Microorganism into which threonine dehydratase derived from arabidopsis thaliana is introduced and method for producing branched-chain amino acid using the same

Abstract

The present disclosure relates to: a microorganism of the genus Corynebacterium, in which threonine dehydratase derived from Arabidopsis thaliana is introduced; a method for producing branched-chain amino acids, which includes a step of culturing the microorganism in a culture medium; a method for improving production of branched-chain amino acids, which includes a step of culturing the microorganism in a culture medium; a composition for producing branched-chain amino acids, which comprises the microorganism, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof; and use of the microorganism for producing branched-chain amino acids.

Description

Technical Field

[0001] This disclosure relates to Corynebacterium microorganisms, wherein the introduction of bacteria derived from Arabidopsis thaliana ( Arabidopsis thaliana The microorganism is a threonine dehydratase; a method for producing branched-chain amino acids, comprising culturing the microorganism in a culture medium; a method for improving the production of branched-chain amino acids, comprising culturing the microorganism in a culture medium; a composition for producing branched-chain amino acids, comprising the microorganism, a culture of the microorganism, a fermentation product of the microorganism, or a combination of at least two of the above; and the use of the microorganism for producing branched-chain amino acids. Background Technology

[0002] L-amino acids are the basic building blocks of proteins and are used as important materials in pharmaceuticals, food additives, animal feed, nutritional supplements, pesticides, and fungicides. In particular, branched-chain amino acids (BCAAs) refer to L-valine, L-leucine, and L-isoleucine; these are essential amino acids, and BCAAs are known to have antioxidant properties and directly promote protein synthesis in muscle cells.

[0003] Meanwhile, the production of branched-chain amino acids using microorganisms is usually carried out by Escherichia coli (Escherichia coli). Escherichia ) belongs to microorganisms or corynebacteria ( Corynebacterium Branched-chain amino acids are produced by microorganisms. For example, L-valine is known to be biosynthesized from pyruvate using 2-oxoisovalerate as a precursor through several steps, but it is difficult to achieve large-scale industrial production of branched-chain amino acids through microorganisms.

[0004] In methods for large-scale microbial production of L-valine, blocking the L-isoleucine biosynthesis pathway is known. However, when the L-isoleucine biosynthesis pathway encodes L-threonine dehydratase... ilvA When the gene is missing, there is a problem of growth being limited due to the need for L-isoleucine.

[0005] Therefore, it is still necessary to study how to improve the production efficiency of branched-chain amino acids while maintaining the sugar consumption rate. Summary of the Invention

[0006] Technical issues This disclosure aims to provide Corynebacterium microorganisms and their uses, wherein the microorganisms are infused with a threonine dehydratase derived from Arabidopsis thaliana.

[0007] Technical solution One aspect of this disclosure provides a Corynebacterium microorganism in which a threonine dehydratase derived from Arabidopsis thaliana is introduced.

[0008] In one specific embodiment, the threonine dehydratase derived from Arabidopsis thaliana may contain an amino acid sequence that has at least 75% identity with SEQ ID NO:15.

[0009] In another specific implementation, the threonine dehydratase derived from Arabidopsis thaliana can be produced by... ilvA Gene encoding.

[0010] In another specific implementation scheme, the thaliana derived from Arabidopsis thaliana ilvA The gene may contain a base sequence that has at least 75% identity with SEQ ID NO: 16.

[0011] As a microorganism according to any one of the foregoing specific embodiments, the microorganism may be a microorganism in which endogenous threonine dehydratase activity is further lacking.

[0012] As a microorganism according to any one of the foregoing specific embodiments, the Corynebacterium genus microorganism may be Corynebacterium glutamicum (… Corynebacterium glutamicum ).

[0013] As a microorganism according to any one of the foregoing specific embodiments, the microorganism may have the ability to produce branched-chain amino acids.

[0014] As a microorganism according to any one of the foregoing specific embodiments, the branched-chain amino acid may be L-valine or L-leucine.

[0015] As a microorganism according to any one of the foregoing specific embodiments, the microorganism may have an enhanced ability to produce branched-chain amino acids compared with unmodified microorganisms.

[0016] Another aspect of this disclosure provides a method for producing branched-chain amino acids, comprising culturing Corynebacterium microorganisms in a culture medium, wherein the microorganisms are inoculated with a threonine dehydratase derived from Arabidopsis thaliana.

[0017] In one specific embodiment, the method may further include recovering branched-chain amino acids from cultured microorganisms, cultures of the microorganisms, fermentation products of the microorganisms, or culture media after culture.

[0018] Another aspect of this disclosure provides a method for improving the production of branched-chain amino acids, comprising culturing Corynebacterium microorganisms in a culture medium, wherein the microorganisms are inoculated with a threonine dehydratase derived from Arabidopsis thaliana.

[0019] Another aspect of this disclosure provides a composition for producing branched-chain amino acids, comprising: a Corynebacterium microorganism introduced with a threonine dehydratase derived from Arabidopsis thaliana; a culture of the microorganism; a fermentation product of the microorganism; or a combination of at least two of these.

[0020] Another aspect of this disclosure provides the use of Corynebacterium microorganisms for the production of branched-chain amino acids, wherein the microorganisms are infused with a threonine dehydratase derived from Arabidopsis thaliana.

