E.co), respectively.

[0188] Example 2-3-2. Evaluation of tryptophan production capacity To confirm the L-tryptophan production ability of the strains prepared in Example 2-3-1, they were cultured and evaluated using the following method. Each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of seed medium and cultured with shaking at 30°C and 200 rpm for 20 hours. After culturing, a new 250 ml corner baffle flask containing 25 ml of production medium was prepared for each strain, and 1 ml of the seed culture solution was inoculated into it. The strains were then cultured with shaking at 30°C and 200 rpm for 24 hours. <Seed culture medium (pH 7.0)> Glucose 20g, Peptone 10g, Yeast extract 5g, Urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4·7H2O 0.5g, Biotin 100μg, Thiamine HCl 1000μg, Calcium pantothenate 2000μg, Nicotinamide 2000μg (per 1L of distilled water) <Production medium (pH 7.0)> Glucose 30g, (NH4)2SO4 15g, MgSO4·7H2O 1.2g, KH2PO4 1g, Yeast extract 5g, Biotin 900μg, Thiamine hydrochloride 4500μg, Calcium pantothenate 4500μg, CaCO3 30g (in 1L of distilled water)

[0189] [Table 9]

[0190] As shown in the table above, introducing the csck wild-type gene from Escherichia coli resulted in increased tryptophan production compared to the parent strain CA04-8405. Furthermore, introducing the cscK mutant gene resulted in an increase in tryptophan production of approximately 12.5% ​​to 25% compared to CA04-8405△Tn1::Pcj7-cscK(WT,E.co) which had the wild-type cscK gene introduced. Thus, it was found that csck gene mutations improve fructokinase activity, thereby enhancing the L-tryptophan production capacity of the strain.

[0191] Example 2-4. Preparation of lysine-producing strains with introduced CscK mutation and evaluation of lysine production capacity. Example 2-4-1. Preparation of a transformed strain by introducing the csck(E.co) mutant gene into a lysine-producing strain. To confirm whether introducing a fructokinase mutant gene derived from Escherichia coli improves lysine production in Corynebacterium glutamicum strains capable of L-lysine production, the recombinant vectors pDC24△Tn1::Pcj7_cscK(E.co), pDC24△Tn1::Pcj7_cscK(W79C,E.co), pDC24△Tn1::Pcj7_cscK(A181V,E.co), and pDC24△Tn1::Pcj7_cscK(W79C / A181V,E.co) prepared in Example 1 were used to transform the lysine-producing strain KCCM11016P (Patent Document 10) using the electropulse method. Subsequently, transformed strains were obtained from a selective medium containing 25 mg / L of kanamycin. These were named KCCM11016P△Tn1::Pcj7_cscK(E.co), KCCM11016P△Tn1::Pcj7_cscK(W79C,E.co), KCCM11016P△Tn1::Pcj7_cscK(A181V,E.co), and KCCM11016P△Tn1::Pcj7_cscK(W79C / A181V,E.co), respectively.

[0192] Example 2-4-2. Evaluation of lysine production capacity To confirm the L-lysine production ability of the strains prepared in Example 2-4-1, they were cultured and evaluated using the following method: Each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of seed medium and cultured with shaking at 37°C and 200 rpm for 20 hours. Next, each strain was inoculated into a 250 ml corner baffle flask containing 25 ml of production medium and cultured with shaking at 37°C and 200 rpm for 48 hours. <Seed culture medium (pH 7.0)> Glucose 20g, Peptone 10g, Yeast extract 5g, Urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4·7H2O 0.5g, Biotin 0.1mg, Thiamine HCl 1mg, Calcium pantothenate 2mg, Nicotinamide 2mg (per 1L of distilled water) <Production medium (pH 7.0)> Glucose 50g, (NH4)2SO4 40g, Corn Steep Solids 5g, BM 10g, KH2PO4 1g, MgSO4·7H2O 0.5g, Biotin 100μg, Thiamine Hydrochloride 1000μg, Calcium Pantothenate 2000μg, Nicotinamide 3000μg, CaCO3 30g (in 1 liter of distilled water)

[0193] [Table 10]

[0194] As shown in the table above, introducing the csck wild-type gene from Escherichia coli resulted in increased lysine production compared to the parent strain KCCM11016P. Furthermore, introducing the cscK mutant gene resulted in an increase in lysine production of approximately 3.8% to 16% compared to KCCM11016P△Tn1::Pcj7-cscK(WT,E.co) which had the wild-type cscK gene introduced. Thus, it was found that csck gene mutations improve fructokinase activity, thereby enhancing the L-lysine production capacity of the strain.

[0195] From the above explanation, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the above embodiments are merely illustrative and not limiting. This application should be interpreted as including all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. The amino acid corresponding to the 79th or 181st position of Sequence ID No. 1 is substituted with another amino acid. Fructogenase mutant polypeptide.

2. The mutant polypeptide is one in which the amino acid corresponding to position 79 of SEQ ID NO: 1 is substituted with cysteine, or the amino acid corresponding to position 181 of SEQ ID NO: 1 is substituted with valine, or a combination thereof. The mutant polypeptide according to claim 1.

3. The amino acid sequence of the mutant polypeptide has 90% or more identity with the amino acid sequence of Sequence ID No.

1. The mutant polypeptide according to claim 1.

4. The mutant polypeptide consists of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:

7. The mutant polypeptide according to claim 1.

5. Encoding a mutant polypeptide according to any one of claims 1 to 4, Polynucleotide.

6. A mutant polypeptide according to any one of claims 1 to 4, and at least one selected from the polynucleotides encoding the mutant polypeptide, Microorganisms.

7. The aforementioned microorganism belongs to the genus Corynebacterium. The microorganism according to claim 6.

8. The aforementioned microorganism of the genus Corynebacterium is Corynebacterium glutamicum. The microorganism according to claim 7.

9. The aforementioned microorganism exhibits improved L-amino acid production capacity compared to microorganisms containing the polypeptide of Sequence ID No. 1 or the polynucleotide encoding it. The microorganism according to claim 6.

10. The L-amino acid is at least one selected from L-histidine, L-isoleucine, L-tryptophan, and L-lysine. The microorganism according to claim 9.

11. The step of culturing the microorganism described in claim 6 in a culture medium, Method for producing L-amino acids.

12. The further step includes recovering L-amino acids from at least one selected from the cultured microorganism, the culture of the microorganism, the fermented product of the microorganism, and the culture medium. The method according to claim 11.

13. The L-amino acid is at least one selected from L-histidine, L-isoleucine, L-tryptophan, and L-lysine. The method according to claim 12.

14. Use of a mutant polypeptide according to any one of claims 1 to 4, or a microorganism containing the mutant polypeptide or a polynucleotide encoding the mutant polypeptide, for the production of L-amino acids.

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