Fructokinase variant and method for producing l-amino acid using same

Abstract

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

Description

Fructokinase mutant and method for producing L-amino acids using the same

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

[0002]

[0003] Coryneform microorganisms are Gram-positive microorganisms frequently used industrially to produce a variety of materials, including L-amino acids and various nucleic acids, for use in feed, pharmaceuticals, and food. Recently, coryneform microorganisms have also been used to produce diamines and keto acids.

[0004] L-amino acids are the basic building blocks of proteins and are used as important raw materials for pharmaceuticals, food additives, animal feed, nutritional supplements, pesticides, and disinfectants. Among them, L-lysine is an essential amino acid that is not synthesized in the body at all. It is known to be necessary for growth promotion, calcium metabolism, gastric juice secretion promotion, and increased resistance to disease. L-lysine is used in various fields such as feed, pharmaceuticals, and food. L-tryptophan is also an essential amino acid and is used as a feed additive, infusion solution, pharmaceutical raw material, and health food ingredient.

[0005] Various studies are being conducted to develop high-efficiency microorganisms and fermentation process technologies for the production of the above amino acids. For example, target-substance-specific approaches, such as increasing the expression of genes encoding enzymes involved in amino acid biosynthesis in Corynebacterium strains or deleting genes unnecessary for amino acid biosynthesis, are primarily utilized (US 9,109,242 B2, US 8,030,036 B2). In addition to these methods, methods for deleting genes not involved in amino acid production and those whose specific functions in amino acid production are unknown are also being utilized. However, research into methods capable of efficiently producing L-amino acids at high yields remains a pressing need.

[0006]

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

[0008]

[0009] One aspect of the present application provides a fructokinase variant polypeptide in which the amino acid corresponding to position 79 or position 181 of SEQ ID NO: 1 is substituted with another amino acid.

[0010] In one specific example, the mutant polypeptide may be one in which the amino acid corresponding to position 79 of SEQ ID NO: 1 is substituted with cysteine, or one in which the amino acid corresponding to position 181 of SEQ ID NO: 1 is substituted with valine, or a combination thereof.

[0011] In another specific example, the mutant polypeptide may be composed of an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

[0012] In another specific example, the fructokinase may be encoded by the cscK gene.

[0013] Another aspect of the present application provides a polynucleotide encoding the mutant polypeptide.

[0014] Another aspect of the present application provides a microorganism comprising at least one selected from the group consisting of a variant polypeptide; a polynucleotide encoding the variant polypeptide; and a vector comprising the same.

[0015] A microorganism according to any one of the preceding specific examples, wherein the microorganism may be a microorganism of the genus Corynebacterium.

[0016] As a microorganism according to any one of the preceding specific examples, the microorganism of the genus Corynebacterium may be Corynebacterium glutamicum.

[0017] A microorganism according to any one of the preceding specific examples, wherein the microorganism may have increased L-amino acid production ability compared to a microorganism comprising a polypeptide of sequence number 1 or a polynucleotide encoding the same.

[0018] A microorganism according to any one of the preceding specific examples, wherein the microorganism may have an increased ability to produce L-histidine, L-isoleucine, L-tryptophan or L-lysine.

[0019] Another aspect of the present application provides a method for producing L-amino acid, comprising the step of culturing the microorganism in a medium.

[0020] As one specific example, the method may further comprise a step of recovering L-amino acid from the cultured microorganism; a culture of the microorganism; a fermented product of the microorganism; or the culture medium.

[0021] As another specific example, the method may be a method for producing L-histidine, L-isoleucine, L-tryptophan or L-lysine.

[0022] Another aspect of the present application provides a composition for producing L-amino acids, comprising at least one selected from the group consisting of a mutant polypeptide; a polynucleotide encoding the mutant polypeptide; a vector comprising the same; a microorganism comprising the vector; a culture of the microorganism; and a fermented product of the microorganism.

[0023] As a specific example, the composition may be a composition for producing L-histidine, L-isoleucine, L-tryptophan or L-lysine.

[0024]

[0025] When culturing a microorganism containing the fructokinase variant polypeptide of the present application, high yield L-amino acid production is possible compared to a microorganism having an existing unmodified polypeptide.

[0026]

[0027] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below. Furthermore, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated into this specification in their entirety by reference to more clearly explain the level of the technical field to which this application belongs and the contents of this application.

[0028]

[0029] One aspect of the present application provides a fructokinase variant polypeptide, wherein the amino acid corresponding to position 79 or position 181 of 1 is substituted with another amino acid.

[0030]

[0031] In the present application, the term "fructokinase (CscK)" may mean an enzyme that catalyzes a reaction that transfers phosphate in the presence of ATP to produce D-fructose-6-phosphate and ADP.

[0032] For the purposes of this application, the protein may also be referred to as "fructose phosphorylase," "fructokinase," or "CscK." The gene encoding the protein may be, for example, the csck gene, but is not limited thereto. In this application, the "csck gene" may be used interchangeably with the "gene encoding fructokinase." In addition, the protein may be derived from, for example, Escherichia coli, but is not particularly limited in type as long as it has an activity corresponding to fructokinase.

[0033] The fructokinase encoded by the csck gene is known in the art, and the amino acid and polynucleotide sequences of the fructokinase can be obtained from known data databases, including, but not limited to, NCBI's GenBank.

[0034]

[0035] The above fructokinase protein may include the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60% or more homology or identity therewith, but is not limited thereto as long as it has fructokinase protein activity. Specifically, it does not exclude meaningless sequence additions before or after the amino acid sequence described in SEQ ID NO: 1, mutations that may occur naturally, or silent mutations thereof, and if it has the same or corresponding activity as a protein including the amino acid sequence of SEQ ID NO: 1, it may correspond to a protein that is the target of mutation introduction in the present application. For example, the protein that is the target of mutation introduction in the present application 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 more homology or identity therewith. In addition, it is obvious that a variant having an amino acid sequence in which some of the sequences are deleted, modified, substituted, conservatively substituted or added is also included within the scope of the polypeptide that is the subject of the mutation of the present application, provided that the amino acid sequence has such homology or identity and the fructokinase exhibits an effect corresponding to the polypeptide.

[0036]

[0037] In the present application, the amino acid sequence before modification of the fructokinase that is the target of mutation, that is, the sequence that can be the parent sequence, the amino acid before modification corresponding to the 79th position of SEQ ID NO. 1 is tryptophan (W), and the amino acid before modification corresponding to the 181st position of SEQ ID NO. 1 is alanine (A).

[0038]

[0039] As used herein, the term "variant polypeptide", "variant protein" or "variant" refers to a polypeptide in which one or more amino acids are conservatively substituted and / or modified, thereby differing from the amino acid sequence of the variant before the mutation, but retaining functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. In addition, some variants may include variants in which one or more portions, such as the N-terminal leader sequence or the transmembrane domain, are deleted. Other variants may include variants in which portions are deleted from the N- and / or C-terminus of the mature protein. The above term "variant" may be used interchangeably with terms such as variant, modification, variant polypeptide, variant protein, variant and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited thereto if the term is used in the meaning of variant.

[0040] Additionally, variants may include deletions or additions of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus of a protein that is involved in co-translational or post-translational protein transfer. Furthermore, the polypeptide may be conjugated to other sequences or linkers to facilitate identification, purification, or synthesis of the polypeptide.

[0041]

[0042] The fructokinase variant polypeptide of the present application refers to a variant polypeptide in which the amino acid corresponding to the 79th position or the 181st position from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid.

[0043] The above “fructokinase mutant polypeptide” may be referred to as “fructokinase mutant,” “mutant CscK,” “CscK mutant,” etc. The fructokinase polypeptide to which the mutation is introduced in the present application may be used interchangeably with the term “CscK” and is not particularly limited, but may be encoded by the cscK gene and may be cscK derived from Escherichia coli, but is not limited thereto.

[0044]

[0045] The fructokinase variant polypeptide of the present application may have an amino acid at a position corresponding to the 79th position or the 181st position in the amino acid sequence of SEQ ID NO: 1 substituted with an amino acid different from the amino acid before substitution.

[0046] In one specific example, the mutant polypeptide may be one in which the amino acid corresponding to position 79 in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid other than tryptophan, which is the amino acid before substitution, or one in which the amino acid corresponding to position 181 in the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid other than alanine, which is the amino acid before substitution, or a combination thereof.

[0047]

[0048] In another specific example, the variant polypeptide may be one in which the amino acid corresponding to position 79 of SEQ ID NO: 1 is substituted with a 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, aspartate, and glutamic acid, but is not limited thereto.

[0049] In another specific example, the variant polypeptide may be one in which the amino acid corresponding to position 181 of SEQ ID NO: 1 is substituted with 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, aspartate, and glutamic acid, but is not limited thereto.

[0050] In any one of the above-described embodiments, the mutant polypeptide provided in the present application may be one in which the amino acid corresponding to the 79th position from the N-terminus of SEQ ID NO: 1 is substituted with cysteine, or the amino acid corresponding to the 181st position from the N-terminus of SEQ ID NO: 1 is substituted with valine, or a combination thereof.

[0051] In another embodiment of the above-described embodiments, the variant polypeptide provided in the present application 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% homology or identity with SEQ ID NO: 1, or less than 100%.