[0021] Beneficial effects The Corynebacterium microorganism disclosed herein (introduced with threonine dehydratase derived from Arabidopsis thaliana) can produce branched-chain amino acids in high yield while maintaining the sugar consumption rate, and therefore can be effectively applied to the industrial production of branched-chain amino acids. Detailed Implementation

[0022] This disclosure will now be described in detail. Furthermore, each description and embodiment described herein can be applied to other descriptions and embodiments. That is, all combinations of the various elements described herein fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited to the specific embodiments described below.

[0023] Furthermore, those skilled in the art can recognize or identify many equivalents of specific aspects of this disclosure by using conventional experiments. Moreover, these equivalents are intended to be included in this disclosure.

[0024] As used in this disclosure and the appended claims, the singular articles (“a,” “an,” and “the”) include plural indicators unless the context clearly indicates otherwise. Furthermore, unless the context otherwise indicates, singular terms include their plural forms, and plural terms include their singular forms. As used in this disclosure and the appended claims, the use of “or” can include the meaning of “and / or” unless otherwise stated.

[0025] As used herein, the term "about" may precede a specific numerical value. As used herein, the term "about" includes not only the exact numerical value specified after the term, but also a range that approximates or approaches that value. Considering the context in which the number appears, it can be determined whether the specific number mentioned is close to or near that number. In one instance, the term "about" may refer to the range of -10% to +10% of the numerical value. In another instance, the term "about" may refer to the range of -5% to +5% of a given numerical value. However, it is not limited to these examples.

[0026] As used herein, terms such as “first, second, third,” “i), ii), iii), or “(a), (b), (c), (d)” are used to distinguish similar elements and do not imply that these elements are performed consecutively or in the listed order. For example, when these terms are used for methods, uses, or analytical steps, these steps may be performed simultaneously without any time intervals, or at intervals of seconds, minutes, hours, days, or months.

[0027] As used herein, the term “consistently made of” may mean that the unspecified component may be present, provided that the characteristics of the object claimed herein are substantially unaffected by the presence of the unspecified component.

[0028] As used herein, the term "composed of" means that the total proportion of a particular component is 100%. The components or features listed in the term "composed of" may be necessary or mandatory. In some specific embodiments, other arbitrary or non-essential components may be excluded in addition to the components or features listed in the term "composed of".

[0029] As used herein, the term "comprising" means the presence of a feature, step, or component that follows the term, and does not exclude the presence or addition of one or more other features, steps, or components. As used herein, the component or feature following the term "comprising" may be necessary or mandatory; however, in some embodiments, other optional or non-essential components or features may also be included.

[0030] One aspect of this disclosure provides a Corynebacterium microorganism in which a threonine dehydratase derived from Arabidopsis thaliana is introduced.

[0031] As used herein, the term "threonine dehydratase (ilvA)" refers to an enzyme involved in the L-isoleucine biosynthesis pathway. It specifically refers to the enzyme that produces 2-ketobutyrate from L-threonine, an amino acid derived from aspartate, in the L-isoleucine biosynthesis pathway. L-isoleucine is produced using 2-ketobutyrate and pyruvate generated in the corresponding process (glycolysis) as precursors. The threonine dehydratase of this disclosure can be used interchangeably with ilvA or L-threonine dehydratase. Specifically, the threonine dehydratase of this disclosure can be characterized by exhibiting the characteristics of… ilvA A gene encodes a protein with threonine dehydratase activity, but its type is not particularly limited as long as it exhibits activity corresponding to threonine dehydratase. ilvAGene-encoded threonine dehydratases are known in the art, and the amino acid and polynucleotide sequences of threonine dehydratases are available from publicly available databases, including, but not limited to, NCBI’s GenBank.

[0032] In one instance, the threonine dehydratase derived from Arabidopsis thaliana of this disclosure may specifically be a threonine dehydratase derived from the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15 (excluding only the transport peptide from SEQ ID NO: 13), but is not limited thereto.

[0033] For example, a threonine dehydratase derived from Arabidopsis thaliana may contain the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 75% homology or identity with it, but is not limited thereto, as long as it retains threonine dehydratase activity. Specifically, a polypeptide exhibiting threonine dehydratase activity may have or contain the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with SEQ ID NO: 15, consisting of said amino acid sequence, or substantially consisting of said amino acid sequence.

[0034] Regarding the amino acid sequences in this disclosure, even if it is disclosed as a polypeptide "comprising" an amino acid sequence described by a specific sequence number, a polypeptide "composed of" an amino acid sequence described by a specific sequence number, or a polypeptide or protein "having" an amino acid sequence described by a specific sequence number, it is apparent that a protein having an amino acid sequence in which some sequences are missing, modified, substituted, conservedly substituted, or added has the same or corresponding activity as a protein composed of an amino acid sequence with the corresponding sequence number. For example, when exhibiting the same or equivalent activity as a variant protein, it is obvious that proteins having sequence additions, naturally occurring mutations, or silent mutations or their conserved substitutions that do not alter protein function are not excluded, and such proteins having sequence additions or mutations fall within the scope of this disclosure.

