Fructokinase variants and methods for producing l-amino acids using same

Abstract

The present disclosure relates to fructokinase variant polypeptides; a polynucleotide encoding the variant polypeptide; a microorganism comprising the same; a method for producing an L-amino acid, said method comprising the step of culturing said microorganism in a culture medium; or to the use of said microorganism in the production of L-amino acids.

Description

Technical Field

[0001] This disclosure relates to a fructokinase variant polypeptide; a polynucleotide encoding the variant polypeptide; a vector comprising the thereof; a microorganism comprising the polypeptide, polynucleotide, and / or vector; a method for producing L-amino acids, the method comprising the step of culturing the microorganism in a culture medium; a composition for producing L-amino acids, the composition comprising any one or more selected from the microorganism, a culture of the microorganism, and a fermentation product of the microorganism; or the use of the microorganism in the production of L-amino acids. Background Technology

[0002] Rod-shaped microorganisms are Gram-positive microorganisms that are frequently used in the industrial production of substances with various applications, such as feed, pharmaceuticals, and food containing L-amino acids and various nucleic acids. In recent years, diamines, keto acids, and other substances have also been produced from rod-shaped microorganisms.

[0003] L-amino acids are the basic structural units of proteins and are important raw materials for pharmaceuticals, food additives, animal feed, nutritional supplements, pesticides, and disinfectants. Among L-amino acids, L-lysine is an essential amino acid that cannot be biosynthesized in living organisms and is known to be essential for promoting growth, calcium metabolism, gastric juice secretion, and disease resistance. L-lysine is widely used in feed, pharmaceutical products, and food. In addition, L-tryptophan is also an essential amino acid and is used as a feed additive, infusion, pharmaceutical raw material, and health food ingredient.

[0004] Various studies have been conducted to develop microbial and fermentation processes for efficient amino acid production. For example, target-specific methods have been developed, such as increasing the expression of genes encoding enzymes involved in amino acid biosynthesis in Corynebacterium strains, or removing genes in Corynebacterium strains that are not needed for amino acid biosynthesis (US 9109242 B2, US 8030036 B2). In addition to these methods, methods for removing genes not involved in amino acid production and removing genes whose specific functions are unknown for amino acid production have also been used. However, methods for efficiently producing L-amino acids in high yields still need to be investigated. Summary of the Invention

[0005] [Technical Issues] The purpose of this disclosure is to provide microorganisms containing fructokinase variants and methods for producing L-amino acids using them.

[0006] [Technical Solution] One aspect of this disclosure provides a fructokinase variant polypeptide wherein the amino acid corresponding to position 79 or 181 of SEQ ID NO: 1 is replaced by a different amino acid.

[0007] In one specific embodiment, the variant polypeptide may have a substitution of amino acid at position 79 of SEQ ID NO: 1 to cysteine, or a substitution of amino acid at position 181 of SEQ ID NO: 1 to valine, or a combination thereof.

[0008] In another specific embodiment, the variant polypeptide may consist of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

[0009] In another specific implementation, fructokinase may be encoded by the cscK gene.

[0010] Another aspect of this disclosure provides a polynucleotide encoding a variant polypeptide.

[0011] Another aspect of this disclosure provides a microorganism comprising any one or more variant polypeptides selected from the variant polypeptides; a polynucleotide encoding the variant polypeptide; and a vector comprising the same.

[0012] Regarding the microorganism described in any of the foregoing specific embodiments, the microorganism may be a microorganism of the genus Corynebacterium.

[0013] Regarding the microorganisms described in any of the foregoing specific embodiments, the Corynebacterium genus microorganisms may be Corynebacterium glutamicum (… Corynebacterium glutamicum ).

[0014] Regarding the microorganisms described according to any of the foregoing specific embodiments, compared with microorganisms containing the polypeptide of SEQ ID NO: 1 or the polynucleotide encoding it, the microorganisms may have an increased ability to produce L-amino acids.

[0015] Regarding the microorganisms described in any of the foregoing specific embodiments, the microorganisms may have an increased ability to produce L-histidine, L-isoleucine, L-tryptophan, or L-lysine.

[0016] Another aspect of this disclosure provides a method for producing L-amino acids, the method comprising the step of culturing microorganisms in a culture medium.

[0017] In one specific implementation, the method may further include the step of recovering L-amino acids from cultured microorganisms, microbial cultures, microbial fermentation products, or culture media.

[0018] In another specific embodiment, the method may be a method for producing L-histidine, L-isoleucine, L-tryptophan, or L-lysine.

[0019] Another aspect of this disclosure provides a composition for producing L-amino acids, the composition comprising any one or more selected from: a variant polypeptide; a polynucleotide encoding the variant polypeptide; a vector containing the variant polypeptide; a microorganism containing the vector; a culture of the microorganism; and a fermentation product of the microorganism.

[0020] In one specific embodiment, the composition may be a composition for producing L-histidine, L-isoleucine, L-tryptophan, or L-lysine.

[0021] [Beneficial Effects] When microorganisms containing the fructokinase variant peptides of this disclosure are cultured, L-amino acids can be produced in high yields compared to microorganisms with existing unmodified peptides. Detailed Implementation

[0022] This disclosure will now be described in detail. Furthermore, the various descriptions and embodiments disclosed herein can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed herein fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited to the specific descriptions below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to further clarify the level and scope of the subject matter to which this disclosure pertains.

[0023] One aspect of this disclosure provides a fructokinase variant polypeptide wherein the amino acid corresponding to position 79 or 181 of SEQ ID NO: 1 is replaced by a different amino acid.

[0024] As used herein, the term "fructokinase (CscK)" can refer to an enzyme that catalyzes phosphate transfer in the presence of ATP to produce D-fructose-6-phosphate and ADP.

[0025] For the purposes of this disclosure, the protein may also be referred to as "fructose phosphorylase," "fructose kinase," or "CscK." The gene encoding the protein may be, for example, the csck gene, but is not limited thereto. In this disclosure, "csck gene" may be used interchangeably with "fructose kinase encoding gene." Furthermore, the protein may be derived from, for example, *Escherichia coli*, but its type is not particularly limited, as long as it possesses activity corresponding to fructose kinase.

[0026] Fructose kinase encoded by the csck gene is known in the art, and the amino acid and polynucleotide sequences of fructokinase can be obtained from known databases, such as NCBI GenBank, but are not limited thereto.

[0027] The fructokinase protein may comprise, but is not limited to, the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60% or higher homology or identity with it, as long as it possesses fructokinase activity. Specifically, the addition of meaningless sequences, naturally occurring mutations, or silent mutations thereof upstream or downstream of the amino acid sequence of SEQ ID NO: 1 is not excluded, and as long as the protein has the same or corresponding activity as a protein comprising the amino acid sequence of SEQ ID NO: 1, it can belong to the protein to which this disclosure introduces mutations. For example, a protein to which this disclosure introduces mutations may be a protein composed 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 higher homology or identity with it. Furthermore, it is evident that any variant of the amino acid sequence having partial sequence deletions, modifications, substitutions, conserved substitutions, or additions may also fall within the scope of polypeptides that are the targets of the introduced mutations of this disclosure, provided that it is an amino acid sequence that has such homology or identity and exhibits efficacy corresponding to the efficacy of fructokinase.

[0028] In the amino acid sequence of the fructokinase before modification, which is the mutant object of this disclosure, i.e. the sequence that serves as the parental sequence, the amino acids corresponding to positions 79 and 181 of SEQ ID NO: 1 before modification are tryptophan (W) and alanine (A), respectively.

[0029] As used herein, the terms “variant peptide,” “variant protein,” or “variant” refer to a peptide in which one or more amino acids have been conservedly substituted and / or modified to form an amino acid sequence different from the unmodified variant, while retaining its function or properties. Such variants are typically identified by modifying one or more amino acids in the peptide’s amino acid sequence and assessing the properties of the modified peptide. In other words, the ability of a variant may be increased, unchanged, or decreased compared to the unmodified peptide. Furthermore, some variants may contain one or more portions of the peptide, such as an N-terminal leader sequence or a transmembrane domain that has been removed. Other variants may include those in which a portion has been removed from the N- and / or C-terminus of the mature protein. The term “variant” may also be used interchangeably with modification, modified peptide, modified protein, mutant, mutant protein, diversifier, etc., and no term is limited thereto, as long as it is used in the sense of variation.

[0030] The variants may also include the deletion or addition of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated to the N-terminal signal (or leader) sequence of a protein involved in protein co-translation or post-translational transfer. Furthermore, the polypeptide may be conjugated to another sequence or linker to identify, purify, or synthesize the polypeptide.

[0031] The fructokinase variant polypeptide disclosed herein refers to a variant polypeptide in which the amino acid corresponding to the 79th or 181st N-terminus of SEQ ID NO: 1 is replaced by another amino acid.

[0032] The "fructokinase variant polypeptide" may be referred to as "fructokinase variant," "variant CscK," "CscK variant," etc. As a target for mutation introduction, the fructokinase polypeptide disclosed herein can be used interchangeably with "CscK," but is not particularly limited thereto. It is encoded by the cscK gene and derived from *E. coli*. cscK However, it is not limited to this.

[0033] Regarding the fructokinase variant polypeptide of this disclosure, the amino acid at position 79 or 181 of the amino acid sequence corresponding to SEQ ID NO: 1 may be replaced by an amino acid different from the amino acid before replacement.