[0052]

[0053] For example, the variant polypeptide of the present application may include an amino acid sequence in which the amino acid corresponding to position 79 in the amino acid sequence described in SEQ ID NO: 1 is fixed as cysteine, and has at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.8% homology or identity with SEQ ID NO: 1. In addition, the variant polypeptide of the present application may include an amino acid sequence in which the amino acid corresponding to the 181st position in the amino acid sequence described in SEQ ID NO: 1 is fixed to valine, and has at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.8% homology or identity with SEQ ID NO: 1. In addition, the variant polypeptide of the present application may include an amino acid sequence in which the amino acid corresponding to the 79th position in the amino acid sequence described in SEQ ID NO: 1 is fixed as cysteine ​​and the amino acid corresponding to the 181st position is fixed as valine, and has at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.8% homology or identity with SEQ ID NO: 1. In addition, it is obvious that a variant polypeptide having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, conservatively substituted or added is also included within the scope of the present application, as long as it has such homology or identity and exhibits an effect corresponding to the variant polypeptide of the present application.

[0054] Meanwhile, a person skilled in the art can identify the amino acid corresponding to the 79th position or the 181st position of the amino acid sequence of SEQ ID NO. 1 of the present application in any amino acid sequence through sequence alignment known in the art, and even if not described separately in the present application, when "an amino acid at a specific position in a specific sequence number" is described, it is self-evident that it includes "an amino acid at a corresponding position" in any amino acid sequence.

[0055]

[0056] In another specific example, the variant polypeptide may be comprised of the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.

[0057] Specifically, the variant polypeptide of the present application may have, comprise, consist of, or consist essentially of an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.

[0058] For example, if the amino acid sequence has sequence additions or deletions, naturally occurring mutations, silent mutations or conservative substitutions that do not alter the function of the variant of the present application at the N-terminus, C-terminus and / or within the amino acid sequence.

[0059]

[0060] The above "other amino acid" is not limited to an amino acid different from the amino acid prior to substitution. Furthermore, when the present application states that "a specific amino acid has been substituted," it is self-evident that the amino acid has been substituted with an amino acid different from the amino acid prior to substitution, even if it is not specifically stated that it has been substituted with another amino acid.

[0061] Amino acids can generally be classified based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of their residues.

[0062] Examples of these classifications 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. As another example, amino acids with charged side chains (electrically charged amino acids) include arginine, lysine, histidine, glutamic acid, and aspartic acid; and amino acids with uncharged side chains (neutral amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. As another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. As another example, valine, leucine, and isoleucine can be classified as branched amino acids. As another example, the 20 amino acids can be classified by size into five groups: glycine, alanine, and serine; cysteine, proline, threonine, aspartic acid, and asparagine; valine, histidine, glutamic acid, and glutamine; isoleucine, leucine, methionine, lysine, and arginine; and phenylalanine, tryptophan, and tyrosine. However, this is not necessarily limited to these groups.

[0063] For example, if it is described that "the amino acid corresponding to position 79 in SEQ ID NO: 1 is replaced with another amino acid," it may mean, but is not limited to, a replacement with asparagine, valine, glycine, alanine, glutamate, phenylalanine, arginine, aspartate, cysteine, glutamine, histidine, proline, isoleucine, tyrosine, lysine, serine, methionine, threonine, or leucine, excluding tryptophan.

[0064] For another example, if it is described that "the amino acid corresponding to position 181 in SEQ ID NO: 1 is replaced with another amino acid", it may mean that the amino acid is replaced with asparagine, valine, glycine, tryptophan, glutamate, phenylalanine, arginine, aspartate, cysteine, glutamine, histidine, proline, isoleucine, tyrosine, lysine, serine, methionine, threonine or leucine, excluding alanine, but is not limited thereto.

[0065] For another example, if it is described that "the amino acids corresponding to positions 79 and 181 in SEQ ID NO: 1 are replaced with other amino acids," it may mean, but is not limited to, that the amino acid corresponding to position 79 is replaced with an amino acid other than tryptophan, and the amino acid corresponding to position 181 is replaced with an amino acid other than alanine.

[0066]

[0067] Even if the present application describes a "protein having an amino acid sequence described by a specific sequence number", it is obvious that a protein having an amino acid sequence in which some sequences are deleted, modified, substituted, conservatively substituted, or added may also be used in the present application, as long as it has the same or corresponding activity as the protein consisting of the amino acid sequence of the corresponding sequence number. For example, if it has the same or corresponding activity as the mutant protein, it does not exclude sequence additions that do not alter the function of the protein before or after the amino acid sequence, mutations that may occur naturally, silent mutations thereof, or conservative substitutions, and it is obvious that even if it has such sequence additions or mutations, it falls within the scope of the present application.

[0068] The "N-position" of the present application may include the N-position and an amino acid position corresponding to the N-position. Specifically, it may include an amino acid position corresponding to any amino acid residue in a mature polypeptide disclosed in a specific amino acid sequence. The specific amino acid sequence may be the amino acid sequence of SEQ ID NO: 1.

[0069] As used herein, the term "corresponding to" refers to an amino acid residue at a position listed in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the residue listed in the polypeptide. Identifying an amino acid at a corresponding position may be determining a specific amino acid in a sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0070] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue of the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described in the present application can identify the position of an amino acid, or the position at which a modification such as a substitution, insertion, or deletion occurs, by comparing it to a query sequence (also referred to as a “reference sequence”).

[0071] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000), Trends Genet. 16: 276-277) can be used, but is not limited thereto, and any sequence alignment program known in the art, pairwise sequence comparison algorithm, etc. can be appropriately used.

[0072]

[0073] The term "conservative substitution" in this application refers to the replacement of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartate; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Additionally, amino acids can be classified into those with electrically charged side chains and those with uncharged side chains. Charged side chain amino acids include aspartic acid, glutamic acid, lysine, arginine, and histidine. Uncharged side chain amino acids can be further classified into nonpolar amino acids or polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conservative substitutions have little or no effect on the activity of the resulting polypeptide. Typically, conservative substitutions may have little or no effect on the activity of a protein or polypeptide.

[0074]

[0075] Another aspect of the present application provides a polynucleotide encoding the mutant polypeptide.

[0076] In this application, the term "polynucleotide" means a polymer of nucleotides in which nucleotide units (monomers) are covalently bonded to form a long chain, a DNA or RNA strand of a certain length or longer, and more specifically, a polynucleotide fragment encoding the variant.

[0077] The polynucleotide encoding the fructokinase variant polypeptide of the present application may include, without limitation, any polynucleotide sequence encoding a variant polypeptide having fructokinase activity.

[0078] For example, the polynucleotide encoding the fructokinase variant polypeptide of the present application may be, but is not limited to, a polynucleotide sequence encoding the amino acid sequence of the fructokinase variant polypeptide of the present application.

[0079] For example, it may comprise a nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7. As an example of the present application, the polynucleotide of the present application may have or comprise SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. Furthermore, the polynucleotide of the present application may consist of, or consist essentially of, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0080]

[0081] The polynucleotide of the present application may undergo various modifications in the coding region without altering the amino acid sequence of the variant of the present application, taking into account codon degeneracy or preferred codons in the organism intended to express the variant of the present application. Therefore, it is self-evident that the polynucleotide of the present application may also include a polynucleotide that can be translated into a polypeptide comprising the amino acid sequence of the variant of the present application, or a polypeptide having homology or identity therewith, due to codon degeneracy. For example, the polynucleotide of the present application may be SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; or a degenerated sequence thereof.

[0082] For example, the polynucleotide of the present application may include a base sequence having a homology or identity of at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.8% with the sequence of SEQ ID NO: 2, wherein the codon encoding tryptophan, which is an amino acid corresponding to positions 235 to 237 of SEQ ID NO: 2, is substituted with a codon encoding an amino acid other than tryptophan, for example, a cysteine ​​amino acid; or the codon encoding alanine, which is an amino acid corresponding to positions 541 to 543, is substituted with a codon encoding an amino acid other than alanine, for example, a valine; or all of the above, but is not limited thereto.

[0083] In addition, it is obvious that a variant having a polynucleotide sequence having a deletion, modification, substitution, conservative substitution or addition of a part of a sequence that has such homology or identity and encodes the amino acid sequence of the fructokinase variant polypeptide of the present application is also included within the scope of the present application.

[0084] As another example, the polynucleotide of the present application may have or include a nucleic acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and less than 100% homology or identity with SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, or may consist of or consist essentially of a nucleic acid sequence having at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and less than 100% homology or identity with SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, but is not limited thereto.

[0085]

[0086] Additionally, the polynucleotide of the present application may include, without limitation, a probe that can be prepared from a known genetic sequence, for example, a sequence that can hybridize under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present application.

[0087]

[0088] The above "stringent conditions" refer to conditions that allow specific hybridization between polynucleotides. These conditions are specifically described 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). For example, conditions in which polynucleotides having high homology or identity hybridize with each other, polynucleotides 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, or 99% or more homology or identity hybridize with each other, and polynucleotides having lower homology or identity do not hybridize with each other, or conditions in which washing is performed once, specifically twice or three times, at a salt concentration and temperature equivalent to 60°C, 1ХSSC, 0.1% SDS, specifically 60°C, 0.1ХSSC, 0.1% SDS, and more specifically 68°C, 0.1ХSSC, 0.1% SDS, which are washing conditions of typical southern hybridization, are performed.

[0089] Hybridization requires that two nucleic acids have complementary sequences, although mismatches between bases are possible depending on the stringency 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 the present application may also include isolated nucleic acid fragments that are complementary in their entirety, as well as substantially similar nucleic acid sequences.