[0035] For example, the amino acid sequence may include sequence additions, naturally occurring mutations, silent mutations, or conserved substitutions that do not alter the function of the variant polypeptide disclosed herein at the N-terminus and / or C-terminus and / or within the amino acid sequence. For example, the polypeptide may be conjugated to a signal (or leader) sequence at the N-terminus of a protein involved in protein transport during or after translation. Furthermore, the polypeptide may be conjugated to another sequence or linker to allow for the identification, purification, or synthesis of the polypeptide.

[0036] As used herein, the term "conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitutions can typically occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In another example, amino acids with charged side chains (charged amino acids) include arginine, lysine, histidine, glutamic acid, and aspartic acid, while amino acids with uncharged side chains (also called uncharged amino acids or neutral amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. In another example, valine, leucine, and isoleucine can be classified as branched-chain amino acids. In yet another example, the 20 amino acids can be divided into five groups based on size, starting with the smaller group: glycine, alanine, and serine; cysteine, proline, threonine, aspartic acid, and asparagine; valine, histidine, glutamic acid, and glutamine; isoleucine, leucine, methionine, lysine, and arginine; and phenylalanine, tryptophan, and tyrosine. However, the classification of amino acids is not limited to this. Generally, conserved substitutions have little or no effect on the activity of peptides.

[0037] Furthermore, the base sequence encoding a threonine dehydratase derived from Arabidopsis thaliana can be the base sequence encoding a protein exhibiting threonine dehydratase activity, and in Corynebacterium microorganisms, this protein confers activity that enhances the production of branched-chain amino acids.

[0038] In one instance, the threonine dehydratase derived from Arabidopsis thaliana of this disclosure may specifically be derived from the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 15 (excluding only the transport peptide from SEQ ID NO: 13). The threonine dehydratase may be encoded by polynucleotides containing the base sequences of SEQ ID NO: 14 and 16, respectively, but is not limited thereto.

[0039] In one example, a threonine dehydratase having the amino acid sequence of SEQ ID NO: 15 may be encoded by a polynucleotide having or containing the sequence of SEQ ID NO: 16 or having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% and less than 100% homology or identity with the sequence of SEQ ID NO: 16, being composed of, or substantially composed of, the base sequence, but not limited thereto.

[0040] In this disclosure, for example, a gene containing the base sequence of SEQ ID NO: 16 may be used interchangeably with: a polynucleotide containing the base sequence of SEQ ID NO: 16; a gene or polynucleotide having the base sequence of SEQ ID NO: 16; a gene or polynucleotide consisting of the base sequence of SEQ ID NO: 16; or a gene or polynucleotide derived from Arabidopsis thaliana. ilvA .

[0041] Considering codon degeneracy or the preferred codons in the organism in which the threonine dehydratase of this disclosure will be expressed, the polynucleotides of this disclosure can be modified in various ways in the coding region without altering the amino acid sequence of the threonine dehydratase of this disclosure. Therefore, it is apparent that polynucleotides that can be translated by codon degeneracy into polypeptides consisting of the amino acid sequence of the threonine dehydratase of this disclosure, or polypeptides homologous to or identical with it, can also be included. For example, the polynucleotides of this disclosure can be the sequence of SEQ ID NO: 16, or its degenerate sequence.

[0042] In another instance, the polynucleotide of this disclosure may have or comprise a base sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% and less than 100% homology or identity with the sequence of SEQ ID NO: 16, or may consist of or substantially consist of a base sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% and less than 100% homology or identity with the sequence of SEQ ID NO: 16, but is not limited thereto.

[0043] Furthermore, the polynucleotides disclosed herein may include, but are not limited to, probes that may be prepared from known gene sequences, such as any polynucleotide sequence that hybridizes under stringent conditions to a sequence that is fully or partially complementary to the polynucleotide sequence of this disclosure to encode the threonine dehydratase of this disclosure.

[0044] As used herein, “homology” or “identity” refers to the degree of correlation between two given amino acid sequences or base sequences, expressed as a percentage. The terms “homology” and “identity” are often used interchangeably.

[0045] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined using standard alignment algorithms, with default gap penalties established by the program used. Essentially, homologous or identical sequences can typically hybridize with at least approximately 50%, 60%, 70%, 80%, or 90% or more of the full-length sequence under moderately or highly stringent conditions. Clearly, hybridization also includes hybridization with polynucleotides containing codons reflecting common codon usage or codon degeneracy within the polynucleotide.

[0046] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined, for example, by a known computer algorithm such as the “FASTA” program (Pearson et al., (1988) [Proc. Natl. Acad. Sci. USA85]: 2444) using default parameters. Alternatively, it can be determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as in the Needleman program (version 5.0.0 or later) of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (including the GCG program package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, SF et al., J MOLEC BIOL 215:403 (1990); Guide to HugeComputers, Martin J. Bishop ed., Academic Press, San Diego, 1994, and CARILLO et al. (1988) SIAM J Applied Math). (48:1073). For example, homology, similarity, or identity can be determined using BLAST or ClustalW from the National Center for Biotechnology Information.