[0034] In one specific embodiment, the variant polypeptide may have a substitution of the amino acid corresponding to position 79 of the amino acid sequence of SEQ ID NO: 1 to an amino acid other than tryptophan, or a substitution of the amino acid corresponding to position 181 of the amino acid sequence of SEQ ID NO: 1 to an amino acid other than alanine, or a combination thereof.

[0035] In another specific embodiment, regarding the variant polypeptide, the amino acid corresponding to position 79 of SEQ ID NO: 1 may be replaced by an amino acid 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 not limited thereto.

[0036] In another specific embodiment, regarding the variant polypeptide, the amino acid corresponding to position 181 of SEQ ID NO: 1 may be replaced by an amino acid 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 not limited thereto.

[0037] In any of the above embodiments, the variant polypeptide provided in this disclosure may have a substitution of cysteine ​​to amino acid at position 79 of the N-terminus of SEQ ID NO: 1, or a substitution of valine to amino acid at position 181 of the N-terminus of SEQ ID NO: 1, or a combination thereof.

[0038] In another embodiment of the above-described embodiments, the variant polypeptide provided in this disclosure 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 higher, or less than 100% homology or identity with SEQ ID NO: 1.

[0039] For example, the variant polypeptide of this disclosure may comprise an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% or higher homology or identity with SEQ ID NO: 1, wherein the amino acid corresponding to position 79 of the amino acid sequence shown in SEQ ID NO: 1 is fixed as cysteine. Furthermore, the variant polypeptide of this disclosure may comprise an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% or higher homology or identity with SEQ ID NO: 1, wherein the amino acid corresponding to position 181 of the amino acid sequence shown in SEQ ID NO: 1 is fixed as valine. Furthermore, the variant peptides of this disclosure may comprise an amino acid sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% or higher homology or identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid corresponding to position 79 of the amino acid sequence shown in SEQ ID NO: 1 is fixed as cysteine, and the amino acid corresponding to position 181 is fixed as valine. Moreover, it is apparent that any variant peptide having amino acid sequences with partial deletions, modifications, substitutions, conservative substitutions, or additions may also fall within the scope of this disclosure, provided that it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the efficacy of the variant peptides of this disclosure.

[0040] Furthermore, those skilled in the art can identify the amino acid corresponding to position 79 or 181 of the amino acid sequence of SEQ ID NO: 1 in this disclosure in any amino acid sequence by sequence alignment known in the art. Unless otherwise described in this disclosure, when describing "amino acid at a specific position of a specific sequence number", it is obvious that the amino acid also includes "amino acid at the corresponding position" in any amino acid sequence.

[0041] In another specific embodiment, the variant polypeptide may consist of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

[0042] Specifically, the variant polypeptides of this disclosure may have or contain the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, or have an amino acid sequence that has at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher homology or identity with the amino acid sequence of SEQ ID NO: 3, 5 or 7, or may be composed of said amino acid sequence, or may be substantially composed of said amino acid sequence.

[0043] Examples may include the addition or deletion of sequences at the N-terminus, C-terminus, and / or interior of an amino acid sequence that do not alter the function of the variants disclosed herein, naturally occurring mutations, silent mutations, or conserved substitutions.

[0044] There is no limitation on "different amino acids," as long as they are different from the amino acids before the substitution. Furthermore, in this disclosure, when it is stated that "a specific amino acid is substituted," it is clear that the amino acid is substituted with an amino acid different from the amino acid before the substitution, even if it is not specifically stated that the amino acid is substituted with a different amino acid.

[0045] Amino acids can usually be classified based on the similarity of their residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity.

[0046] Examples of this classification can include positively charged (basic) amino acids, such as arginine, lysine, and histidine; negatively charged (acidic) amino acids, such as glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids), such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids), such as serine, threonine, cysteine, tyrosine, asparagine, and glutamine. For example, amino acids can also be classified into amino acids with charged side chains (charged amino acids), such as arginine, lysine, histidine, glutamic acid, and aspartic acid, and amino acids without charged side chains (uncharged amino acids are also called neutral amino acids), such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. For example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. For example, valine, leucine, and isoleucine can be classified as branched-chain amino acids. For example, classifying the 20 amino acids according to size, starting with the smaller groups, amino acids can be divided into five groups: glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, and tyrosine. However, they are not necessarily limited to these categories.

[0047] For example, when describing "the amino acid corresponding to position 79 of SEQ ID NO: 1 is replaced by a different amino acid", this may mean that the amino acid is replaced by asparagine, valine, glycine, alanine, glutamic acid (glutamate), phenylalanine, arginine, aspartic acid, cysteine, glutamine, histidine, proline, isoleucine, tyrosine, lysine, serine, methionine, threonine, or leucine (excluding tryptophan), but is not limited thereto.

[0048] For example, when describing "the amino acid corresponding to position 181 of SEQ ID NO: 1 is replaced by a different amino acid", this may mean that the amino acid is replaced by asparagine, valine, glycine, tryptophan, glutamic acid (glutamate), phenylalanine, arginine, aspartic acid, cysteine, glutamine, histidine, proline, isoleucine, tyrosine, lysine, serine, methionine, threonine, or leucine (excluding alanine), but is not limited thereto.

[0049] In another instance, when it is described that "the amino acids corresponding to positions 79 and 181 of SEQ ID NO: 1 are replaced by different amino acids", it may mean that the amino acid corresponding to position 79 is replaced by an amino acid other than tryptophan, and the amino acid corresponding to position 181 is replaced by an amino acid other than alanine, but is not limited thereto.

[0050] As used herein, although the phrase "a protein having an amino acid sequence described by a specific sequence number" is used, it is apparent that any protein having an amino acid sequence comprising deletions, modifications, substitutions, conserved substitutions, or additions of certain sequences may also be used in this disclosure, provided that the protein has the same or equivalent activity as the protein composed of the amino acid sequence of the corresponding sequence number. Examples do not exclude the addition of sequences upstream or downstream of the amino acid sequence that do not alter protein function, possibly naturally occurring mutations, their silent mutations, or conserved substitutions, provided that they have the same or equivalent activity as the variant protein, and it is obvious that even proteins with such sequence additions or mutations fall within the scope of this disclosure.

[0051] The term "Nth position" in this disclosure may include the Nth position and the amino acid position corresponding to the Nth position. Specifically, the Nth position may include the amino acid position disclosed in a particular amino acid sequence that corresponds to any amino acid residue in the mature polypeptide. The particular amino acid sequence may be the amino acid sequence of SEQ ID NO: 1.

[0052] As used herein, the term "corresponding to" refers to an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar to, identical to, or homologous to a residue listed in the polypeptide. Identifying an amino acid at a corresponding position can be done by determining a specific amino acid in a specific sequence, where the specific amino acid refers to a specific sequence. As used herein, "corresponding region" typically refers to a similar or corresponding position in a related protein or a reference protein.

[0053] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue in the amino acid sequence can be numbered by referring to the amino acid residue number corresponding to the amino acid residue in SEQ ID NO: 1. For example, the sequence alignment algorithm described in this disclosure can determine the position of an amino acid or the location of a modification such as substitution, insertion, or deletion by comparing it with a query sequence (also called a “reference sequence”).

[0054] In such alignments, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needleman program in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), etc., can be used, but not limited to these, as well as sequence alignment programs, pairwise sequence comparison algorithms, etc., which are all known in the art and can be used appropriately.

[0055] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitutions typically occur based on the similarity of residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity. 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 can be classified into amino acids with charged side chains and amino acids without charged side chains. Amino acids with charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine, while amino acids without charged side chains can be further classified into nonpolar amino acids and polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, while polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Generally, conserved substitutions may have little or no effect on the activity of the resulting peptide.

[0056] Another aspect of this disclosure provides a polynucleotide encoding a variant polypeptide.

[0057] As used herein, the term "polynucleotide" is a DNA or RNA chain of a certain length or longer, which is a polymer of nucleotides in which nucleotide monomers are linked together by covalent bonds to form a long chain, and more specifically, refers to a polynucleotide segment encoding the variant.

[0058] The polynucleotide encoding the fructokinase variant polypeptide disclosed herein may include any polynucleotide sequence without limitation, as long as it is a polynucleotide sequence encoding a variant polypeptide having fructokinase activity.

[0059] For example, the polynucleotide encoding the fructokinase variant polypeptide disclosed herein may be a polynucleotide sequence encoding the amino acid sequence of the fructokinase variant polypeptide disclosed herein, but is not limited thereto.

[0060] For example, the polynucleotides of this disclosure may comprise a nucleotide sequence encoding an amino acid sequence represented by SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. For example, the polynucleotides of this disclosure may have or comprise SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. Furthermore, the polynucleotides of this disclosure may consist of or substantially consist of the sequences of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0061] Considering codon degeneracy or preferred codons in organisms intending to express variants of this disclosure, the polynucleotides of this disclosure may undergo various modifications in the coding region without altering the amino acid sequence of the variants of this disclosure. Therefore, it is apparent that, due to codon degeneracy, they may also contain polynucleotides intended to be translated into polypeptides composed of the amino acid sequence of variants of this disclosure, or polypeptides homologous to or identical with them. For example, the polynucleotides of this disclosure may be sequences of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; or their degenerate sequences.