[0090] Specifically, a polynucleotide having homology or identity with the polynucleotide of the present application can be detected using hybridization conditions including a hybridization step at a Tm value of 55°C and using the conditions described above. In addition, the Tm value may be, but is not limited to, 60°C, 63°C, or 65°C, and can be appropriately adjusted by a person skilled in the art depending on the purpose.

[0091] The appropriate stringency for hybridizing the polynucleotides depends on the length and degree of complementarity of the polynucleotides, variables which are well known in the art (e.g., J. Sambrook et al., supra).

[0092] As used herein, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid sequences or base sequences, which may be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0093] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms, and may be combined with default gap penalties established by the program being used. In practice, homologous or identical sequences can generally hybridize with all or part of the sequence under moderate or high stringency conditions. It should be appreciated that hybridization also includes hybridization with polynucleotides containing common codons or codons that take codon degeneracy into account.

[0094] Whether any two polynucleotide or polypeptide sequences are homologous, similar or identical can be determined using known computer algorithms such as the "FASTA" program using default parameters, for example 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) as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) can be determined using the GCG program 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); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO ETA / .](1988) SIAM J Applied Math 48: 1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW of the National Center for Biotechnology Information Database.

[0095] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information, for example, using a GAP computer program such as that of Needleman et al. (1970), J Mol Biol. 48:443, as disclosed, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In brief, the GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and (2) a comparison matrix as disclosed by Gribskov et al. (1986) Nucl. Acids Res. 48:443, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979). 14: 6745 weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Accordingly, the term "homology" or "identity" as used herein refers to the relevance between sequences.

[0096]

[0097] Another aspect of the present application provides a vector comprising the polynucleotide of the present application. The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a microorganism.

[0098] The term "vector" in this application may include a DNA construct comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide in a suitable host. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating the termination of transcription and translation. After being transformed into a suitable microorganism, the vector may replicate or function independently of the host genome, or may be integrated into the genome itself.

[0099] The vector used in the present application is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or cosmid vectors, and pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and pET series can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, PDC24, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors can be used.

[0100] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a vector for intracellular chromosomal insertion. The insertion of the polynucleotide into the chromosome can be achieved by any method known in the art, for example, homologous recombination, but is not limited thereto. A selection marker for confirming the chromosomal insertion can be additionally included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the insertion of the target nucleic acid molecule. Markers that confer a selectable phenotype, such as drug resistance, nutrient requirement, cytotoxic agent resistance, or expression of a surface polypeptide, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypic traits, so that transformed cells can be selected.

[0101] The term "transformation" in this application refers to introducing a vector containing a polynucleotide encoding a target polypeptide into a microorganism or into a microorganism, so that the polypeptide encoded by the polynucleotide can be expressed in the microorganism. The transformed polynucleotide can be located either within the chromosome of the microorganism or outside the chromosome, as long as it can be expressed in the microorganism. In addition, the polynucleotide includes DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form as long as it can be introduced into the microorganism and expressed. For example, the polynucleotide can be introduced into the microorganism in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression. The expression cassette can typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, all of which are operably linked to the polynucleotide. The expression cassette can be in the form of a self-replicating expression vector. Additionally, the polynucleotide may be introduced into a microorganism in its own form and operably linked to a sequence required for expression in the microorganism, but is not limited thereto.

[0102] Additionally, the term "operably linked" as used herein means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target variant of the present application.

[0103] The method for transforming the vector of the present application includes any method for introducing nucleic acids into cells, and can be performed by selecting an appropriate standard technique known in the art depending on the host cell. Examples thereof include, but are not limited to, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method.

[0104]

[0105] Another aspect of the present application provides a microorganism comprising at least one selected from the group consisting of the mutant polypeptide; a polynucleotide encoding the mutant polypeptide; and a vector comprising the same.

[0106] In one specific example, the microorganism of the present application may be a microorganism having L-amino acid production ability.

[0107]

[0108] In this application, the term "microorganism (or strain)" includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification. It may be a microorganism that has a specific mechanism weakened or strengthened due to causes such as the insertion of an external gene or the enhancement or inactivation of the activity of an endogenous gene, and may be a microorganism that includes genetic modification for the production of a desired polypeptide, protein, or product. In this application, "microorganism," "strain," and "microorganism" may be used interchangeably without limitation with the same meaning.

[0109] As used herein, the term "recombinant microorganism" refers to a microorganism that has been genetically modified to exhibit a different genotype and / or phenotype compared to a naturally occurring microorganism (e.g., when the genetic modification affects the nucleic acid sequence coding of the microorganism), and may include all progeny or potential progeny of the microorganism. The terms "recombinant microorganism," "genetically modified microorganism," "recombinant host cell," "recombinant cell," and "recombinant strain" may be used interchangeably in this application. The recombinant microorganism may, for example, express a gene not found in its native (non-recombinant) form; may not express a gene expressed in its native form; or may express a native gene in a manner different from that in which it is expressed in its native form.

[0110]

[0111] In this application, the term "microorganism producing L-amino acid" refers to a prokaryotic or eukaryotic microbial strain capable of producing L-amino acid within the organism, and may include both a microorganism in which the ability to produce L-amino acid has been conferred on a parent strain that does not have the ability to produce L-amino acid, and a microorganism that inherently has the ability to produce L-amino acid. The ability to produce L-amino acid may be conferred or enhanced through species improvement.

[0112]

[0113] The term "unmodified microorganism" in this application does not exclude strains that contain mutations that can occur naturally in microorganisms, and may refer to wild-type strains or natural strains themselves, or strains before their characteristics are changed by genetic mutations caused by natural or artificial factors. For example, the unmodified microorganism may refer to a strain into which the fructokinase mutant polypeptide described herein is not introduced or before it is introduced. The "unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "unmutated strain," "unmodified microorganism," or "reference microorganism."

[0114]

[0115] The microorganism having L-amino acid production ability of the present application may be, but is not limited to, a microorganism comprising at least one of the mutant polypeptide of the present application, the polynucleotide of the present application, and a vector comprising the polynucleotide of the present application; a microorganism modified to express the mutant polypeptide of the present application or the polynucleotide of the present application; a microorganism (e.g., a recombinant strain) expressing the mutant polypeptide of the present application or the polynucleotide of the present application; or a microorganism (e.g., a recombinant strain) having fructokinase mutant polypeptide activity of the present application.

[0116] For example, the strain of the present application is a cell or microorganism that is transformed with a vector containing a polynucleotide encoding the variant polypeptide of the present application and expresses a variant fructokinase containing the variant polypeptide of the present application, and the strain of the present application may include all microorganisms capable of producing L-amino acids, including the variant polypeptide of the present application.

[0117] For example, the microorganism of the present application may be a recombinant strain having increased L-amino acid productivity by introducing a polynucleotide encoding the mutant polypeptide of the present application into a natural wild-type microorganism or a microorganism having L-amino acid productivity, thereby expressing a fructokinase mutant polypeptide. The recombinant strain having increased L-amino acid productivity may be a microorganism having increased L-amino acid productivity compared to a natural wild-type microorganism or a fructokinase-free microorganism (e.g., a microorganism expressing wild-type fructokinase or a microorganism not expressing the mutant polypeptide of the present application), but is not limited thereto. As an example, the microorganism of the present application having increased L-amino acid productivity may be a microorganism having increased L-amino acid productivity compared to a microorganism comprising the polypeptide of SEQ ID NO: 1 or a polynucleotide encoding the same, but is not limited thereto. For example, the non-modified microorganism, which is a target strain for comparing whether the L-amino acid production ability increases, may be, but is not limited to, a Corynebacterium glutamicum strain CA14-0114; KCCM12739P; CA04-8405; or KCCM11016P.

[0118]

[0119] The microorganism of the present application may include any microorganism capable of expressing the fructokinase variant polypeptide of the present application by various known methods in addition to the introduction of the nucleic acid or vector.

[0120] For example, the L-amino acid production ability of the microorganism with increased L-amino acid production ability is about 1% or more, specifically, about 1% or more, about 2% or more, about 3% or more, about 4% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 16% or more, about 17% or more, about 18% or more, about 19% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 50% or more (the upper limit is not particularly limited, for example, about 200% or less, about 150% or less, about It may be increased by 100% or less, about 90% or less, about 80% or less, about 70% or less, about 60% or less, about 55% or less, or about 50% or less), but is not limited thereto, as long as it has a positive value of increase compared to the productivity of the parent strain or unmodified microorganism before the mutation. In another example, the recombinant strain with increased L-amino acid production ability has an L-amino acid production ability of about 1.01 times or more, about 1.02 times or more, about 1.03 times or more, about 1.04 times or more, about 1.05 times or more, about 1.06 times or more, about 1.07 times or more, about 1.08 times or more, about 1.09 times or more, about 1.1 times or more, about 1.11 times or more, about 1.12 times or more, about 1.13 times or more, about 1.14 times or more, about 1.15 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, or about It may be increased by, but is not limited to, 1.40 times or more, about 1.45 times or more, or about 1.50 times or more (the upper limit is not particularly limited, and may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, or about 1.5 times or less).The term “about” above includes all ranges including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all ranges of values ​​equal to or similar to the value following the term “about,” but is not limited thereto.