[0047] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, a GAP computer program (such as Needleman et al., (1970), J Mol Biol. 48:443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482). In short, the GAP program can be defined as a value obtained by dividing the number of similarly aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP procedure may include: (1) a unitary matrix (containing a value of 1 for identity and 0 for non-identity), a PAM matrix (see Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation (1978)) or a weighted comparison matrix of Gribskov et al., (1986) Nucl. Acids Res. 14:6745 (or an alternative matrix of EDNAFULL (EMBOSS version of NCBI NUC4.4)); (2) a penalty of 3.0 for each vacancy and an additional penalty of 0.10 for each symbol in each vacancy (or a penalty of 10 for vacancy opening and 0.5 for vacancy extension); and (3) no penalty for terminal vacancy.

[0048] Furthermore, whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined by comparing the sequences in a Southern hybridization experiment under strictly defined conditions. Suitable hybridization conditions are within the scope of the art and can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., *Molecular Cloning*, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratorypress, Cold Spring Harbor, New York, 1989; FM Ausubel et al., *Current Protocols in Molecular Biology*, John Wiley & Sons, Inc., New York). However, the methods for identifying homology, similarity, or identity, and suitable hybridization conditions, are not limited thereto.

[0049] As used herein, the term “strict conditions” refers to conditions that enable specific hybridization between polynucleotides. These conditions are described in detail in the literature (see Sambrook et al., ibid., 9.50–9.51, 11.7–11.8). For example, strict conditions may include polynucleotides with high homology or identity, i.e., polynucleotides with 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%, or at least 99% homology or identity hybridizing with each other, while polynucleotides with less than the above homology or identity do not hybridize with each other; or may include conventional washing conditions for Southern hybridization, i.e., washing once, and more specifically twice or three times, at salt concentrations and temperatures corresponding to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0050] Hybridization requires two nucleotides to have complementary sequences, although base mismatches are permitted due to the strictness of hybridization. The term "complementary" is used to describe the relationship between the bases of nucleotides that can hybridize with each other. For example, in DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this disclosure may also comprise separate nucleic acid fragments complementary to the whole sequence, as well as a substantially similar base sequence thereto.

[0051] For example, polynucleotides that are homologous or identical to the polynucleotides disclosed herein can be detected using hybridization conditions, said hybridization conditions including the conditions described above, T. m The hybridization step is at a temperature of 55°C. Furthermore, T... m The value can be 60°C, 63°C or 65°C, but is not limited to these, and can be appropriately adjusted by those skilled in the art for their purposes.

[0052] The appropriate stringency of polynucleotide hybridization depends on the length and complementarity of the polynucleotides, variables that are well known in the field (e.g., Sambrook et al., ibid.).

[0053] As used herein, the term "microorganism (or strain)" includes wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modifications. It can be a microorganism whose specific mechanism is weakened or enhanced due to the insertion of a foreign gene or the enhancement or inactivation of an endogenous gene, and it can be a microorganism containing genetic modifications for the production of peptides, proteins, or target products. In this disclosure, the terms "microorganism" and "strain" have the same meaning and can be used interchangeably without limitation.

[0054] For example, the microorganisms disclosed herein may be microorganisms incorporating threonine dehydratase derived from Arabidopsis thaliana (e.g., recombinant strains), but are not limited thereto. The microorganisms may also be microorganisms capable of producing branched-chain amino acids.

[0055] As used herein, the term "microorganism capable of producing branched-chain amino acids" refers to a prokaryotic or eukaryotic microbial strain capable of producing branched-chain amino acids in a living organism, and may include microorganisms that confer the ability to produce branched-chain amino acids to parental strains that do not possess this ability, and microorganisms that inherently exhibit the ability to produce branched-chain amino acids. The ability to produce branched-chain amino acids can be conferred or enhanced through strain modification.

[0056] As used herein, the term "branched-chain amino acid" refers to an amino acid having a branched alkyl group on its side chain, including valine, leucine, and isoleucine. Specifically, in this disclosure, the branched-chain amino acid may be an L-branched-chain amino acid, and the L-branched-chain amino acid may be L-valine, L-leucine, or L-isoleucine, more specifically L-valine or L-leucine. As used herein, the term "unmodified microorganism" does not exclude strains that may naturally occur in microorganisms, and may refer to wild-type strains or unchanged natural strains, or strains whose traits have been altered due to genetic mutations caused by natural or artificial factors. "Unmodified microorganism" may be used interchangeably with "pre-modified strain," "pre-modified microorganism," "unmutated strain," "unmodified strain," "unmutated microorganism," "pre-mutated parental strain," "wild-type microorganism," "reference microorganism," or "control microorganism." As used herein, unmodified microorganism may refer to, but is not limited to, strains in which the threonine dehydratase of this disclosure has not been introduced or has not been introduced with the threonine dehydratase of this disclosure. In addition, in this disclosure, the unmodified microorganism may be a microorganism that does not contain the amino acid sequence of SEQ ID NO: 13 or 15, or the base sequence of SEQ ID NO: 14 or 16, but is not limited thereto.