[0062] For example, the polynucleotides disclosed herein may be contained in nucleotide sequences having 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.8% or higher homology or identity with the sequence of SEQ ID NO: 2, wherein the codon encoding tryptophan (which is the amino acid corresponding to positions 235-237 of SEQ ID NO: 2) is replaced by a codon encoding an amino acid other than tryptophan (e.g., cysteine); the codon encoding alanine (which is the amino acid corresponding to positions 541-543) is replaced by a codon encoding an amino acid other than alanine (e.g., valine); or both, but not limited thereto.

[0063] It is equally apparent that variants of polynucleotide sequences with deletions, modifications, substitutions, conserved substitutions, or additions of certain sequences also fall within the scope of this disclosure, provided that the polynucleotide sequence has such homology or identity and encodes the amino acid sequence of the fructokinase variant polypeptide of this disclosure.

[0064] For example, the polynucleotides disclosed herein may have or contain nucleotide sequences having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequences of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, or may be composed of or substantially composed of nucleotide sequences having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the sequences of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, but are not limited thereto.

[0065] Furthermore, the polynucleotides disclosed herein may contain probes prepared from known gene sequences; for example, they may contain any sequence without limitation, as long as it is a sequence capable of hybridizing with all or part of the complementary sequence of the polynucleotide sequence disclosed herein under stringent conditions.

[0066] “Strict conditions” refer to conditions that enable specific hybridization between polynucleotides. These conditions are described in detail in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). Examples include polynucleotides with high homology or identity, i.e., polynucleotides with 60% or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology or identity, hybridizing with each other, while polynucleotides with lower homology or identity do not hybridize with each other, or conditions in which a single wash is performed at a salt concentration and temperature equivalent to 60°C, 1×SSC, 0.1% SDS, specifically, 60°C, 0.1×SSC, 0.1% SDS, more specifically, 68°C, 0.1×SSC, 0.1% SDS, or specifically, two to three washes, which are the washing conditions for ordinary Southern hybridization.

[0067] Hybridization requires two nucleic acids to have complementary sequences, although mismatches between bases are possible depending on the strictness of hybridization. The term "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 disclosure can further include not only substantially similar nucleic acid sequences, but also separate nucleic acid fragments complementary to the complete sequence.

[0068] Specifically, polynucleotides homologous to or identical with the polynucleotides disclosed herein can be detected using hybridization conditions, which include a Tm value of 55°C and a hybridization step under the above conditions. Furthermore, the Tm value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art for their purposes.

[0069] The appropriate stringency of polynucleotide hybridization depends on the length and complementarity of the polynucleotides, and the variables are well known in the art (e.g., J. Sambrook et al., see above).

[0070] As used herein, the terms “homology” or “identity” refer to the correlation between two given amino acid sequences or nucleotide sequences, expressed as a percentage. The terms “homology” and “identity” are generally used interchangeably.

[0071] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms and can be used together with default gap penalties established by the program used. Essentially, homologous or identical sequences can generally hybridize with whole or partial sequences under moderately or highly stringent conditions. Clearly, hybridization also includes hybridization of polynucleotides with polynucleotides containing codons in general or with codons that take codon degeneracy into account.

[0072] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms such as the “FASTA” program, for example using default parameters as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine this, as in the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (including the GCG package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN1, FASTA (Atschul, [S.] [F.,][ET AL, J MOLEC BIOL 215]: 403 (1990); Guideto Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO] This is performed as described in ETA / .](1988) SIAM J Applied Math 48: 1073). For example, BLAST or ClustalW from the National Center for Biotechnology can be used to determine homology, similarity, or identity.

[0073] Homology, similarity, or identity of polynucleotides or polypeptides can be determined, for example, by comparing sequence information using a computer program such as GAP (e.g., Needleman et al., (1970), J Mol Biol. 48:443), as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In short, the GAP program defines homology, similarity, or identity as a value obtained by dividing the number of similarly arranged symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter sequence of the two sequences. The default parameters of the GAP procedure may include: (1) a binary alignment matrix (containing a value of 1 for identity and a value of 0 for non-identity) and a weighted alignment matrix (or an EDNAFULL permutation matrix (EMBOSS version of NCBI NUC4.4) as disclosed in Schwartz and Dayhoff's Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp.353–358 (1979)); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a penalty of 10 for the beginning of a gap and a penalty of 0.5 for the extension of a gap); and (3) no penalty for end gaps. Therefore, the terms “homology” or “identity” used herein refer to the correlation between sequences.

[0074] Another aspect of this disclosure provides a vector comprising the polynucleotides of this disclosure. The vector may be an expression vector for expressing polynucleotides in microorganisms, but is not limited thereto.

[0075] As used herein, the term "vector" can encompass a DNA construct containing a nucleotide sequence of a polynucleotide encoding a desired polypeptide, operatively linked to a suitable expression regulatory region (or expression control sequence) such that the desired polypeptide can be expressed in a suitable host. The expression regulatory region may contain a promoter capable of initiating transcription, any operator sequence controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences controlling the termination of transcription and translation. Once transformed into a suitable microorganism, the vector can replicate or function independently of the host genome, or it can integrate into the host genome.

[0076] There are no particular limitations on the vectors that can be used in this disclosure, and any vector known in the art may be used. Examples of commonly used vectors may include natural or recombinant plasmids, granules, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or granule vectors, while those based on pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pDC24, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, and other vectors may be used.

[0077] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosome insertion. The polynucleotide can be inserted into the chromosome by any method known in the art, such as homologous recombination, but not limited thereto. The vector may also include a selection marker to confirm insertion into the chromosome. The selection marker is used to select cells transformed by the vector, i.e., to confirm the insertion of the target nucleic acid molecule, and may use markers that provide selectable phenotypes (e.g., drug resistance, auxotrophic phenotype, resistance to cytotoxic agents, or expression of surface polypeptides). Only cells expressing the selection marker are able to survive or exhibit different phenotypes under conditions treated with a selection agent, thus allowing for the selection of transformed cells.

[0078] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a microorganism, enabling the expression of the polypeptide encoded by the polynucleotide in the microorganism. Regardless of location, the transformed polynucleotide can be positioned either by insertion into the chromosome of the microorganism or outside the chromosome, as long as it can be expressed in the microorganism. Furthermore, the polynucleotide contains DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form, as long as it can be introduced into and expressed by the microorganism. For example, the polynucleotide can be introduced into the microorganism in the form of an expression cassette, which is a gene construct containing all the elements required for self-expression. Expression cassettes typically contain a promoter, transcription termination signal, ribosome binding site, and translation termination signal operably linked to the polynucleotide. The expression cassette can be in the form of a self-replicating expression vector. Furthermore, the polynucleotide can be introduced into the microorganism in its own form and operably linked to the sequence required for expression in the microorganism, but is not limited thereto.

[0079] Furthermore, the term "operably linked" refers to the functional linking of a polynucleotide sequence to a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding a target variant of this disclosure.

[0080] The methods for transforming the vectors disclosed herein include any method of introducing nucleic acids into cells, and can be performed by selecting appropriate standard techniques known in the art according to the host cell. For example, transformation methods may include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) technology, DEAE-dextran technology, cationic liposome technology, lithium acetate-DMSO technology, etc.

[0081] Another aspect of this disclosure provides a microorganism comprising any one or more variant polypeptides selected from the variant polypeptides; a polynucleotide encoding the variant polypeptide; and a vector comprising the same.

[0082] In one specific embodiment, the microorganism disclosed herein may be a microorganism capable of producing L-amino acids.

[0083] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and can be a microorganism that weakens or enhances a particular mechanism due to the insertion of a foreign gene or the enhancement or inactivation of an endogenous gene, and can be a microorganism containing genetic modifications for the production of a target polypeptide, protein, or product. As used herein, the terms "microorganism" and "strain" have the same meaning and can be used interchangeably without limitation.

[0084] As used herein, the term "recombinant microorganism" refers to a microorganism that has been genetically modified and exhibits a genotype and / or phenotype different from that of naturally occurring microorganisms (e.g., when the genetic modification affects the coding nucleic acid sequence of the microorganism), and may include the offspring or all potential offspring of said microorganism. As used herein, the terms "recombinant microorganism," "genetically modified microorganism," "recombinant host cell," "recombinant cell," and "recombinant strain" are used interchangeably. For example, a recombinant microorganism may express genes not present in its natural (non-recombinant) form; or may not express genes expressed in their natural form; or may express natural genes in a manner different from their natural form.

[0085] As used herein, the term "L-amino acid-producing microorganism" refers to a prokaryotic or eukaryotic microbial strain capable of producing L-amino acids in a living organism, and may include microorganisms prepared by providing the ability to produce L-amino acids to a parent strain that does not possess the ability to produce L-amino acids, or microorganisms that inherently possess the ability to produce L-amino acids. The ability to produce L-amino acids can be conferred or enhanced through species modification.

[0086] As used herein, the term "unmodified microorganism" does not exclude strains that may have naturally occurring mutations in the microorganism and can be a wild-type strain or the natural strain itself, or a strain whose traits were altered by genetic variation due to natural or artificial factors. For example, an unmodified microorganism can be a strain in which no fructokinase variant peptides described herein have been introduced or have not yet been introduced. The term "unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "unmutated strain," "unmodified strain," "unmutated microorganism," or "reference microorganism."