[0121]

[0122] As a microorganism according to any one of the preceding specific examples, the microorganism of the present application may be a microorganism belonging to the genus Corynebacteria sp., Escherichia sp., Erwinia sp., Serratia sp., Providencia sp., Pseudomonas sp., Leptospira sp., Salmonella sp., Brevibacteria sp., Hypomononas sp., Chromobacterium sp., and Norcardia sp., or a microorganism belonging to the genus fungi or yeast, specifically, a microorganism belonging to the genus Corynebacterium, but is not limited thereto.

[0123] As an example of the present application, the microorganism of the present application is Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium It may be Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganism of the present application may be a microorganism of the genus Corynebacterium, more specifically, Corynebacterium glutamicum, but is not limited thereto.

[0124] Specifically, the microorganism of the present application may be a microorganism of the genus Corynebacterium, more specifically, Corynebacterium glutamicum, but is not limited thereto.

[0125]

[0126] Meanwhile, the Corynebacterium genus microorganism with improved L-amino acid productivity of the present application may include a natural wild-type microorganism itself, a Corynebacterium genus microorganism with improved L-amino acid productivity by increasing or decreasing the activity of a gene related to the L-amino acid production mechanism, or a Corynebacterium genus microorganism with improved L-amino acid productivity by introducing or increasing the activity of an external gene. As a specific example, the present application may include a Corynebacterium genus microorganism with improved L-amino acid productivity by increasing the fructokinase activity through introduction of the mutant polypeptide, but is not limited thereto.

[0127]

[0128] In this application, the term "increase" of protein (polypeptide) activity means that the activity of the protein (polypeptide) within a host cell (microorganism) is increased compared to its intrinsic activity. The increase may be used interchangeably with terms such as activation, up-regulation, overexpression, and enhancement. The host cell (microorganism) may be a prokaryotic or eukaryotic microorganism.

[0129] The increase in the above protein (polypeptide) activity may include the display of a protein (polypeptide) activity that the host cell (microorganism) did not inherently possess, or the display of an enhanced protein (polypeptide) activity compared to the inherent activity or activity before modification.

[0130] For example, the above “exhibiting a protein (polypeptide) activity that was not inherently present” or “exhibiting an improved protein (polypeptide) activity” may be due to, but is not limited to, “introduction of a protein (polypeptide).”

[0131] In this application, the term "introduction" of a protein (polypeptide) means that a gene that a microorganism did not originally possess is expressed within the microorganism, thereby causing the activity of a specific protein to be exhibited, or that the activity of the polypeptide is strengthened, increased, or improved compared to the intrinsic activity of the protein or the activity before modification. For example, this may be due to the introduction of a gene encoding the protein (polypeptide) into a host cell (microorganism). For example, a polynucleotide encoding a specific protein (polypeptide) may be introduced into the chromosome of a host cell (microorganism), or a vector containing a polynucleotide encoding a specific protein (polypeptide) may be introduced into the host cell (microorganism), thereby causing the activity of the protein (polypeptide) to be exhibited or improved.

[0132] The above "intrinsic activity" refers to the activity of a specific protein (polypeptide) originally possessed by a host cell (microorganism) or an untransformed host cell (microorganism) before transformation, when the trait changes due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with "activity before transformation."

[0133] An increase in the activity of a protein (polypeptide) compared to the intrinsic activity means that the activity and / or concentration (expression amount) of the protein (polypeptide) of the host cell (microorganism) is enhanced compared to the activity and / or concentration (expression amount) of the protein (polypeptide) originally present in the host cell (microorganism) before transformation or in the non-transformed host cell (microorganism).

[0134] For example, the increase may be, but is not limited to, an increase in the activity or concentration of the corresponding protein (polypeptide) from a previous state, or an increase in the activity or concentration thereof, typically by at least about 1%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%, up to at least about 1000% or at least about 2000%, relative to the activity or concentration in the host cell (microorganism) before transformation or in the untransformed host cell (microorganism).

[0135] An increase in the activity of the above protein (polypeptide) can be achieved by introducing an exogenous protein (polypeptide) or by increasing the activity of an endogenous protein (polypeptide). Whether the activity of the above protein (polypeptide) has increased can be confirmed by an increase in the activity level of the protein (polypeptide), the expression level, or the amount of a product resulting from the activity of the protein (polypeptide).

[0136] The increase in the activity of the above protein (polypeptide) can be achieved by various methods well known in the art, and is not limited thereto, as long as the activity of the target protein (polypeptide) can be increased compared to the host cell (microorganism) before transformation. Specifically, it may be, but is not limited to, genetic engineering and / or protein engineering, which are routine methods of molecular biology and are well known to those skilled in the art (e.g., SiTn1icka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0137] Specifically, the increase in activity of the protein (polypeptide) of the present application is

[0138] 1) Increase in the intracellular copy number of a polynucleotide encoding a protein (polypeptide);

[0139] 2) Modification of the gene expression control region on the chromosome that codes for a protein (polypeptide) (e.g., introduction of a mutation in the expression control region, replacement with a sequence having stronger activity, or insertion of a sequence having stronger activity);

[0140] 3) Modification of the base sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a protein (polypeptide);

[0141] 4) Modification of the amino acid sequence of the protein (polypeptide) so as to increase the activity of the protein (polypeptide);

[0142] 5) Modification of a polynucleotide sequence encoding a protein (polypeptide) such that the activity of the protein (polypeptide) is increased (e.g., modification of a polynucleotide sequence of a protein (polypeptide) encoding gene such that the protein (polypeptide) is modified such that the activity of the protein (polypeptide) is increased);

[0143] 6) Introduction of a foreign protein (polypeptide) that exhibits the activity of a protein (polypeptide) or a foreign polynucleotide encoding the same;

[0144] 7) Codon optimization of polynucleotides encoding proteins (polypeptides);

[0145] 8) Analyzing the tertiary structure of a protein (polypeptide) and selecting the exposed portion to modify or chemically modify; or

[0146] 9) Control of cellular localization of proteins (polypeptides); or

[0147] 10) It may be a combination of two or more selected from 1) to 9), but is not particularly limited thereto.

[0148] for example,

[0149] The increase in the intracellular copy number of the polynucleotide encoding the protein (polypeptide) described above 1) may be caused by introducing a vector containing a polynucleotide encoding the protein (polypeptide) operably linked to an appropriate regulatory sequence into a host cell (microorganism). Alternatively, one copy or two or more copies of the polynucleotide encoding the protein (polypeptide) operably linked to an appropriate regulatory sequence may be introduced into a chromosome within the host cell (microorganism). The introduction into the chromosome may be performed by introducing a vector capable of inserting the polynucleotide into the chromosome within the host cell (microorganism), but is not limited thereto. The vector is as described above. The regulatory sequence may be a natural sequence (same in origin) or a foreign sequence (derived from another gene) to the polynucleotide sequence it encodes, or a mutant sequence thereof, or another artificial sequence, and may induce expression of the polynucleotide within the host cell (microorganism).

[0150] 2) The replacement of the gene expression control region (or expression control sequence) on the chromosome encoding the protein (polypeptide) with a sequence having strong activity may be, for example, introducing a mutation in the sequence by deletion, insertion, substitution, or a combination thereof to further increase the activity of the expression control region, or replacement with a sequence having stronger activity. The expression control region may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. As an example, the original promoter may be replaced with a strong promoter, but is not limited thereto.

[0151] Examples of known strong promoters include, but are not limited to, the cj1 to cj7 promoters (US Patent No. US 7662943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (US Patent No. US 10584338 B2), the O2 promoter (US Patent No. US 10273491 B2), the tkt promoter, and the yccA promoter.

[0152] The above 3) modification of the base sequence of the region encoding the initiation codon or 5'-UTR of the gene encoding the protein (polypeptide) may be, for example, a modification that encodes another initiation codon having a higher protein (polypeptide) expression rate than the endogenous initiation codon, or an RBS sequence having a higher protein (polypeptide) expression rate than the endogenous RBS (ribosome binding site) sequence, but is not limited thereto.

[0153] The modification of the amino acid sequence or polynucleotide sequence of the protein (polypeptide) of the above 4) and 5) may be, but is not limited to, introducing a sequence mutation by deletion, insertion, substitution, or a combination thereof in the amino acid sequence of the protein (polypeptide) or the polynucleotide sequence encoding the protein (polypeptide) so as to increase the activity of the protein (polypeptide), or replacing it with an amino acid sequence or polynucleotide sequence modified to increase the activity. The replacement may be performed, for example, by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited thereto.

[0154] The introduction of the foreign polynucleotide exhibiting the activity of the above 6) protein (polypeptide) may be the introduction into the host cell (microorganism) of a foreign polynucleotide encoding a protein (polypeptide) exhibiting the same / similar activity as the protein (polypeptide). The foreign polynucleotide is not limited in its origin or sequence as long as it exhibits the same / similar activity as the protein (polypeptide). The method used for the above introduction can be performed by a person skilled in the art appropriately selecting a known transformation method, and the protein (polypeptide) can be produced by expressing the introduced polynucleotide in the host cell, thereby increasing its activity.

[0155] The above 7) codon optimization of a polynucleotide encoding a protein (polypeptide) may be codon optimization of an endogenous polynucleotide to increase transcription or translation within a host cell (microorganism), or codon optimization of a foreign polynucleotide to achieve optimized transcription or translation within a host cell (microorganism).