[0057] For the purposes of this disclosure, the microorganisms of this disclosure may include any microorganism incorporating a threonine dehydrating enzyme derived from Arabidopsis thaliana. Furthermore, the microorganisms of this disclosure may include any microorganism incorporating a threonine dehydrating enzyme derived from Arabidopsis thaliana, wherein the incorporation enables the production of desired branched-chain amino acids. For example, the microorganisms of this disclosure are characterized by an enhanced ability to produce branched-chain amino acids due to the incorporation of a threonine dehydrating enzyme derived from Arabidopsis thaliana, and may be genetically modified microorganisms or recombinant microorganisms, but are not limited thereto. Specifically, recombinant strains with enhanced branched-chain amino acid production capacity may be microorganisms with enhanced branched-chain amino acid production capacity compared to natural wild-type microorganisms, unmodified microorganisms with endogenous threonine dehydrating enzyme activity, or unmodified microorganisms without endogenous threonine dehydrating enzyme activity, but are not limited thereto.

[0058] In one instance, a microorganism capable of producing branched-chain amino acids refers to a prokaryotic or eukaryotic microbial strain capable of producing branched-chain amino acids in a living organism, and may include microorganisms that inherently exhibit the ability to produce branched-chain amino acids and microorganisms in which the ability to produce branched-chain amino acids is conferred upon a parent strain that does not possess the ability to produce branched-chain amino acids by introducing a threonine dehydratase derived from Arabidopsis thaliana according to this disclosure. The ability to produce branched-chain amino acids can be conferred or enhanced through strain modification.

[0059] The microorganisms disclosed herein may include any microorganism in which threonine dehydratase derived from Arabidopsis thaliana is introduced by various known methods.

[0060] As used herein, the term “enhanced” peptide activity refers to an increase in peptide activity compared to its endogenous activity. Enhancement can be used interchangeably with terms such as activation, upregulation, overexpression, and increase.

[0061] "Enhancement" can include exhibiting activities that were not initially present, as well as exhibiting increased activity compared to endogenous activity or activity before modification. For example, "exhibiting activities that were not initially present" could be "introducing protein," but is not limited to this.

[0062] As used herein, the term "introduction" of activity refers to the expression in a microorganism of a gene that was not originally present in the microorganism, thereby exhibiting the activity of a specific protein, or an increase, enhancement, or improvement in the activity of a polypeptide compared to its endogenous activity or its activity before modification. For example, activity can be exhibited by introducing a polynucleotide encoding a specific protein into the chromosome of a microorganism or by introducing a vector containing a polynucleotide encoding a specific protein into the microorganism.

[0063] The term "endogenous activity" refers to the activity of a specific polypeptide originally possessed by the parental strain before the trait was altered, or the activity of a specific polypeptide originally possessed by an unmodified microorganism when the trait is altered due to genetic variation caused by natural or artificial factors. This term can be used interchangeably with "activity before modification".

[0064] Enhanced peptide activity relative to endogenous activity refers to the increase in the activity and / or concentration (expression level) of a specific peptide naturally present in the parental strain or unmodified microorganism before transformation.

[0065] In one instance, enhancement may refer to the presence of the activity of a corresponding protein (peptide) that is not present, or an enhancement of the activity or concentration of a protein relative to the activity or concentration of the unmodified wild-type protein or in a microbial strain, typically by about 1%, about 10%, about 25%, about 50%, about 75%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500%, up to about 1,000%, or about 2,000% or more, but not limited thereto.

[0066] Enhanced peptide activity can be achieved by introducing exogenous peptides or enhancing the activity of endogenous peptides. This enhancement can be confirmed by increasing peptide activity or expression levels, or by increasing the amount of peptide secretion products.

[0067] Enhancement of peptide activity can be achieved by applying a variety of methods well known in the art, and such methods are not limited, as long as they enhance the activity of the desired peptide compared to the unmodified microorganism. Specifically, it can be achieved using genetic engineering and / or protein engineering techniques well known to those skilled in the art, which are routine molecular biology techniques, but are not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al., Molecular Cloning 2012, etc.).

[0068] Specifically, the enhancement of peptide activity can be achieved in the following ways: 1) Increase the intracellular copy number of the polynucleotide encoding the polypeptide; 2) Modify the gene expression regulatory region on the chromosome that encodes the polypeptide (e.g., modify the expression regulatory region by replacing it with a sequence that exhibits stronger activity, or inserting a sequence that exhibits stronger activity). 3) Modify the start codon or 5'-UTR sequence of the gene transcript (which encodes the polypeptide); 4) Modify the amino acid sequence of the polypeptide to enhance its activity; 5) Modify the polynucleotide sequence encoding the polypeptide to enhance its activity (e.g., modify the polynucleotide sequence of the polypeptide gene so that the polynucleotide sequence encodes a polypeptide that exhibits enhanced polypeptide activity). 6) Introduce an exogenous polypeptide that exhibits the activity of the polypeptide or an exogenous polynucleotide encoding the polypeptide; 7) Codon optimization was performed on the polynucleotide encoding the polypeptide; 8) By analyzing the tertiary structure of the polypeptide, select and modify or chemically modify the exposed regions of the polypeptide; 9) Regulate the cellular localization of the polypeptide; or 10) A combination of at least two of the above 1) to 9), but not particularly limited thereto.

[0069] More specifically, 1) Increasing the intracellular copy number of the polynucleotide encoding the polypeptide can be achieved by introducing a vector operatively linked to the polynucleotide encoding the polypeptide into a host cell, the vector being capable of replicating and functioning independently of the host. Alternatively, this method can be achieved by introducing one or at least two copies of the polynucleotide encoding the polypeptide into the chromosome of a host cell. Introduction into the chromosome can be performed by introducing a vector capable of inserting the polynucleotide into the host cell's chromosome, but is not limited thereto. The vector is as described above.