[0087] Microorganisms capable of producing the L-amino acids of this disclosure may be microorganisms containing any one or more variant polypeptides, polynucleotides of this disclosure, and vectors containing polynucleotides of this disclosure; microorganisms modified to express variant polypeptides or polynucleotides of this disclosure; microorganisms expressing variant polypeptides or polynucleotides of this disclosure (e.g., recombinant strains); or microorganisms having fructokinase variant polypeptide activity of this disclosure (e.g., recombinant strains), but are not limited thereto.

[0088] For example, the strains of this disclosure are cells or microorganisms transformed with a vector containing a polynucleotide encoding a variant polypeptide of this disclosure to express a variant fructokinase containing a variant polypeptide of this disclosure, and the strains of this disclosure may comprise any microorganism capable of producing L-amino acids by containing a variant polypeptide of this disclosure.

[0089] For example, by introducing a polynucleotide encoding a variant polypeptide of this disclosure into a natural wild-type microorganism or a microorganism capable of producing L-amino acids, the microorganism of this disclosure can be a recombinant strain with increased L-amino acid production capacity due to the expression of the fructokinase variant polypeptide. A recombinant strain with increased L-amino acid production capacity can be, but is not limited to, a microorganism with increased L-amino acid production capacity compared to a natural wild-type microorganism or a microorganism unmodified by fructokinase (e.g., a microorganism expressing wild-type fructokinase or a microorganism not expressing the variant polypeptide of this disclosure). For example, a microorganism with increased L-amino acid production capacity can be, but is not limited to, a microorganism containing the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding it. For example, an unmodified microorganism (the target strain being compared to determine whether the ability to produce L-amino acids is increased) can be *Corynebacterium glutamicum* strains CA14-0114, KCCM12739P, CA04-8405, or KCCM11016P, but is not limited to these.

[0090] In addition to introducing nucleotides or vectors, the microorganisms disclosed herein may include any microorganism capable of expressing the fructokinase variant polypeptides of this disclosure by a variety of known methods.

[0091] For example, microorganisms with increased ability to produce L-amino acids, compared to their unmodified parental strains or unmodified microorganisms, may have an increase of approximately 1% or more, specifically, 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, approximately 19% or more. The ability to produce L-amino acids at approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, or approximately 50% or more (there is no particular upper limit, but it can be, for example, 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), but the increase is not limited to this, as long as the production capacity has a positive value for the increase compared to the production capacity of the parent strain before modification or the unmodified microorganism. In another embodiment, a recombinant strain having increased L-amino acid production capacity, compared to the unmodified parental strain or the unmodified microorganism, may have an increase 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, approximately 1... The ability to produce L-amino acids by 0.15 times or more, about 1.16 times or more, about 1.17 times or more, about 1.18 times or more, about 1.19 times or more, about 1.20 times or more, about 1.25 times or more, about 1.30 times or more, about 1.35 times or more, about 1.40 times or more, about 1.45 times or more, or about 1.50 times or more (there is no particular upper limit, but it can be, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, or about 1.5 times or less), but is not limited thereto. As used herein, the term “about” means a range that includes all ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values ​​that are equivalent to or similar to the value following the term “about,” but the range is not limited thereto.

[0092] Regarding the microorganisms according to any of the foregoing specific embodiments, the microorganisms disclosed herein may be of the genus Corynebacterium (…). Corynebacteria sp.), Escherichia coli ( Escherichia sp.), Erwinia sp., Serratia sp. Serratia sp.), Providencia genus ( Providencia sp.), Pseudomonas spp. Pseudomonas sp.), Leptospira ( Leptospira sp.), Salmonella spp. Salmonella sp.), genus *Brugia* ( Brevibacteria sp.), Submonobacteria ( Hypomononas sp.), Chromobacterium spp. Chromobacterium sp.) and Nocardia spp. Norcardia Microorganisms, fungi, or yeasts (sp.), particularly those of the genus Corynebacterium, but not limited thereto.

[0093] For example, the microorganisms disclosed herein may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium desertti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganisms disclosed herein may be microorganisms of the genus Corynebacterium, more specifically Corynebacterium glutamicum, but are not limited thereto.

[0094] Specifically, the microorganisms disclosed herein may be microorganisms of the genus Corynebacterium, more specifically Corynebacterium glutamicum, but are not limited thereto.

[0095] Furthermore, the Corynebacterium genus microorganisms of this disclosure (in which the ability to produce L-amino acids is enhanced) may include any naturally occurring wild-type microorganism itself, Corynebacterium genus microorganisms with enhanced L-amino acid production capacity by increasing or decreasing the activity of genes related to L-amino acid production mechanisms, and Corynebacterium genus microorganisms with enhanced L-amino acid production capacity by introducing or increasing the activity of exogenous genes. In one specific embodiment, the microorganism may include a Corynebacterium genus microorganism, wherein the ability to produce L-amino acids is enhanced by increasing fructokinase activity due to the introduction of the variant polypeptide of this disclosure, but is not limited thereto.

[0096] As used herein, the term “increased protein (peptide) activity” refers to an increase in the activity of a protein (peptide) in a host cell (microorganism) compared to its endogenous activity. This increase can be used interchangeably with terms such as activation, upregulation, overexpression, and enhancement. The host cell (microorganism) can be a prokaryotic or eukaryotic microorganism.

[0097] Increased protein (peptide) activity may include exhibiting protein (peptide) activity that was not originally present in the host cell (microbe), as well as exhibiting protein (peptide) activity that is enhanced compared to endogenous activity or pre-modified activity.

[0098] For example, "exhibiting protein (peptide) activity that was not initially present" or "exhibiting enhanced protein (peptide) activity" can be caused by "introducing a protein (peptide)," but is not limited to this.

[0099] As used herein, the term "introduced protein (peptide)" refers to the expression of a gene in a microorganism that was not originally present in the microorganism, thereby exhibiting the activity of a specific protein, or exhibiting enhanced, increased, or improved peptide activity, compared to endogenous activity or the activity of the corresponding protein before modification. For example, this can be caused by introducing a gene encoding a protein (peptide) into a host cell (microorganism). For example, a polynucleotide encoding a specific protein (peptide) can be introduced into the chromosome of a host cell (microorganism), or a vector containing a polynucleotide encoding a specific protein (peptide) can be introduced into a host cell (microorganism), thereby presenting or enhancing its activity.

[0100] "Endogenous activity" refers to the activity of a specific protein (peptide) that was originally present in the host cell (microorganism) or the unmodified host cell (microorganism) before the trait change due to genetic variation caused by natural or artificial factors. This can be used interchangeably with "pre-modification activity".

[0101] The fact that protein (peptide) activity is increased compared to endogenous activity means that the activity and / or concentration (expression level) of the protein (peptide) in the host cell (microorganism) is higher than the activity and / or concentration (expression level) of the protein (peptide) originally present in the host cell (microorganism) before the phenotypic change or in the unmodified host cell (microorganism).

[0102] For example, the increase indicates the presence of activity of the corresponding protein (peptide) that was not initially present, or its activity or concentration, based on the activity or concentration in the host cell (microorganism) before the phenotypic change or in the unmodified host cell (microorganism), typically increasing by about 1% or more, about 10% or more, about 25% or more, about 50% or more, about 75% or more, about 100% or more, about 150% or more, about 200% or more, about 300% or more, about 400% or more, or about 500% or more, up to about 1000% or about 2000% or more, but not limited thereto.

[0103] Increased protein (peptide) activity can be achieved by introducing exogenous proteins (peptides) or increasing the activity of endogenous proteins (peptides). This increase can be confirmed by enhanced activity levels and expression levels of the corresponding protein (peptide), or by an increase in the amount of products generated by the activity of the corresponding protein (peptide).

[0104] Increased protein (peptide) activity can be achieved by a variety of methods well known in the art, and such methods are not limited, as long as the activity of the target protein (peptide) can be increased compared to the host cell (microbe) before modification. Specifically, genetic engineering and / or protein engineering, which are well known to those skilled in the art and are routine methods in molecular biology, can be used, but the methods are not limited thereto (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0105] Specifically, the increase in protein (peptide) activity disclosed herein can be: 1) Increase the intracellular copy number of polynucleotides encoding proteins (peptides); 2) Modify gene expression regulatory regions on chromosomes that encode proteins (peptides) (e.g., introduce mutations into expression regulatory regions, replace with more active sequences, or insert more active sequences); 3) Modify the nucleotide sequence of the start codon or 5′-UTR region of the transcript of a gene encoding a protein (peptide); 4) Modify the amino acid sequence of proteins (peptides) to increase their activity; 5) Modifying the polynucleotide sequence encoding a protein (peptide) to increase its activity (e.g., modifying the polynucleotide sequence of a protein (peptide) encoding gene to encode a protein (peptide) that has been modified to increase its activity). 6) Introduce a foreign protein (peptide) that exhibits protein (peptide) activity or a foreign polynucleotide encoding said protein (peptide); 7) Optimize the codons of polynucleotides encoding proteins (peptides); 8) Analyze the tertiary structure of proteins (peptides) to select and modify or chemically modify exposed sites; 9) Controlling the intracellular localization of proteins (peptides); or 10) Selected from two or more of 1) to 9), but not limited thereto.