[0156] The above 8) analyzing the tertiary structure of a protein (polypeptide) and selecting an exposed portion to modify or chemically modify may be done by, for example, comparing the sequence information of the protein (polypeptide) to be analyzed with a database storing the sequence information of known proteins, determining a template protein candidate based on the degree of sequence similarity, confirming the structure based on this, and selecting an exposed portion to modify or chemically modify, and modifying or modifying it.

[0157] The above 9) regulation of the intracellular location of a protein (polypeptide) may target the protein (polypeptide) to a specific organelle or specific intracellular space within the cell. For example, targeting to the periplasm or cytoplasm may be achieved by adding or removing a leader sequence that functions in targeting the protein (polypeptide), but is not limited thereto.

[0158] Such an increase in protein (polypeptide) activity may be, but is not limited to, an increase in the activity or concentration of the corresponding protein (polypeptide) relative to the activity or concentration of the protein (polypeptide) expressed in the wild type or pre-transformed host cell (microorganism), or an increase in the amount of a product resulting from the activity of the corresponding protein (polypeptide).

[0159]

[0160] Modification of part or all of the polynucleotide in the microorganism of the present application may be induced by, but is not limited to, (a) a method using homologous recombination using a vector for chromosome insertion into the microorganism, or genome editing using genetic scissors (e.g., CRISPR-Cas9) and / or (b) treatment with light and / or chemicals such as ultraviolet rays and radiation.

[0161]

[0162] The microorganism of the present application may have improved L-amino acid production ability.

[0163] In the present application, “L-amino acid” includes all L-amino acids that can be produced by microorganisms through metabolic processes from various carbon sources, but specifically, may include, but is not limited to, L-histidine, L-isoleucine, L-tryptophan, or L-lysine.

[0164] In one specific example of the present application, the microorganism of the genus Corynebacterium may have an increased ability to produce at least one amino acid selected from L-histidine, L-isoleucine, L-tryptophan, and L-lysine compared to a non-modified microorganism.

[0165]

[0166] Another aspect of the present application provides a method for producing an L-amino acid, comprising the step of culturing a microorganism comprising a variant polypeptide of the present application; a polynucleotide encoding the same; or a vector comprising the polynucleotide in a medium.

[0167]

[0168] The above microorganisms are as described in other aspects.

[0169] In this application, the term "cultivation" refers to growing the microorganism of this application under appropriately controlled environmental conditions. The culturing process in this application can be performed using any suitable medium and culture conditions known in the art. Such culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing process may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0170] The microorganism of the present application can be cultured under aerobic conditions in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorus, inorganic compounds, amino acids, and / or vitamins, while controlling temperature, pH, etc.

[0171] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugar) may be used, and other appropriate amounts of carbon sources may be used in various ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0172] The nitrogen source may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc.; peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0173] The above-mentioned components may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the medium in batch or continuous manner, but are not limited thereto.

[0174] During the cultivation of the microorganism of the present invention, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, an antifoaming agent such as fatty acid polyglycol ester can be used to suppress bubble formation. In addition, to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas can be injected into the medium, or to maintain anaerobic and microaerobic states, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, but the present invention is not limited thereto.

[0175] Additionally, the culture medium may contain metal salts, such as magnesium sulfate or iron sulfate, necessary for growth. Finally, in addition to the above substances, essential growth substances, such as amino acids and vitamins, may be used. Appropriate precursors may also be used in the culture medium. The above-mentioned raw materials may be added to the culture in a batch or continuous manner during the culture process, but are not limited thereto.

[0176] In the present application, during the cultivation of microorganisms, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. can be appropriately added to the culture to adjust the pH of the culture. In addition, during the cultivation, an antifoaming agent such as fatty acid polyglycol ester can be used to suppress bubble formation. Furthermore, to maintain the aerobic state of the culture, oxygen or an oxygen-containing gas can be injected into the culture, or to maintain anaerobic or microaerobic states, nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection, but is not limited thereto.

[0177] In the culture of the present application, the culture temperature can be maintained at 20 to 35°C, specifically 25 to 35°C, and the culture period can be continued until the amount of useful material produced is obtained, and the culture can be performed for about 10 to 160 hours, about 20 to 130 hours, about 24 to 120 hours, about 36 to 120 hours, about 48 to 120 hours, about 48 hours or more, or about 48 hours, about 72 hours, or about 120 hours, but is not limited thereto.

[0178]

[0179] L-amino acids produced by the culture of the present invention may be secreted into the medium or remain within the cells.

[0180] In one specific example of the present application, the L-amino acid may be at least one selected from L-histidine, L-isoleucine, L-tryptophan, and L-lysine, but is not limited thereto.

[0181]

[0182] In one specific example, the method for producing L-amino acids of the present application may further include, for example, prior to the culturing step, a step of preparing a microorganism of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order).

[0183] The L-amino acid production method of the present application may further include a step of recovering a target substance, specifically an L-amino acid, from the cultured microorganism, a culture of the microorganism, a fermented product of the microorganism, or the culture medium. The recovering step may be additionally included after the culturing step.

[0184] The above recovery may be performed by collecting the target L-amino acid using a suitable method known in the art according to the culture method of the microorganism of the present application, such as a batch, continuous or fed-batch culture method. For example, various chromatographies such as centrifugation, filtration, treatment with a crystallized protein precipitant (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC or a combination thereof may be used, and the target substance, specifically, the L-amino acid, may be recovered from the medium or microorganism using a suitable method known in the art.

[0185] Additionally, the L-amino acid production method of the present application may additionally include a purification step. The purification may be performed using any suitable method known in the art. In one example, if the L-amino acid production method of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or discontinuously, regardless of order, or may be performed simultaneously or integrated into a single step, but is not limited thereto.

[0186] In the method of the present application, the mutant polypeptide, introduction and L-amino acid, etc. are as described in the other embodiments above.

[0187]

[0188] Another aspect of the present application provides a composition for producing L-amino acids, comprising at least one selected from the group consisting of: a variant polypeptide of the present application; a polynucleotide encoding the variant polypeptide; a vector comprising the same; a microorganism comprising the vector; a culture of the microorganism; and a fermented product of the microorganism.

[0189] The composition of the present application may further comprise any suitable excipient commonly used in compositions for producing L-amino acids, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffering agents, stabilizers, or isotonic agents.

[0190] In one specific example, each component present in the composition of the present application may be included in a microbiologically effective amount, or an amount that can be suitably present in the composition for production.

[0191] In the composition of the present application, the mutant polypeptide, introduction and L-amino acid, etc. are as described in the other embodiments above.

[0192]

[0193] Another aspect of the present application provides a use for producing L-amino acids by a microorganism having improved L-amino acid production ability, wherein at least one selected from the mutant polypeptide of the present application and a polynucleotide encoding the mutant polypeptide is introduced.

[0194] In the purposes of the present application, the mutant polypeptide, introduction and L-amino acid, etc. are as described in the other embodiments above.

[0195]

[0196] Another aspect of the present application provides a method for producing a microorganism with improved L-amino acid production ability, comprising the step of introducing at least one selected from the mutant polypeptide of the present application and a polynucleotide encoding the mutant polypeptide into a Corynebacterium microorganism having L-amino acid production ability.

[0197] Another aspect of the present application provides a method for increasing L-amino acid production capacity, comprising the step of introducing at least one selected from the group consisting of a mutant polypeptide of the present application and a polynucleotide encoding the mutant polypeptide into a Corynebacterium microorganism having L-amino acid production capacity.

[0198] In the method of the present application, the mutant polypeptide, introduction and L-amino acid, etc. are as described in the other embodiments above.

[0199]

[0200] Hereinafter, this application will be described in more detail through examples and experimental examples. However, these examples and experimental examples are intended to exemplify this application and the scope of this application is not limited to these examples and experimental examples.

[0201]

[0202] Example 1: Construction of a recombinant vector for introducing a fructokinase mutation.

[0203]

[0204] We aimed to produce a Corynebacterium glutamicum mutant with improved fructokinase activity. Specifically, to produce a vector for deletion of Tn1, a gene encoding a transposon in Corynebacterium glutamicum, and for inserting a target gene, PCR was performed using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template and primer pairs of SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively. PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase for the PCR reaction, and 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, and these denaturation, annealing, and polymerization conditions were repeated 28 times. As a result, DNA fragments of 511 bp and 526 bp were obtained, respectively.

[0205] SEQ ID NO NAME Sequence 9PrimerttcgagctcggtacccCAAATGCTCCAACCGTCCGT10PrimerCGCTGGGATGTTTCTGAACTCATTCCTTCTGCT11PrimerACAAGAACTGGAATAGGACATCTAATAACCGGGC12PrimerctctagaggatccccTACACCTCCACCTGCCCAG

[0206] Next, PCR was performed using the genomic DNA of wild-type Escherichia coli ATCC 9637 as a template and the primer pair of SEQ ID NOs: 13 and 14 to obtain a wild-type csck (E. co) gene fragment. Information on the gene and surrounding base sequences (accession number CP002967.1) was obtained from the National Institutes of Health GenBank (NIH GenBank).