[0070] 2) Replacing the expression regulatory region (expression regulatory sequence) of the gene encoding the polypeptide on the chromosome with a highly active sequence can be achieved, for example, by introducing modifications to the sequence through deletion, insertion, non-conserved or conserved substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or by replacing the sequence with a more active sequence. The expression regulatory region may include, but is not specifically limited to, promoters, operon sequences, sequences encoding ribosome binding sites, sequences regulating transcription and translation termination, etc. In one instance, it may specifically include replacing the original promoter with a strong promoter, but is not limited to this.

[0071] Examples of known strong promoters include CJ1 to CJ7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, λ phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US 10584338 B2), O2 promoter (US 10273491 B2), tkt promoter, and yccA promoter, but strong promoters are not limited to these.

[0072] 3) Modifying the start codon or 5'-UTR of the gene transcript (which encodes the polypeptide) may, for example, replace the start codon with a start codon that has a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0073] 4) and 5) Modification of amino acid sequences or polynucleotide sequences can be achieved by inducing mutations in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide through deletion, insertion, non-conservative or conserved substitution, or a combination thereof, thereby enhancing the activity of the polypeptide, or by replacing the sequence with a modified amino acid sequence or polynucleotide sequence that has enhanced activity, but is not limited thereto. Substitution can be specifically carried out by inserting polynucleotides into chromosomes through homologous recombination, but is not limited thereto. The vectors used herein may further contain selection markers for confirming chromosomal insertion. The selection markers are as described above.

[0074] 6) Introduction of exogenous polynucleotides exhibiting the activity of the polypeptide can be achieved by introducing exogenous polynucleotides encoding polypeptides exhibiting the same or similar activity as the polypeptide into host cells. The exogenous polynucleotide is not limited by its source or sequence, as long as it exhibits the same or similar activity as the polypeptide. Introduction can be carried out using transformation methods known in the art, appropriately selected by those skilled in the art, and the expression of the polynucleotide introduced into the host cell can generate the polypeptide, thereby enhancing its activity.

[0075] 7) Codon optimization of the polynucleotide encoding the polypeptide can be performed on endogenous polynucleotides to enhance their transcription or translation in the host cell, or on exogenous polynucleotides to optimize their transcription or translation in the host cell.

[0076] 8) Analyzing the tertiary structure of the polypeptide and thereby selecting and modifying the exposed sites or chemically modifying them can, for example, compare the sequence information of the polypeptide to be analyzed with a database storing the sequence information of known proteins to identify template protein candidates based on the degree of sequence similarity, confirm the structure based on the information, and thereby select and transform or modify the exposed sites to be modified or chemically modified.

[0077] 9) Regulating the cellular localization of the peptide can be achieved by targeting the peptide to specific organelles or specific spaces within the cell. For example, the peptide can be targeted to the periplasm or cytoplasm by adding or removing a leader sequence that plays a role in protein targeting, but is not limited to this.

[0078] This increase in peptide activity, relative to the activity or concentration of the peptide expressed in the unmodified wild-type strain or microorganism, can be an enhancement of the activity or concentration of the corresponding peptide, or an enhancement of the amount of peptide produced, but is not limited to these.

[0079] For example, the recombinant microorganisms of this disclosure capable of producing branched-chain amino acids may include any microorganism transformed with a vector that is capable of producing branched-chain amino acids by introducing a foreign gene encoding a threonine dehydratase derived from Arabidopsis thaliana of this disclosure.

[0080] The vector disclosed herein may comprise a DNA construct containing the base sequence of a polynucleotide encoding a desired polypeptide, which is operatively linked to an expression regulatory region suitable for expressing the target polypeptide in a suitable host. The expression regulatory sequence may include a promoter capable of initiating transcription, any operon sequence regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating transcription and translation termination. Once transformed into a suitable host cell, the vector may replicate or function independently of the host genome, or it may integrate into the host genome.

[0081] There are no particular limitations on the vectors used in this disclosure, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, granules, viruses, and bacteriophages in their native or recombinant states. For example, as phage vectors or granule vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc., may be used; as plasmid vectors, those based on pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pSK, pSKH, pET, etc., may be used. Specifically, pDZ, pDC, pDC24, pACYC177, pACYC184, pCL, pSK, pSKH130, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, etc., may be used.

[0082] In one embodiment, a polynucleotide encoding a desired polypeptide can be inserted into a chromosome using a vector for intracellular chromosome insertion. The polynucleotide can be inserted into the chromosome using any method known in the art, such as homologous recombination, but is not limited thereto. A selection marker for confirming chromosome insertion may be further included. The selection marker is used to screen cells transformed with the vector, i.e., to confirm the insertion of the desired nucleic acid molecule; a marker conferring a selectable phenotype (such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or expression of a surface polypeptide) may be used. In an environment treated with a selection agent, only cells expressing the selection marker survive or exhibit a different phenotype, thereby allowing selection of transformed cells.