[0106] For example, 1) Increasing the intracellular copy number of a polynucleotide encoding a protein (peptide) can be achieved by introducing a vector containing the polynucleotide encoding the protein (peptide) into a host cell (microorganism), the polynucleotide being operatively linked to an appropriate regulatory sequence. Alternatively, this increase can be achieved by introducing one or two or more copies of the polynucleotide encoding the protein (peptide) into the chromosome of the host cell (microorganism), the polynucleotide being operatively linked to an appropriate regulatory sequence. Chromosomal introduction can be performed by, but is not limited to, introducing a vector capable of inserting the polynucleotide into the host cell (microorganism) chromosome. The vector is as described above. The regulatory sequence can be a natural (from the same source) or exogenous (from a different gene) sequence encoding the polynucleotide sequence, or a mutant or other artificial sequence thereof, and can induce the expression of the polynucleotide in the host cell (microorganism).

[0107] 2) Replacing the gene expression regulatory region (or expression regulatory sequence) encoding a protein (peptide) on a chromosome with a more active sequence can, for example, involve introducing mutations into the sequence through deletion, insertion, substitution, or a combination thereof, or by replacing it with a more active sequence to further increase the activity of the expression regulatory region. The expression regulatory region may include, but is not limited to, promoters, operon sequences, sequences encoding ribosome binding sites, sequences controlling transcription and translation termination, etc. For example, it can be replacing the original promoter with a stronger promoter, but it is not limited to this.

[0108] Examples of known stronger promoters may include, but are not limited to, the CJ1 to CJ7 promoters (US Patent No. 7,662,943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the λ phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13(sm3) promoter (US Patent No. 10,584,338 B2), the O2 promoter (US Patent No. 10,273,491 B2), the tkt promoter, the yccA promoter, etc.

[0109] 3) The nucleotide sequence of the gene encoding the start codon or 5'-UTR region of the protein (peptide) can be, for example, replaced with a start codon of another protein (peptide) with a higher expression rate than the endogenous start codon, or modified to encode a ribosome binding site (RBS) sequence with a higher protein (peptide) expression rate than the endogenous RBS sequence, but is not limited thereto.

[0110] 4) and 5) Modification of the amino acid sequence or polynucleotide sequence of a protein (peptide) can be carried out by introducing mutations into the amino acid sequence of the protein (peptide) or the polynucleotide sequence encoding the protein (peptide), said mutations increasing the activity of the protein (peptide) through deletion, insertion, substitution, or a combination thereof, or by replacing it with a modified amino acid sequence or polynucleotide sequence to increase activity, but not limited thereto. Substitution can be carried out, for example, by inserting polynucleotides into the chromosome through homologous recombination, but not limited thereto.

[0111] 6) Introduction of exogenous polynucleotides exhibiting protein (peptide) activity can be achieved by introducing exogenous polynucleotides encoding proteins (peptides) that exhibit the same or similar activity as the protein (peptide) into host cells (microorganisms). There are no restrictions on the source or sequence of the exogenous polynucleotide, as long as it exhibits the same or similar activity as the protein (peptide). The introduction process can be carried out using known transformation methods appropriately selected by those skilled in the art. When the introduced polynucleotide is expressed in the host cells, a protein (peptide) can be produced, and its activity can be increased.

[0112] 7) Codon optimization of polynucleotides encoding proteins (peptides) can be either codon optimization of endogenous polynucleotides to enhance transcription or translation in host cells (microorganisms) or codon optimization of exogenous polynucleotides to optimize transcription and translation in host cells (microorganisms).

[0113] 8) Analyzing the tertiary structure of a protein (peptide) to select and modify or chemically modify exposed sites can, for example, involve comparing the sequence information of the protein (peptide) to be analyzed with a database storing known protein sequence information, determining template protein candidates based on sequence similarity, confirming the structure based on this, and selecting and modifying or chemically modifying the exposed sites to be modified or chemically modified.

[0114] 9) Controlling the intracellular localization of proteins (peptides) can involve targeting the protein (peptide) to specific organelles or intracellular spaces within the cell. For example, targeting the periplasm or cytoplasm can be achieved by adding or removing a leader sequence that functions in the target protein (peptide), but is not limited to this.

[0115] This increase in protein (peptide) activity may be based on the activity or concentration of the protein (peptide) expressed in the wild type or host cell (microorganism) before modification, an increase in the activity or concentration of the corresponding protein (peptide), or an increase in the amount of product generated by the activity of the corresponding protein (peptide), but is not limited thereto.

[0116] Modifications of some or all polynucleotides in the microorganisms disclosed herein can be induced by: (a) inserting microbial chromosomes using vector-mediated homologous recombination technology or editing the genome using engineered nucleases (e.g., CRISPR-Cas9), and / or (b) treating with light (e.g., ultraviolet light and radiation) and / or chemicals, but not limited thereto.

[0117] The microorganism disclosed herein can have an enhanced ability to produce L-amino acids.

[0118] As used herein, “L-amino acid” includes all L-amino acids that can be produced by microorganisms from various carbon sources through metabolic processes, specifically including but not limited to L-histidine, L-isoleucine, L-tryptophan, or L-lysine.

[0119] In one specific embodiment of this disclosure, compared with unmodified microorganisms, Corynebacterium microorganisms may have an increased ability to produce any one or more amino acids selected from L-histidine, L-isoleucine, L-tryptophan and L-lysine.

[0120] Another aspect of this disclosure provides a method for producing L-amino acids, the method comprising culturing microorganisms in a culture medium containing a variant polypeptide of this disclosure, a polynucleotide encoding the variant polypeptide, or a vector containing the polynucleotide.

[0121] The microorganisms are as described elsewhere.

[0122] As used herein, the term "culture" refers to the growth of the microorganisms of this disclosure under appropriately controlled environmental conditions. In this disclosure, the culture process can be carried out according to suitable culture media or culture conditions known in the art. Depending on the strain to be selected, those skilled in the art can readily adapt such a culture process for use. Specifically, the culture can be batch, continuous, and / or fed-batch, but is not limited thereto.

[0123] The microorganisms disclosed herein can be cultured under aerobic conditions in a conventional culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins, while controlling temperature, pH and other parameters.

[0124] In this disclosure, carbon sources may include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Furthermore, natural organic nutrient sources such as starch hydrolysate, molasses, molasses, rice bran, cassava, bagasse, and corn steep liquor may be used. Specifically, carbohydrates such as glucose and aseptically pretreated molasses (i.e., molasses converted to reducing sugars) may be used, and various other carbon sources may be used in unlimited quantities. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0125] For nitrogen sources, inorganic nitrogen sources can be used, such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; amino acids, such as glutamic acid, methionine, glutamine, etc.; and organic nitrogen sources, such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, defatted soybean meal or its degradation products, etc. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0126] Phosphorus sources may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and their corresponding sodium-containing salts. For inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc., may be used. In addition, amino acids, vitamins, and / or suitable precursors may also be included. These components or precursors may be added to the culture medium in batches or continuously, but are not limited to this.

[0127] The pH of the culture medium can be adjusted during the cultivation of the microorganisms disclosed herein by adding compounds, such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc., to the medium in an appropriate manner. Furthermore, antifoaming agents, such as polyethylene glycol esters of fatty acids, can be used to inhibit foam formation during cultivation. Additionally, oxygen or oxygen-containing gases can be injected into the culture medium to maintain its aerobic state, or no gas can be injected, or nitrogen, hydrogen, or carbon dioxide gases can be injected to maintain its anaerobic or non-aerobic state, but the method is not limited thereto.

[0128] In addition, the culture medium may contain metal salts necessary for growth, such as magnesium sulfate or ferric sulfate. Finally, besides the substances mentioned above, essential growth substances such as amino acids and vitamins may also be used. Suitable precursors may also be used in the culture medium. The above-mentioned materials may be added to the culture in batches or continuously during the culture process in a suitable manner, but are not limited to this.

[0129] In this disclosure, the pH of the culture can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. These compounds are added to the culture in an appropriate manner during the microbial culture process. Furthermore, antifoaming agents such as fatty acid polyethylene glycol esters can be used to inhibit foam formation during culture. Additionally, oxygen or oxygen-containing gases can be injected into the culture to maintain its aerobic state, or no gas may be injected, or nitrogen, hydrogen, or carbon dioxide gases may be injected to maintain an anaerobic or non-aerobic state, but the disclosure is not limited to these methods.

[0130] In the cultivation of this disclosure, the cultivation temperature can be maintained between 20°C and 35°C, specifically between 25°C and 35°C, and the cultivation can continue until the desired amount of useful material is obtained, and can be carried out for about 10 hours to about 160 hours, about 20 hours to about 130 hours, about 24 hours to about 120 hours, about 36 hours to about 120 hours, about 48 hours to about 120 hours, about 48 hours or longer, or about 48 hours, about 72 hours, or about 120 hours, but not limited thereto.

[0131] The L-amino acids produced by the culture according to this disclosure can be released into the culture medium or retained in the cells.

[0132] In one specific embodiment of this disclosure, the L-amino acid may be any one or more selected from L-histidine, L-isoleucine, L-tryptophan and L-lysine, but is not limited thereto.

[0133] In one specific embodiment, the method for producing L-amino acids disclosed herein may further include the steps of preparing the microorganisms of the present disclosure, preparing a culture medium for culturing the strains, or a combination thereof (in any order, regardless of the order), for example prior to the culturing step.

[0134] The method for producing L-amino acids disclosed herein may further include a step of recovering the target substance (particularly L-amino acids) from cultured microorganisms, microbial cultures, microbial fermentation products, or culture media. The recovery step may be additionally included after the culturing step.