[0207] In addition, PCR was performed using the genomic DNA of Escherichia coli ATCC 9637 as a template with the primer pairs of SEQ ID NOs: 13 and 15 and 16 and 14, respectively, and then overlapping PCR was performed using the mixture of these obtained PCR fragments as a template again with the primer pairs of SEQ ID NOs: 13 and 14, thereby obtaining the 'cscK (W79C, E.co)' gene fragment. Similarly, PCR was performed using the genomic DNA of Escherichia coli ATCC 9637 as a template with primer pairs of SEQ ID NOs: 13 and 17 and primer pairs of SEQ ID NOs: 18 and 14, respectively, and a mixture of the two obtained fragments was used as a template with primer pairs of SEQ ID NOs: 13 and 14 to perform overlapping PCR again, thereby obtaining the 'cscK (A181V, E.co)' gene fragment. In addition, in order to produce a combination mutant, PCR was performed using the genomic DNA of Escherichia coli ATCC 9637 as a template with primer pairs of SEQ ID NOs: 13 and 15, primer pairs of SEQ ID NOs: 16 and 17, and primer pairs of SEQ ID NOs: 18 and 14, respectively, and a mixture of the three obtained fragments was used as a template with primer pairs of SEQ ID NOs: 13 and 14 to perform overlapping PCR again. The 'cscK(W79C / A181V, E.co)' gene fragment was obtained. PCR was performed under the same conditions as above.

[0208] SEQ ID NO: Name Sequence 13PrimerCCCAACGAAAGGAAACACTCATGTCAGCCAAAGTATGGGT14PrimerTGCCCGGTTATTAGATGTCCTATTCCAGTTCTTGTC15PrimerATGTCCGGTGACATTCATCTTGC16PrimerAAGATGAATGTCACCGGACATCC17PrimerACATCCACCAGTTGTAGCGCCTGCCGCAAA18PrimerAACTGGTGGATGTCGTCAAGCTCTCGGAAG

[0209]

[0210] Next, in order to use the CJ7 promoter derived from Corynebacterium ammoniagenes (SEQ ID NO: 19, Patent Registration No. 10-0620092), PCR was performed in the same manner as above using the primers of SEQ ID NO: 20 and SEQ ID NO: 21 using Corynebacterium ammoniagenes genomic DNA as a template to obtain a CJ7 promoter fragment.

[0211] Sequence number name sequence 20PrimerGCAGAAGGAATGAGTTCAGAAACATCCCAGCGCTACT 21PrimerTACTTTGGCTGACATGAGTGTTTCCTTTCGTTGGGT

[0212] Each obtained DNA product was purified using a PCR purification kit (PCR Purification kit, QUIAGEN), and the purified DNA product and the chromosomal transformation vector pDC24 (SEQ ID NO: 85) digested with SmaI restriction enzyme were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY 2009, NEBuilder HiFi DNA Assembly 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 the calculated molar number and storing at 50°C for 1 hour.

[0213]

[0214] Example 2. Evaluation of the productivity of an L-amino acid producing strain into which a cscK mutant gene has been introduced.

[0215]

[0216] Example 2-1. Production of histidine-producing strain and evaluation of histidine production capacity.

[0217]

[0218] Example 2-1-1. Production of histidine-producing strain

[0219]

[0220] In order to evaluate the L-histidine production ability of the strain into which the cscK mutant gene constructed in Example 1 was introduced, a CA14-0114 strain was first constructed, using the wild-type Corynebacterium glutamicum ATCC13032 as a starting microorganism, with enhanced genes in the histidine biosynthetic pathway. Specifically, in order to resolve the feedback inhibition of the HisG protein, the first enzyme in the L-histidine biosynthetic pathway, the codon of the hisG gene was modified so that a protein (SEQ ID NO: 22) (ACS Synth. Biol., 2014, 3 (1), pp 21-29) was expressed in which the 233rd and 235th amino acids from the N-terminus of HisG were simultaneously substituted from glycine to histidine and from threonine to glutamine, respectively. In addition, the start codon was substituted from GTG to ATG to enhance the activity of the hisE gene, which exists in the same operon as hisG. Additionally, to strengthen the L-histidine biosynthetic pathway, the promoters of the biosynthetic genes hisN, hisH, hisD, hisA, and hisB were replaced with strong promoters, and the pathway was strengthened by introducing additional copies of the hisE(g1a)G(G233H / T235Q) operon and hisD gene.

[0221]

[0222] Example 2-1-1-1. Production of a histidine-producing strain with resolved feedback limitations.

[0223]

[0224] To produce a histidine-producing strain free of feedback limitations, PCR was performed using the Corynebacterium glutamicum ATCC13032 genomic DNA as a template and the primer pairs of SEQ ID NO: 23 and SEQ ID NO: 24, and the primer pairs of 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 and the primers of SEQ ID NO: 23 and SEQ ID NO: 26, PCR was performed in the same manner as in Example 1 to obtain the 'hisE(g1a)G(G233H / T235Q)' gene fragment. In addition, the upstream region of the hisE gene was obtained by performing PCR using the primers of SEQ ID NO: 27 and 28 using the ATCC13032 chromosomal DNA as a template.

[0225] Sequence number name sequence (5'-> 3')23PrimergaggagatcaaaacaATGAAGACATTTGAC24PrimerACACCAAGCTTGATGGATACCTCTACTGCA25PrimerCAGTAGAGGTATCCATCAAGCTTGGTGTC26PrimerACTCTAGAGGATCCCCCTAGATGCGGGC27PrimerTCGAGCTCGGTACCCACCGAA CTCCTGACAGAGT28PrimeracatgaagcgccTCGGTACATTCTTCCACA30PrimerAAGAATGTACCGAggcgcttcatgtcaaca31PrimerCAAATGTCTTCATtgttttgatctcctcca32PrimerACTGCCTGGTACCACCAGA33PrimerCTGCCTCTCACAAGTTGAAG

[0226] To replace the strong promoter, PCR was performed in the same manner as in Example 1 using the synthetic promoter Pspl13 promoter (SEQ ID NO: 29, Republic of Korea Patent No. 10-1783170) as a template and primers of SEQ ID NO: 30 and 31.

[0227] After treating the pDC24 vector with the restriction enzyme Sma1, the upstream DNA fragment of the amplified hisE gene, the Pspl13 promoter, and the 'hisE(g1a)G(G233H / T235Q)' gene fragment were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named pDC24△Pn_hisEG:: Pspl13_hisE(g1a)G(G233H / T235Q). Gibson cloning was performed in the same manner as in Example 1.

[0228] The constructed pDC24△Pn_hisEG:: Pspl13_hisE(g1a)G(G233H / T235Q) vector was transformed into Corynebacterium glutamicum ATCC13032 by electroporation, and then a second crossover process was performed to introduce a mutation into the existing hisG gene, thereby eliminating feedback restriction, and a strain with enhanced hisE activity was obtained by replacing the initiation codon of the hisE gene. The genetic manipulation was confirmed by PCR using primers of SEQ ID NOs: 32 and 33 that can amplify the external regions of the upstream and downstream regions of the homologous recombination site where the gene was inserted, respectively, and by genome sequencing. The strain thus obtained was named CJ-HIS1.

[0229]

[0230] Example 2-1-1-2. Production of a histidine-producing strain with an enhanced biosynthetic pathway through promoter replacement.

[0231]

[0232] Next, plasmids were constructed to replace the wild-type promoter of each gene with a strong promoter to enhance the activity of the biosynthetic genes hisN, hisH, hisD, hisA, and hisB.

[0233] Specifically, PCR was performed in the same manner as in Example 1 using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template and the primers of 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, and the upstream regions of the hisN, hisH, hisD, hisA, and hisB genes were obtained. In addition, using the chromosomal DNA of Corynebacterium glutamicum ATCC13032 as a template, the downstream regions of the hisN, hisH, hisD, hisA, and hisB genes were obtained using primers of 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, respectively.

[0234] To replace the endogenous promoters of the hisN, hisH, and hisD genes with the strong promoter, the Pcj7 promoter, PCR was performed in the same manner as in Example 1 using the primers of SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57, and SEQ ID NO: 58 and SEQ ID NO: 59 using Corynebacterium ammoniagenes genomic DNA as a template.

[0235] In addition, in order to replace the endogenous promoters of the hisA and hisB genes with the strong promoter, the Pspl13 promoter, PCR was performed in the same manner as in Example 1 using the primers of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63, using the Pspl13 promoter as a template.

[0236] After treating the pDC24 vector with the restriction enzyme Sma1, the amplified upstream DNA fragments of the 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, thereby obtaining recombinant plasmids, which were designated pDC24△Pn:: cj7_hisN, pDC24△Pn:: cj7_hisH, and pDC24△Pn:: cj7_hisD. In addition, after treating the pDC24 vector with the restriction enzyme Sma1, the amplified upstream DNA fragments of the 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, thereby obtaining recombinant plasmids, which were designated pDC24△Pn:: Pspl13_hisA, and pDC24△Pn:: Pspl13_hisB. Gibson cloning was performed in the same manner as in Example 1.

[0237] The constructed pDC24△Pn:: cj7_hisN vector was transformed into CJ-HIS1 constructed in Example 2-1-1-1 by electroporation, and then a second crossover process was performed to replace the promoter in the existing hisN gene, thereby obtaining a strain with an enhanced gene. The genetic manipulation was confirmed through PCR using primers of SEQ ID NO: 64 and SEQ ID NO: 65 that can amplify the external regions of the upstream and downstream regions of the homologous recombination region where the gene was inserted, respectively, and genome sequencing. The strain thus obtained was named CJ-HIS2.