[0083] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell so that the polypeptide encoded by the polynucleotide can be expressed within the host cell. The transformed polynucleotide may be located within the host cell's chromosome or exist extrachromosomally, provided it can be expressed within the host cell. Furthermore, the polynucleotide comprises DNA and / or RNA encoding the polypeptide. The polynucleotide can be introduced in any form, as long as it can be introduced into and expressed within the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene construct containing all the elements necessary for self-expression. The expression cassette may contain a promoter, transcription termination signal, ribosome binding site, and translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. Furthermore, the polynucleotide may be introduced into the host cell as is and operably linked to sequences necessary for expression in the host cell, but is not limited thereto.

[0084] As used herein, the term "operably linked" refers to an arrangement in which a regulatory sequence is positioned appropriately to regulate the expression of a coding sequence. Therefore, the term "operably linked" includes attaching or linking a regulatory region with a functional domain having known or desired activity, such as a promoter, stop codon, signal sequence, or enhancer region, to a target (gene or polypeptide) to regulate the expression, secretion, or function of the target according to known or desired activity. For example, this means that a polynucleotide encoding a desired variant polypeptide of this disclosure is functionally linked to a promoter sequence that initiates and mediates polynucleotide transcription.

[0085] As used herein, the term “expression” includes, but is not limited to, any step involved in peptide production, such as transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0086] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule that contains a coding sequence and an operable linked expression regulatory sequence.

[0087] As used herein, the term "regulatory sequence" refers to a polynucleotide sequence essential for the expression of a coding sequence. Each regulatory sequence can be native (from the same source) or exogenous (from a different gene) to the coding sequence. Examples of regulatory sequences can include leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, operon sequences, sequences encoding ribosome-binding domains, and sequences that regulate the termination of transcription and translation. The smallest unit of a regulatory sequence can contain a promoter as well as sequences that terminate transcription and translation.

[0088] As used herein, the term "recombinant" in relation to cells, nucleic acids, peptides, or vectors means that the cells, polynucleotides, peptides, or vectors have been modified by introducing heterologous polynucleotides or peptides, or by altering native polynucleotides or peptides, or that the cells are derived from such modified cells. Thus, for example, recombinant cells can express genes not found in the natural (non-recombinant) form of the cell, or can express native genes that would otherwise be abnormally expressed, underexpressed, or not expressed at all.

[0089] For example, the microorganisms that produce branched-chain amino acids may be microorganisms that introduce the following: a protein encoding an amino acid sequence of SEQ ID NO: 15 or a protein containing an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or higher homology or identity with the amino acid sequence of SEQ ID NO: 15.

[0090] For example, the microorganisms that produce branched-chain amino acids may be microorganisms that introduce the following: a base sequence capable of encoding a protein containing an amino acid sequence having at least 75% homology with the amino acid sequence of SEQ ID NO: 15; the base sequence of SEQ ID NO: 16; or a base sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, or at least 98% and less than 100% homology or identity with the base sequence of SEQ ID NO: 16.

[0091] In one example, the microorganism with enhanced branched-chain amino acid production capacity disclosed herein may be, but is not limited to, a microorganism with enhanced branched-chain amino acid production capacity compared to an unmodified microorganism. In one example, the unmodified microorganism used as a reference strain for comparing enhanced L-valine production capacity may be *Corynebacterium glutamicum* strain KCCM11201P (US 8,465,962 B) or strain CJ7V (US 2020-0362374 A1). In another example, the unmodified microorganism used as a reference strain for comparing enhanced L-leucine production capacity may be *Corynebacterium glutamicum* strain CJL8109 (WO 2022 / 163981 A1), but the unmodified microorganism is not limited to this.

[0092] In one instance, a recombinant microorganism with enhanced ability to produce branched-chain amino acids may exhibit an enhancement of at least about 1% compared to the ability of its parental strain before modification or the unmodified microorganism to produce branched-chain amino acids. Specifically, this enhancement may be at least about 1%, at least about 2.5%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 26%, or at least about 27% (with no particular upper limit, e.g., up to about 200%, up to about 150%, up to about 100%, up to about 50%, up to about 45%, up to about 40%, up to about 35%, or up to about 30%). However, the ability of the recombinant microorganism to produce branched-chain amino acids is not limited to this, as long as it exhibits a positive increase compared to the production capacity of its parental strain before modification or the unmodified microorganism. In another instance, a microorganism with enhanced ability to produce branched-chain amino acids may have an enhancement of at least about 1.1 times, at least about 1.15 times, at least about 1.20 times, at least about 1.25 times, at least about 1.26 times, or at least about 1.27 times (with no particular upper limit, e.g., up to about 10 times, up to about 5 times, up to about 3 times, up to about 2 times, up to about 1.5 times, up to about 1.4 times, or up to about 1.3 times) compared to the ability of the parental strain before modification or the unmodified microorganism to produce branched-chain amino acids, but is not limited thereto.

[0093] In one instance, the microorganism may be a Corynebacterium genus.