[0135] Recovery involves collecting the desired L-amino acids using the methods for culturing microorganisms disclosed herein, for example, using suitable methods known in the art, depending on whether it is batch culture, continuous culture, or fed-batch culture. For example, methods such as centrifugation, filtration, treatment with a protein crystallizing precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatography methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, and combinations thereof can be used to recover the desired substances, particularly L-amino acids, from the culture medium or microorganisms using suitable methods known in the art.

[0136] Furthermore, the method for producing L-amino acids disclosed herein may further include a purification step. Purification can be performed using suitable methods known in the art. In one exemplary embodiment, when the method for producing L-amino acids disclosed herein includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously without regard to the order, or they may be performed simultaneously or integrated into a single step, but are not limited thereto.

[0137] Regarding the methods of this disclosure, the variant peptides, introductions, and L-amino acids, etc., are as described in other aspects.

[0138] Another aspect of this disclosure provides compositions for the production of L-amino acids, the compositions comprising any one or more selected from: variant polypeptides of this disclosure; polynucleotides encoding said variant polypeptides; a vector comprising said vector; a microorganism comprising said vector; a culture of said microorganism; and a fermentation product of said microorganism.

[0139] The compositions disclosed herein may further comprise any suitable excipients commonly used in the production of amino acids, and such excipients may comprise, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, isotonic agents, etc., but are not limited thereto.

[0140] In one specific embodiment, each component present in the compositions of this disclosure may be contained in a microbially effective amount or in an amount that may be suitably present in the compositions used for production.

[0141] Regarding the compositions disclosed herein, the variant peptides, introduced peptides, and L-amino acids, etc., are as described in other aspects.

[0142] Another aspect of this disclosure provides the use of microorganisms with enhanced L-amino acid production capabilities in the production of L-amino acids, wherein the microorganisms are introduced with any one or more variant polypeptides selected from the present disclosure, and polynucleotides encoding the variant polypeptides.

[0143] Regarding the uses of this disclosure, the variant peptides, introductions, and L-amino acids, etc., are as described in other aspects.

[0144] Another aspect of this disclosure provides a method for preparing microorganisms with enhanced L-amino acid production capabilities, the method comprising the step of introducing one or more of the following into a Corynebacterium microorganism with L-amino acid production capabilities: a variant polypeptide of this disclosure; and a polynucleotide encoding the variant polypeptide.

[0145] Another aspect of this disclosure provides a method for increasing the ability to produce L-amino acids, the method comprising the step of introducing one or more of the following into a Corynebacterium microorganism having the ability to produce L-amino acids: a variant polypeptide of this disclosure; and a polynucleotide encoding the variant polypeptide.

[0146] Regarding the methods of this disclosure, the variant peptides, introductions, and L-amino acids, etc., are as described in other aspects. Detailed Implementation

[0147] The present disclosure will be described in more detail below with reference to embodiments and examples. However, these embodiments and examples are for illustrative purposes only, and the scope of the present disclosure is not intended to be limited by these embodiments and examples.

[0148] Example 1: Construction of a recombinant vector for introducing a fructokinase mutation The aim was to prepare a *Corynebacterium glutamicum* mutant strain with enhanced fructokinase activity. Specifically, to construct a vector in *Corynebacterium glutamicum* for deleting the Tn1 transposon coding gene and inserting the target gene, genomic DNA of *Corynebacterium glutamicum* ATCC13032 was used as a template, and primer pairs SEQ ID NO: 9 and SEQ ID NO: 10, and SEQ ID NO: 11 and SEQ ID NO: 12 were used for PCR. PfuUltra TM 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. The denaturation, annealing, and polymerization conditions were repeated for 28 cycles. As a result, DNA fragments of 511 bp and 526 bp were obtained, respectively.

[0149] [Table 1] Subsequently, PCR was performed using genomic DNA from wild-type Escherichia coli ATCC 9637 as a template and primer pairs SEQ ID NOS: 13 and 14 to obtain the wild-type csck (Escherichia coli) gene fragment. Information on the corresponding gene and surrounding nucleotide sequences was obtained from the National Institutes of Health GenBank (NIH GenBank) (accession number CP002967.1).

[0150] In addition, using genomic DNA of *E. coli* ATCC 9637 as a template, PCR was performed using primer pairs SEQ ID NO: 13 and 15 and primer pairs SEQ ID NO: 16 and 14, respectively. Using a mixture of these obtained PCR fragments as a template, overlap PCR was performed using primer pairs SEQ ID NO: 13 and 14 to obtain the 'cscK(W79C, *E. coli*)' gene fragment. Similarly, using genomic DNA of *E. coli* ATCC 9637 as a template, PCR was performed using primer pairs SEQ ID NO: 13 and 17 and primer pairs SEQ ID NO: 18 and 14, respectively. A mixture of the two obtained fragments was used as a template, and overlap PCR was performed again using primer pairs SEQ ID NO: 13 and SEQ ID NO: 14 to obtain the 'cscK(W79C, *E. coli*)' gene fragment. cscK (A181V, E.co)' gene fragment. Furthermore, to prepare combinatorial variants, genomic DNA of *E. coli* ATCC 9637 was used as a template, and PCR was performed using primer pairs SEQ ID NO: 13 and SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, and SEQ ID NO: 18 and SEQ ID NO: 14, respectively. A mixture of the obtained three fragments was used as a template, and overlap PCR was performed again using primer pairs SEQ ID NO: 13 and SEQ ID NO: 14 to obtain '...' gene fragment. cscK (W79C / A181V, E.co)' gene fragment. PCR was performed under the same conditions as described above.

[0151] [Table 2] Subsequently, in order to use the CJ7 promoter derived from Corynebacterium ammoniagenes (SEQ ID NO: 19, Korean Patent No. 10-0620092), genomic DNA of Corynebacterium ammoniagenes was used as a template, and PCR was performed using primers of SEQ ID NO: 20 and SEQ ID NO: 21 in the same manner as described above, thereby obtaining the CJ7 promoter fragment.

[0152] [Table 3] Each DNA product was purified using a PCR purification kit (QUIAGEN). The purified DNA products and the chromosome transformation vector pDC24 (SEQ ID NO: 85) digested with SmaI restriction enzyme were cloned using the Gibson assembly method (DGGibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNAAssembly Master Mix) to obtain recombinant plasmids, which 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 and each gene fragment in calculated molar amounts and storing the mixture at 50°C for 1 hour.

[0153] Example 2. Evaluation of the production capacity of L-amino acid producing strains infused with the cscK mutant gene Example 2-1. Preparation of histidine-producing strains and evaluation of their histidine production capacity Example 2-1-1. Preparation of histidine-producing strains To evaluate the ability of the strain incorporating the cscK mutant gene prepared in Example 1 to produce L-histidine, the wild-type Corynebacterium glutamicum ATCC13032 was first used as the starting microorganism to prepare the gene-enhanced CA14-0114 strain in the histidine biosynthesis pathway. Specifically, to overcome the feedback inhibition of the HisG protein (the first enzyme in the L-histidine biosynthesis pathway), hisGThe codons of the genes were modified to express a protein (SEQ ID NO: 22) (ACS Synth. Biol., 2014, 3 (1), pp 21-29), in which the N-terminal amino acids at positions 233 and 235 of HisG were simultaneously replaced by histidine from glycine and glutamine from threonine, respectively. Furthermore, the start codon was replaced by ATG from GTG to enhance the activity of the hisE gene, which resides in the same operon as hisG. In addition, to enhance the L-histidine biosynthesis pathway, the promoters of the biosynthetic genes hisN, hisH, hisD, hisA, and hisB were replaced with strong promoters, and the corresponding pathways were further enhanced by introducing additional copies of the hisE(g1a)G(G233H / T235Q) operon and the hisD gene.

[0154] Example 2-1-1-1. Preparation of histidine-producing strains with feedback inhibition relieved To prepare a histidine-producing strain with feedback inhibition lifted, genomic DNA of *Corynebacterium glutamicum* ATCC13032 was used as a template, and PCR was performed using primer pairs SEQ ID NO: 23 and SEQ ID NO: 24, and primer pairs SEQ ID NO: 25 and SEQ ID NO: 26. The PCR reaction was performed in the same manner as in Example 1. Using the two amplified DNA fragments as templates, PCR was performed using primers SEQ ID NO: 23 and SEQ ID NO: 26 in the same manner as in Example 1 to obtain… hisE(g1a)G(G233H / T235Q) 'Gene fragment.' In addition, the upstream region of the hisE gene was obtained by PCR using chromosomal DNA from ATCC13032 as a template and primers SEQ ID NOS: 27 and 28.

[0155] [Table 4] To replace the strong promoter, the synthesized Pspl13 promoter (SEQ ID NO: 29, Korean Patent No. 10-1783170) was used as a template, and PCR was performed in the same manner as in Example 1 using primers SEQ ID NO: 30 and 31.

[0156] After treating the pDC24 vector with the restriction enzyme SmaI, the upstream DNA fragment of the hisE gene, the Pspl13 promoter, and '…' were cloned using the Gibson assembly method. hisE(g1a)G(G233H / T235Q)Gene fragments were extracted to obtain a recombinant plasmid named pDC24△Pn_hisEG::Pspl13_hisE(g1a)G(G233H / T235Q). Gibson cloning was performed in the same manner as in Example 1.