[0238] Next, the constructed pDC24△Pn:: cj7_hisH vector was transformed into the constructed CJ-HIS2 by electroporation, and then the promoter of the existing hisH gene was replaced through a second crossover process to obtain a strain with an enhanced gene. The genetic manipulation was confirmed through PCR using primers of SEQ ID NOs: 66 and 67 that can amplify the external regions of the upstream and downstream regions of the homologous recombination region where the gene was inserted, respectively, and genome sequencing. The strain thus obtained was named CJ-HIS3.

[0239] Next, the constructed pDC24△Pn:: cj7_hisD vector was transformed into the constructed CJ-HIS3 by electroporation, and then a second crossover process was performed to replace the promoter of the existing hisD gene, thereby obtaining a strain with an enhanced gene. The genetic manipulation was confirmed by PCR using primers of SEQ ID NO: 68 and SEQ ID NO: 69 and genome sequencing. The strain thus obtained was named CJ-HIS4.

[0240] Next, the constructed pDC24△Pn:: Pspl13_hisA vector was transformed into the constructed CJ-HIS4 by electroporation, and then a second crossover process was performed to replace the promoter of the existing hisA gene, thereby obtaining a strain with an enhanced gene. The genetic manipulation was confirmed by PCR using primers of SEQ ID NO: 70 and SEQ ID NO: 71 and genome sequencing. The strain thus obtained was named CJ-HIS5.

[0241] Next, the constructed pDC24△Pn:: Pspl13_hisB vector was transformed into the constructed CJ-HIS5 by electroporation, and then a second crossover process was performed to replace the promoter of the existing hisB gene, thereby obtaining a strain with an enhanced gene. The genetic manipulation was confirmed by PCR using primers of SEQ ID NO: 72 and SEQ ID NO: 73 and genome sequencing. The strain thus obtained was named CJ-HIS6.

[0242]

[0243]

[0244] Example 2-1-1-3. Production of a histidine-producing strain with an enhanced biosynthetic pathway through additional gene insertion.

[0245]

[0246] Next, NCgl1021, known as a gene encoding a transposon in Corynebacterium glutamicum, was used as an insertion site to additionally insert the hisE(g1a)G(G233H / T235Q) operon and the hisD gene. Specifically, to construct a NCgl1021 (SEQ ID NO: 74) deletion and target gene insertion vector, the chromosome of ATCC13032 was used as a template and primer pairs of SEQ ID NO: 75 and SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78 were used to perform PCR in the same manner as in Example 1 to amplify the left homologous arm region of NCgl1021 and the right homologous arm region of NCgl1021. PCR was performed in the same manner as in Example 1 using the vector pDC24 △Pn:: Pspl13_ hisE(g1a)G(G233H / T235Q) produced in the above Example 2-1-1-1 as a template and the primers of SEQ ID NO: 79 and SEQ ID NO: 80, and the 'Pspl13_ hisE(g1a)G(G233H / T235Q)' gene fragment was obtained.

[0247] In addition, PCR was performed in the same manner as in Example 1 using the vector pDC24 △Pn:: cj7_hisD produced in Example 2-1-1-2 as a template and primers of SEQ ID NO: 81 and SEQ ID NO: 82, and the 'Pcj7_hisD' gene fragment was obtained.

[0248] After treating the pDC24 vector with the restriction enzyme Sma1, the amplified left homology arm of NCgl1021, the right homology arm of NCgl1021, 'Pspl13_ hisE(g1a)G(G233H / T235Q)' and 'Pcj7_hisD' gene fragments were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named 'pDC24△NCgl1021:: Pspl13_ hisE(g1a)G(G233H / T235Q)- Pcj7_hisD'. Gibson cloning was performed in the same manner as in Example 1. The constructed 'pDC24△NCgl1021:: Pspl13_ hisE(g1a)G(G233H / T235Q)- Pcj7_hisD' vector was transformed into CJ-HIS6 constructed in Example 2-1-1-2 by electroporation, and then a second crossing process was performed to obtain a strain with an enhanced histidine biosynthetic pathway through additional gene insertion. The genetic manipulation was confirmed through PCR using primers of SEQ ID NOs: 83 and 84 that can amplify the external regions of the upstream and downstream regions of the homologous recombination region where the gene was inserted, respectively, and genome sequencing. The strain thus obtained was named CA14-0114.

[0249] Sequence number name sequence (5'-> 3')75PrimerTTCGAGCTCGGTACCCATGAAGTCTACCGGC 76PrimergacatgaagcgccGACATCTAATAACCGGG 77PrimerCCGACGAGGCCTAAGAACTCATTCCTTCTGCT 78PrimerCTCTAGAGGATCCCCTTAGAGTGCATTGATC 79PrimerCCGGTTATTAGATGTCggcgcttcatgtca 80PrimerggatgtttctCTAGATGCGGGCGAT 81PrimerGCCCGCATCTAGagaaacatcccagcgct 82PrimerAGAAGGAATGAGTTCTTAGGCCTCGTCGG 83PrimerCTTTCAGCTTTCCCTCCCG 84PrimerGCCTGTACTTTTAGTACA

[0250] Example 2-1-2. Production of a transformed strain with a csck (E.co) mutant gene introduced into a histidine-producing strain.

[0251]

[0252] 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) produced in the above Example 1 were transformed into the histidine-producing strain CA14-0114 produced in the above Example 2-1-1 by the electric pulse method, and then the transformed strains were obtained in a selection medium containing 25 mg / L of kanamycin, and these were respectively transformed into CA14-0114△Tn1::Pcj7_cscK(E.co), CA14-0114△Tn1:: They were named Pcj7_cscK(W79C, E.co), CA14-0114△Tn1:: Pcj7_cscK(A181V, E.co), and CA14-0114△Tn1::Pcj7_cscK(W79C / A181V, E.co).

[0253]

[0254] Example 2-1-3. Evaluation of histidine production capacity

[0255]

[0256] To confirm the L-histidine production ability of the strains produced in the above Example 2-1-2, they were cultured and evaluated using the following method. Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of the following seed medium and cultured at 30°C for 20 hours with shaking at 200 rpm. Then, 1 ml of the seed culture was inoculated into a 250 ml corner-baffle flask containing 25 ml of the production medium and cultured at 30°C for 48 hours with shaking at 200 rpm.

[0257]

[0258] <Seed medium (pH 7.0)>

[0259] Glucose 5%, Bactopeptone 1%, Sodium Chloride 0.25%, Yeast Extract 1%, Urea 0.4% (based on 1 liter of distilled water)

[0260]

[0261] <Production medium (pH 7.0)>

[0262] 6% raw sugar, 2% ammonium sulfate, 0.1% monobasic potassium phosphate, 0.05% magnesium sulfate heptahydrate, 2.0% CSL (corn steep liquor), 200 μg / L biotin, 30 g / L calcium carbonate (based on 1 liter of distilled water)

[0263] Comparison of L-histidine production ability of L-histidine-producing strains derived from Corynebacterium glutamicum ATCC 13032 (48H) Strain name L-histidine concentration (g / L) CA14-0114 4.3 CA14-0114△Tn1:: Pcj7-cscK (WT, E.co) 4.5 CA14-0114△Tn1:: Pcj7-cscK (W79C, E.co) 4.9 CA14-0114△Tn1:: Pcj7-cscK (A181V, E.co) 4.6 CA14-0114△Tn1:: Pcj7-cscK (W79C / A181V, E.co) 4.9

[0264] As shown in the table above, when the csck wild-type gene derived from Escherichia coli was introduced, it was confirmed that histidine production increased by about 4.6% compared to the parent strain CA14-0114. In addition, when the cscK mutant gene was introduced, it was confirmed that histidine production increased by about 2.2% to about 8.8% compared to CA14-0114△Tn1:: Pcj7-cscK (WT, E.co) into which the wild-type cscK gene was introduced. This indicates that the csck gene mutation improves the L-histidine production ability of the strain by improving the activity of fructokinase.

[0265]

[0266] Example 2-2. Production of an isoleucine-producing strain with a CscK mutation and evaluation of isoleucine production capacity.

[0267]

[0268] Example 2-2-1. Production of a transformed strain with a csck (E.co) mutant gene introduced into an isoleucine-producing strain.

[0269]

[0270] In order to confirm whether there is an effect of increasing isoleucine production by introducing a fructokinase mutant gene derived from Escherichia coli into a Corynebacterium glutamicum strain having L-isoleucine production ability, the recombinant vectors pDC24△Tn1::Pcj7_cscK(E.co), pDC24△Tn1:: Pcj7_cscK(W79C, E.co), pDC24△Tn1:: Pcj7_cscK(A181V, E.co), and pDC24△Tn1:: Pcj7_cscK(W79C / A181V, E.co) prepared in Example 1 were transformed into the isoleucine-producing strain KCCM12739P (Korean Patent No. 10-2363913) by the electric pulse method, and then kanamycin was added. Transformant strains were obtained from the selection medium containing 25 mg / L, 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.

[0271]

[0272] Example 2-2-2. Evaluation of isoleucine production capacity

[0273]

[0274] In order to confirm the L-isoleucine production ability of the strain produced in the above Example 2-2-1, it was cultured and evaluated using the following method. After inoculating the parent strain and the mutant strain into a 250 ml corner-bottom flask containing 25 ml of isoleucine production medium, the strain was cultured with shaking at 200 rpm for 60 hours at 32°C.