[0157] The constructed pDC24△Pn_hisEG::Pspl13_hisE(g1a)G(G233H / T235Q) vector was transformed into *Corynebacterium glutamicum* ATCC13032 via electroporation. Then, through a second crossover recombination, the mutation was introduced into the existing hisG gene, resulting in a strain that enhances hisE activity by releasing feedback inhibition through replacing the start codon of the hisE gene. The genetic manipulation was confirmed by PCR and genome sequencing using primers SEQ ID NO: 32 and SEQ ID NO: 33, which amplified the external regions of the upstream and downstream homologous recombination regions of the inserted genes, respectively. The resulting strain was named CJ-HIS1.

[0158] Example 2-1-1-2. Preparation of histidine-producing strains with enhanced biosynthetic pathways via promoter substitution Next, in order to enhance the activity of the biosynthetic genes hisN, hisH, hisD, hisA, and hisB, plasmids were constructed that replaced the wild-type promoters of each gene with strong promoters.

[0159] Specifically, using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template, and employing primers SEQ ID NO: 34 and SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41, and SEQ ID NO: 42 and SEQ ID NO: 43, PCR was performed in the same manner as in Example 1 to obtain... hisN , hisH , hisD , hisA and hisB The upstream region of the gene. Furthermore, using chromosomal DNA from Corynebacterium glutamicum ATCC13032 as a template, and primers SEQ ID NO: 44 and SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51, and SEQ ID NO: 52 and SEQ ID NO: 53 were used to obtain... hisN , hisH , hisD , hisA and hisB Downstream regions of genes.

[0160] To replace the endogenous promoters of the hisN, hisH, and hisD genes with the strong Pcj7 promoter, genomic DNA of *Corynebacterium ammoniagenes* was used as a template, and PCR was performed in the same manner as in Example 1 using primers SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, SEQ ID NO: 58 and SEQ ID NO: 59.

[0161] In addition, in order to replace the endogenous promoters of the hisA and hisB genes with the strong Pspl13 promoter, the Pspl13 promoter was used as a template, and PCR was performed in the same manner as in Example 1 using primers SEQ ID NO: 60 and SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63.

[0162] After treating the pDC24 vector with the restriction enzyme SmaI, 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 were cloned using the Gibson assembly method to obtain recombinant plasmids, named pDC24△Pn::cj7_hisN, pDC24△Pn::cj7_hisH, and pDC24△Pn::cj7_hisD, respectively. Furthermore, after treating the pDC24 vector with the restriction enzyme SmaI, the upstream DNA fragments of the amplified hisA and hisB genes, the Pspl13 promoter fragment, and the downstream DNA fragments of the hisA and hisB genes were cloned using the Gibson assembly method to obtain recombinant plasmids, named pDC24△Pn::Pspl13_hisA and pDC24△Pn::Pspl13_hisB, respectively. Gibson cloning was performed in the same manner as in Example 1.

[0163] The constructed pDC24△Pn::cj7_hisN vector was transformed into CJ-HIS1 prepared in Example 2-1-1-1 via electroporation. A second crossover recombination was performed to replace the promoter in the existing hisN gene, thereby obtaining a strain with enhanced genes. The genetic manipulation was confirmed by PCR and genome sequencing using primers SEQ ID NO: 64 and SEQ ID NO: 65, which amplified the external regions of the upstream and downstream homologous recombination regions of the inserted gene, respectively. The resulting strain was named CJ-HIS2.

[0164] Next, the constructed pDC24△Pn::cj7_hisH vector was transformed into the CJ-HIS2 strain prepared above via electroporation. A second crossover recombination was performed to replace the promoter in the existing hisH gene, thereby obtaining a strain with enhanced genes. The genetic manipulation was confirmed by PCR and genome sequencing using primers SEQ ID NO: 66 and SEQ ID NO: 67, which were able to amplify the external regions of the upstream and downstream homologous recombination regions of the inserted genes, respectively. The resulting strain was named CJ-HIS3.

[0165] Next, the constructed pDC24△Pn::cj7_hisD vector was transformed into the CJ-HIS3 prepared above via electroporation. A second cross-recombination was then performed to replace the existing vector. hisD The promoter in the gene was selected to obtain a strain with corresponding gene enhancement. The corresponding genetic manipulation was confirmed by PCR and genome sequencing using primers SEQ ID NO: 68 and SEQ ID NO: 69. The resulting strain was named CJ-HIS4.

[0166] Next, the constructed pDC24△Pn::Pspl13_hisA vector was transformed into the CJ-HIS4 prepared above via electroporation. A second crossover recombination was then performed to replace the existing vector. hisA The promoter in the gene was selected to obtain a strain with corresponding gene enhancement. The corresponding genetic manipulation was confirmed by PCR and genome sequencing using primers SEQ ID NO: 70 and SEQ ID NO: 71. The resulting strain was named CJ-HIS5.

[0167] Next, the constructed pDC24△Pn::Pspl13_hisB vector was transformed into the CJ-HIS5 prepared above via electroporation. A second cross-recombination was then performed to replace the existing vector. hisBThe promoter in the gene was selected to obtain a strain with corresponding gene enhancement. The corresponding genetic manipulation was confirmed by PCR and genome sequencing using primers SEQ ID NO: 72 and SEQ ID NO: 73. The resulting strain was named CJ-HIS6.

[0168] [Table 5] Example 2-1-1-3. Preparation of histidine-producing strains with enhanced biosynthetic pathways via additional gene insertion. Next, the gene NCgl1021, known to encode transposons in Corynebacterium glutamicum, was used as the insertion site to additionally insert the hisE(g1a)G(G233H / T235Q) operon and the hisD gene. Specifically, to construct a vector for NCgl1021 (SEQ ID NO: 74) deletion and target gene insertion, PCR was performed in the same manner as in Example 1, using the chromosome of ATCC13032 as a template, and primer pairs SEQ ID NO: 75 and SEQ ID NO: 76 and SEQ ID NO: 77 and SEQ ID NO: 78 to amplify the left and right homologous arm regions of NCgl1021. Using the vector pDC24 △Pn:: Pspl13_ hisE(g1a)G(G233H / T235Q) constructed in Example 2-1-1-1 as a template, and primers of SEQ ID NO: 79 and SEQ ID NO: 80, PCR was performed in the same manner as in Example 1 to obtain ' Pspl13_ hisE(g1a)G(G233H / T235Q) 'Gene fragment'.

[0169] In addition, using the vector pDC24 △Pn::cj7_hisD constructed in Example 2-1-1-2 as a template, and primers SEQ ID NO: 81 and SEQ ID NO: 82, PCR was performed in the same manner as in Example 1 to obtain ' Pcj7_ hisD 'Gene fragment'.

[0170] After treating the pDC24 vector with the restriction endonuclease Smal, the amplified NCgl1021, Pspl13_hisE(g1a)G(G233H / T235Q)' and ' were cloned using the Gibson assembly method. Pcj7_hisDThe left and right homologous arm regions of the gene fragment were used to obtain a recombinant plasmid named 'pDC24△NCgl1021:: Pspl13_hisE(g1a)G(G233H / T235Q)-Pcj7_hisD'. Gibson cloning was performed in the same manner as in Example 1. The constructed 'pDC24△NCgl1021:: Pspl13_hisE(g1a)G(G233H / T235Q)-Pcj7_hisD' vector was transformed into CJ-HIS6 prepared in Example 2-1-1-2 by electroporation, and then a strain was obtained through a second crossover recombination, in which the histidine biosynthesis pathway was enhanced by additional gene insertion. The corresponding genetic manipulation was confirmed by PCR and genome sequencing using primers SEQ ID NO: 83 and SEQ ID NO: 84, which were able to amplify the outer regions of the upstream and downstream homologous recombination regions of the inserted gene, respectively. The strain obtained in this way was named CA14-0114.

[0171] [Table 6] Example 2-1-2. Preparation of transformant strains by introducing the csck(E.co) mutant gene into histidine-producing strains. 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) constructed in Example 1 were transformed into the histidine-producing strain CA14-0114 prepared in Example 2-1-1 by electroporation. Transformed strains were then obtained on selective medium containing 25 mg / L kanamycin and 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.

[0172] Example 2-1-3. Evaluation of histidine production capacity To assess the L-histidine production capacity of the strains prepared in Example 2-1-2, the strains were cultured as follows for evaluation: Each strain was inoculated into a 250 mL corner-baffled flask containing 25 mL of the following seed culture medium and cultured at 30°C with shaking at 200 rpm for 20 hours. Next, 1 mL of the seed culture was inoculated into a 250 mL corner-baffled flask containing 25 mL of production culture medium and cultured at 30°C with shaking at 200 rpm for 48 hours.

[0173] Seed culture medium (pH 7.0) 5% glucose, 1% bacterial peptone, 0.25% sodium chloride, 1% yeast extract, 0.4% urea (based on 1 liter of distilled water) <Production medium (pH 7.0)> 6% raw sugar, 2% ammonium sulfate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 2.0% corn steep liquor (CSL), 200 μg / L biotin, 30 g / L calcium carbonate (based on 1 liter of distilled water) [Table 7] Comparison of L-histidine production capacity among L-histidine-producing strains derived from Corynebacterium glutamicum ATCC 13032 (48H) As shown in the table above, compared with the parental strain CA14-0114, the introduction of the wild-type csck gene from *E. coli* increased histidine production by approximately 4.6%. Furthermore, compared with CA14-0114△Tn1::Pcj7-cscK(WT, E.co), which contains the wild-type cscK gene, the introduction of the cscK variant gene increased histidine production by approximately 2.2% to approximately 8.8%. These results indicate that the csck gene mutation enhances fructokinase activity, thereby enhancing the strain's L-histidine production capacity.

[0174] Example 2-2. Preparation of isoleucine-producing strains with CscK mutant and evaluation of isoleucine production capacity. Example 2-2-1. Preparation of transformant strains by introducing the csck(E.co) mutant gene into isoleucine-producing strains. To determine whether the introduction of a fructose kinase variant gene from *E. coli* increased isoleucine production in *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) constructed in Example 1 were transformed into the isoleucine-producing strain KCCM12739P (Korean Patent No. 10-2363913) by electroporation. Transformed strains were obtained on selective medium containing 25 mg / L kanamycin and 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.

[0175] Example 2-2-2. Evaluation of isoleucine production capacity To determine the L-isoleucine production capacity of the strain prepared in Example 2-2-1, the strain was cultured for evaluation as follows: The parental strain and the mutant strain were each inoculated into 250 mL corner-baffled flasks containing 25 mL of isoleucine production medium and cultured at 32°C with shaking at 200 rpm for 60 hours.

[0176] <Production medium (pH 7.2)> 10% glucose, 0.2% yeast extract, 1.6% ammonium sulfate, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate heptahydrate, 10 mg / L ferric sulfate heptahydrate, 10 mg / L manganese sulfate monohydrate, 200 μg / L biotin (based on 1L distilled water) [Table 8] Comparison of L-isoleucine production capacity among L-isoleucine-producing strains derived from Corynebacterium glutamicum ATCC 13032 (60H) As shown in the table above, compared with the parental strain KCCM12739P, the introduction of the wild-type csck gene from *E. coli* increased isoleucine production by approximately 9.5%. Furthermore, compared with KCCM12739P△Tn1::Pcj7-cscK(WT, E.co) which incorporated the wild-type cscK gene, the introduction of the cscK variant gene increased isoleucine production by approximately 8.6% to approximately 30.4%. These results indicate that the csck gene mutation enhances fructokinase activity, thereby increasing the strain's L-isoleucine production capacity.

[0177] Examples 2-3. Preparation of tryptophan-producing strains incorporating CscK mutants and evaluation of tryptophan production capacity. Example 2-3-1. Preparation of transformant strains by introducing the csck(E.co) mutant gene into tryptophan-producing strains. To determine whether the introduction of a fructose kinase variant gene from *E. coli* increased 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) constructed in Example 1 were transformed into tryptophan-producing strain CA04-8405 (Korean Patent No. 10-1968317) by electroporation. Transformed strains were then obtained on selective medium containing 25 mg / L kanamycin and 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.

[0178] Example 2-3-2. Evaluation of tryptophan production capacity To assess the L-tryptophan production capacity of the strains prepared in Example 2-3-1, the strains were cultured as follows for evaluation: Each strain was inoculated into a 250 mL corner-baffled flask containing 25 mL of seed culture medium and cultured at 30°C with shaking at 200 rpm for 20 hours. After culture, a new 250 mL corner-baffled flask containing 25 mL of production culture medium was prepared for each strain, and 1 mL of the above seed culture was inoculated into it, and cultured at 30°C with shaking at 200 rpm for 24 hours.

[0179] Seed culture medium (pH 7.0) 20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH2PO4, 8 g, 0.5 g MgSO47H2O, 100 μg biotin, 1000 μg thiamine hydrochloride, 2000 μg calcium pantothenate, 2000 μg nicotinamide (based on 1 L of distilled water) <Production medium (pH 7.0)> 30g glucose, 15g (NH4)2SO4, 1.2g MgSO4 7H2O, 1g KH2PO4, 5g yeast extract, 900μg biotin, 4500μg thiamine hydrochloride, 4500μg calcium pantothenate, 30g CaCO3 (based on 1 L of distilled water) [Table 9] Comparison of L-tryptophan production capacity among L-tryptophan-producing strains derived from Corynebacterium glutamicum ATCC 13869 (24H) As shown in the table above, the introduction of the wild-type csck gene from *E. coli* was confirmed to increase tryptophan production compared to the parental strain CA04-8405. Furthermore, the introduction of the cscK variant gene was confirmed to increase tryptophan production by approximately 12.5% ​​to approximately 25% compared to CA04-8405△Tn1::Pcj7-cscK(WT, E.co), in which the wild-type cscK gene was introduced. These results indicate that the csck gene mutation enhances fructokinase activity, thereby enhancing the strain's L-tryptophan production capacity.

[0180] Examples 2-4. Preparation of lysine-producing strains infused with CscK mutant and evaluation of lysine production capacity. Example 2-4-1. Preparation of transformant strains by introducing the csck(E.co) mutant gene into lysine-producing strains. To determine whether the introduction of a fructose kinase variant gene from *E. coli* increased lysine production in *Corynebacterium glutamicum* strains capable of producing L-lysine, 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) constructed in Example 1 were transformed into the lysine-producing strain KCCM11016P (Korean Patent No. 10-0159812) by electroporation. Transformed strains were then obtained on selective medium containing 25 mg / L kanamycin and 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.

[0181] Example 2-4-2. Evaluation of Lysine Production Capacity To assess the L-lysine production capacity of the strains prepared in Example 2-4-1, the strains were cultured as follows for evaluation: Each strain was inoculated into a 250 mL corner-baffled flask containing 25 mL of seed medium and cultured at 37°C with shaking at 200 rpm for 20 hours. Each strain was then inoculated into a 250 mL corner-baffled flask containing 25 mL of production medium and cultured at 37°C with shaking at 200 rpm for 48 hours.

[0182] Seed culture medium (pH 7.0) 20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH2PO4, 8 g, 0.5 g MgSO47H2O, 0.1 mg biotin, 1 mg thiamine hydrochloride, 2 mg calcium pantothenate, 2 mg nicotinamide (based on 1 L of distilled water) <Production medium (pH 7.0)> 50g glucose, 40g (NH4)2SO4, 5g corn steep liquor solids, 10g Bm, 1g KH2PO4, 0.5g MgSO4·7H2O, 100μg biotin, 1000μg thiamine hydrochloride, 2000μg calcium pantothenate, 3000μg nicotinamide, 30g CaCO3 (based on 1 L of distilled water) [Table 10] Comparison of L-lysine production capacity of L-lysine-producing strains derived from Corynebacterium glutamicum ATCC 13869 (48H) As shown in the table above, the introduction of the wild-type csck gene from *E. coli* was confirmed to increase lysine production compared to the parental strain KCCM11016P. Furthermore, the introduction of the cscK variant gene was confirmed to increase lysine production by approximately 3.8% to approximately 16% compared to KCCM11016P △Tn1:: Pcj7-cscK(WT, E.co), in which the wild-type cscK gene was introduced. These results indicate that the csck gene mutation enhances fructokinase activity, thereby enhancing the strain's L-lysine production capacity.

[0183] Based on the foregoing description, those skilled in the art will understand that this disclosure may be implemented in different specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the above embodiments are not restrictive but illustrative in all respects. The scope of this disclosure is defined by the appended claims and not by the preceding description; therefore, all changes and modifications falling within the boundaries and scope of the claims, or equivalents thereof, are included in the claims.

Claims

1. A fructose kinase variant polypeptide, wherein the amino acid corresponding to position 79 or 181 of SEQ ID NO: 1 is replaced by a different amino acid.

2. The variant polypeptide of claim 1, wherein the variant polypeptide has a substitution of amino acid at position 79 of SEQ ID NO: 1 to cysteine, or a substitution of amino acid at position 181 of SEQ ID NO: 1 to valine, or a combination thereof.

3. The variant polypeptide according to claim 1, wherein the amino acid sequence of the variant polypeptide has 90% or higher identity with the amino acid sequence of SEQ ID NO:

1.

4. The variant polypeptide according to claim 1, wherein the variant polypeptide consists of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO:

7.

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

6. A microorganism comprising one or more of a variant polypeptide selected from any one of claims 1 to 4 and a polynucleotide encoding said variant polypeptide.

7. The microorganism according to claim 6, wherein the microorganism is a member of the genus Corynebacterium.

8. The microorganism according to claim 7, wherein the Corynebacterium genus microorganism is Corynebacterium glutamicum.

9. The microorganism according to claim 6, wherein the microorganism has an increased ability to produce L-amino acids compared to microorganisms containing the polypeptide of SEQ ID NO: 1 or the polynucleotide encoding it.

10. The microorganism according to claim 9, wherein the L-amino acid is selected from any one or more of L-histidine, L-isoleucine, L-tryptophan and L-lysine.

11. A method for producing L-amino acids, the method comprising culturing the microorganism according to claim 6 in a culture medium.

12. The method of claim 11, further comprising the step of recovering L-amino acids from any one or more of the following: cultured microorganisms; cultures of said microorganisms; fermentation products of said microorganisms; and culture media.

13. The method according to claim 12, wherein the L-amino acid is selected from any one or more of L-histidine, L-isoleucine, L-tryptophan and L-lysine.

14. Use of the variant polypeptide of any one of claims 1 to 4, or of a microorganism comprising the variant polypeptide or encoding the polynucleotide of the variant polypeptide, in the production of L-amino acids.

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