[0275]

[0276] <Production medium (pH 7.2)>

[0277] Glucose 10%, yeast extract 0.2%, ammonium sulfate 1.6%, potassium phosphate monobasic 0.1%, magnesium sulfate heptahydrate 0.1%, iron sulfate heptahydrate 10 mg / ℓ, manganese sulfate monohydrate 10 mg / ℓ, biotin 200 ㎍ / ℓ (based on 1L of distilled water)

[0278]

[0279] Comparison of L-isoleucine production ability of L-isoleucine-producing strains derived from Corynebacterium glutamicum ATCC 13032 (60H) Strain name L-isoleucine concentration (g / L) KCCM12739P 2.1 KCCM12739P △Tn1:: Pcj7-cscK (WT, E.co) 2.3 KCCM12739P △Tn1:: Pcj7-cscK (W79C, E.co) 2.9 KCCM12739P △Tn1:: Pcj7-cscK (A181V, E.co) 2.5 KCCM12739P △Tn1:: Pcj7-cscK (W79C / A181V, E.co) 3.0

[0280] As shown in the table above, when the csck wild-type gene derived from Escherichia coli was introduced, it was confirmed that the isoleucine production increased by about 9.5% compared to the parent strain KCCM12739P. In addition, when the cscK mutant gene was introduced, it was confirmed that the isoleucine production increased by about 8.6% to about 30.4% compared to KCCM12739P△Tn1:: Pcj7-cscK (WT, E.co) into which the wild-type cscK gene was introduced. This indicates that the csck gene mutation improves the L-isoleucine production ability of the strain by improving the activity of fructokinase.

[0281]

[0282] Example 2-3. Production of a tryptophan-producing strain with a CscK mutation and evaluation of tryptophan production capacity.

[0283]

[0284] Example 2-3-1. Production of a transformed strain with a csck (E.co) mutant gene introduced into a tryptophan-producing strain.

[0285]

[0286] In order to confirm whether there is an effect of increasing tryptophan production by introducing a fructokinase mutant gene derived from Escherichia coli into a Corynebacterium glutamicum strain having L-tryptophan production ability, the recombinant vectors pDC24△Tn1:: Pcj7_cscK(E.co), pDC24△Tn1:: Pcj7_cscK(W79C, E.co), pDC24△Tn1:: Pcj7_cscK(A181V, E.co), and pDC24△Tn1:: Pcj7_cscK(W79C / A181V, E.co) prepared in Example 1 were transformed into the tryptophan-producing strain CA04-8405 (Registered Patent No. 10-1968317 of the Republic of Korea) by the electric pulse method, Transformant strains were obtained from a selective medium containing 25 mg / L of 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.

[0287]

[0288] Example 2-3-2. Evaluation of tryptophan production capacity

[0289]

[0290] In order to confirm the L-tryptophan production ability of the strains produced in the above Example 2-3-1, they were cultured and evaluated using the following method. Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of seed medium and cultured at 30°C for 20 hours with shaking at 200 rpm. After culture, a new 250 ml corner-baffle flask containing 25 ml of production medium for each strain was prepared, 1 ml of the above seed culture solution was inoculated into it, and cultured at 30°C for 24 hours with shaking at 200 rpm.

[0291] <Seed medium (pH 7.0)>

[0292] Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4 7H2O 0.5g, biotin 100㎍, thiamine HCl 1000㎍, calcium-pantothenic acid 2000㎍, nicotinamide 2000㎍ (based on 1L of distilled water)

[0293] <Production medium (pH 7.0)>

[0294] Glucose 30g, (NH4)2SO4 15g, MgSO4 7H2O 1.2g, KH2PO4 1g, yeast extract 5g, biotin 900㎍, thiamine hydrochloride 4500㎍, calcium-pantothenic acid 4500㎍, CaCO3 30g (based on 1L of distilled water)

[0295]

[0296] Comparison of L-tryptophan production ability of L-tryptophan-producing strains derived from Corynebacterium glutamicum ATCC 13869 (24H) Strain name L-tryptophan concentration (g / L) CA04-8405 1.5 CA04-8405 △Tn1:: Pcj7-cscK (WT, E.co) 1.6 CA04-8405 △Tn1:: Pcj7-cscK (W79C, E.co) 1.9 CA04-8405 △Tn1:: Pcj7-cscK (A181V, E.co) 1.8 CA04-8405 △Tn1:: Pcj7-cscK (W79C / A181V, E.co) 2.0

[0297] As shown in the table above, when the csck wild-type gene derived from Escherichia coli was introduced, it was confirmed that the tryptophan production increased compared to the parent strain CA04-8405. In addition, when the cscK mutant gene was introduced, it was confirmed that the tryptophan production increased by about 12.5% ​​to about 25% compared to CA04-8405 △Tn1:: Pcj7-cscK (WT, E.co) into which the wild-type cscK gene was introduced. This indicates that the csck gene mutation improves the L-tryptophan production ability of the strain by improving the activity of fructokinase.

[0298]

[0299] Example 2-4. Production of a lysine-producing strain with a CscK mutation and evaluation of lysine production capacity.

[0300]

[0301] Example 2-4-1. Production of a transformed strain with a csck (E. co) mutant gene introduced into a lysine-producing strain.

[0302]

[0303] In order to confirm whether there is an effect of increasing lysine production by introducing a fructokinase mutant gene derived from Escherichia coli into a Corynebacterium glutamicum strain having L-lysine production ability, the recombinant vectors pDC24△Tn1:: Pcj7_cscK(E.co), pDC24△Tn1:: Pcj7_cscK(W79C, E.co), pDC24△Tn1:: Pcj7_cscK(A181V, E.co), and pDC24△Tn1:: Pcj7_cscK(W79C / A181V, E.co) prepared in Example 1 were transformed into the lysine-producing strain KCCM11016P (Korean Patent No. 10-0159812) by the electric pulse method, and then 25 mg / L of kanamycin was added. Transformant strains were obtained from the selection medium containing the strain, 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.

[0304]

[0305] Example 2-4-2. Evaluation of lysine production capacity

[0306]

[0307] To confirm the L-lysine production ability of the strains produced in Example 2-4-1, they were cultured and evaluated using the following method. Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of seed medium and cultured at 37°C for 20 hours with shaking at 200 rpm. Each strain was inoculated into a 250 ml corner-baffle flask containing 25 ml of production medium and cultured at 37°C for 48 hours with shaking at 200 rpm.

[0308]

[0309] <Seed medium (pH 7.0)>

[0310] Glucose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4·7H2O 0.5g, biotin 0.1mg, thiamine HCl 1mg, calcium-pantothenic acid 2mg, nicotinamide 2mg (based on 1L of distilled water)

[0311]

[0312] <Production medium (pH 7.0)>

[0313] Glucose 50g, (NH4)2SO4 40g, Corn Steep Solids 5g, BM 10g, KH2PO4 1g, MgSO4 7H2O 0.5g, Biotin 100㎍, Thiamine Hydrochloride 1000㎍, Calcium Pantothenate 2000㎍, Nicotinamide 3000㎍, CaCO3 30g (based on 1 liter of distilled water)

[0314]

[0315] Comparison of L-lysine production ability of L-lysine producing strains derived from Corynebacterium glutamicum ATCC 13869 (48H) Strain name L-lysine concentration (g / L) KCCM11016P 9.45 KCCM11016P △Tn1:: Pcj7-cscK (WT, E.co) 10.4 KCCM11016P △Tn1:: Pcj7-cscK (W79C, E.co) 11.6 KCCM11016P △Tn1:: Pcj7-cscK (A181V, E.co) 10.8 KCCM11016P △Tn1:: Pcj7-cscK (W79C / A181V, E.co) 12.1

[0316] As shown in the table above, when the csck wild-type gene derived from Escherichia coli was introduced, it was confirmed that the lysine production increased compared to the parent strain KCCM11016P. In addition, when the cscK mutant gene was introduced, it was confirmed that the lysine production increased by about 3.8% to about 16% compared to KCCM11016P △Tn1:: Pcj7-cscK (WT, E.co) into which the wild-type cscK gene was introduced. This indicates that the csck gene mutation improves the L-lysine production ability of the strain by improving the activity of fructokinase.

[0317]

[0318] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering its technical concept or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalents, rather than the detailed description above.

Claims

1. A fructokinase mutant polypeptide in which the amino acid corresponding to position 79 or position 181 of sequence number 1 is substituted with another amino acid.

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

3. A mutant polypeptide according to claim 1, wherein the amino acid sequence of the mutant polypeptide has at least 90% identity with the amino acid sequence of SEQ ID NO:

1.

4. In the first paragraph, the mutant polypeptide is a mutant polypeptide consisting of an amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO:

7.

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

6. A microorganism comprising a mutant polypeptide according to any one of claims 1 to 4; and at least one polynucleotide encoding the mutant polypeptide.

7. In paragraph 6, the microorganism is a microorganism of the genus Corynebacterium.

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

9. In the 6th paragraph, the microorganism has an increased ability to produce L-amino acids compared to a microorganism containing the polypeptide of sequence number 1 or a polynucleotide encoding the same.

10. A microorganism in claim 9, wherein the L-amino acid is at least one selected from L-histidine, L-isoleucine, L-tryptophan, and L-lysine.

11. A method for producing L-amino acid, comprising a step of culturing the microorganism of clause 6 in a medium.

12. A method according to claim 11, further comprising a step of recovering L-amino acid from at least one selected from the cultured microorganism; a culture of the microorganism; a fermented product of the microorganism; and the culture medium.

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

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