[0094] In one instance, the microorganism disclosed herein may be *Corynebacterium glutamicum*, *Corynebacterium crudeum*, or... Corynebacterium crudilactis ), Corynebacterium desertis ( Corynebacterium deserti ), High-efficiency Corynebacterium ( Corynebacterium efficiens ), Corynebacterium tumefaciens ( Corynebacterium callunae ), Corynebacterium stasis ( Corynebacterium stationis ), single rod-shaped bacteria ( Corynebacterium singulare ), salt-tolerant Corynebacterium ( Corynebacterium halotolerance ), Corynebacterium bandingense ( Corynebacterium striatum ), Corynebacterium ammoniagenicus ( Corynebacterium ammoniagenes ), contaminating Corynebacterium ( Corynebacterium pollutisoli ), mimicking Corynebacterium ( Corynebacterium imitans ), Corynebacterium testis ( Corynebacterium testudinoris ) or Corynebacterium fulvidracosum ( Corynebacterium flavescens ).

[0095] Specifically, the microorganisms disclosed herein may be Corynebacterium genus, and more specifically Corynebacterium glutamicum, but are not limited thereto.

[0096] The microorganisms disclosed herein may be Corynebacterium genus microorganisms, wherein endogenous threonine dehydratase activity is further lacking, but are not limited thereto.

[0097] As used herein, the term “attenuation” of peptide activity is a concept that includes reduced activity and loss of activity compared to its intrinsic activity. Attenuation can be used interchangeably with terms such as lack, inactivation, deletion, destruction, downregulation, reduction, decrease, weakening, inhibition, and reduction.

[0098] For example, attenuation refers to a state where a protein exhibits activity but is not completely inactivated due to its absence. This could mean that the protein activity is reduced relative to the unmodified microorganism, the wild-type strain, or the parent strain, but it is not limited to these.

[0099] For example, attenuation can be inactivation or deletion, but is not limited to these. Inactivation may mean that the protein is not expressed, or even if it is expressed, no activity is observed, or its activity is attenuated compared to the parent strain or the unmodified strain.

[0100] Attenuation can also include the following situations: the activity of the polypeptide is weakened or removed compared to the original polypeptide activity of the microorganism due to mutations in the polynucleotide encoding the polypeptide; the overall activity level and / or concentration (expression level) of the intracellular polypeptide is reduced compared to the natural strain due to the inhibition of gene expression of the polynucleotide encoding the polypeptide, or the inhibition of translation into polypeptide; the polynucleotide is not expressed at all; and / or even when the polynucleotide is expressed, no polypeptide activity is observed.

[0101] Compared to endogenous activity, reduced peptide activity refers to a decrease in the activity of a specific peptide naturally present in the parental strain or unmodified microorganism before transformation. Reduced peptide activity can be confirmed by a decrease in peptide activity or expression level, or a decrease in the amount of peptide secreted products.

[0102] In one instance, attenuation can be a situation where the protein activity is less than approximately 100%, approximately 90% or less, approximately 80% or less, approximately 70% or less, approximately 60% or less, approximately 50% or less, approximately 40% or less, approximately 30% or less, approximately 20% or less, approximately 10% or less, approximately 5% or less, or 0% of the protein activity in the untransformed parental strain or the unmodified microorganism, but is not limited to this.

[0103] For example, inactivation or absence may mean that the protein is not expressed, or even if it is expressed, no activity is observed or the activity is reduced compared to unmodified microorganisms.

[0104] The attenuation of peptide activity can be achieved by any method known in the art, but is not limited to this method, and can be achieved by applying a variety of methods well known in the art (e.g., Nakashima N et al., Bacterialcellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al., Molecular Cloning 2012).

[0105] Specifically, the reduced activity of the polypeptide disclosed herein may be due to: 1) The gene encoding the polypeptide is missing in whole or in part; 2) Modify the expression control region (or expression control sequence) to reduce the expression of the gene encoding the polypeptide; 3) Modify the amino acid sequence that makes up the polypeptide so that the activity of the polypeptide is eliminated or weakened (e.g., by deleting / replacing / adding at least one amino acid in the amino acid sequence). 4) Modify the polynucleotide sequence encoding the polypeptide to eliminate or reduce the activity of the polypeptide (e.g., delete / replace / add at least one nucleic acid in the nucleic acid sequence of the polypeptide gene to encode a modified polypeptide, thereby eliminating its activity or exhibiting reduced polypeptide activity). 5) Modify the start codon or 5'-UTR sequence of the gene encoding the polypeptide; 6) Introduce an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the polypeptide. 7) Add a sequence complementary to the SD sequence upstream of the Shine-Dalgarno (SD) sequence of the gene encoding the polypeptide to form a secondary structure that prevents ribosome attachment; 8) Reverse transcription engineering (RTE), which adds the promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) encoding the polynucleotide sequence of the polypeptide; 9) Regulate the cellular localization of the polypeptide; or 10) A combination of at least two of the above 1) to 9), but not particularly limited thereto.

[0106] For example, 1) The deletion of part or all of the gene encoding the polypeptide can be achieved by deleting all polynucleotides encoding the desired endogenous polypeptide within the chromosome, or by replacing the polynucleotide with a polynucleotide that has a partially deleted nucleotide sequence or by replacing the polynucleotide with a marker gene.

[0107] Methods for deleting part or all of a polynucleotide can be performed by, but are not limited to, methods such as: deleting polynucleotides via homologous recombination using a chromosome integration vector in a microorganism, or inducing mutations using light such as ultraviolet light or chemical reagents, and selecting strains from the obtained mutants that have deleted the target gene. Methods for deleting part or all of a gene can include methods based on DNA recombination technology. For example, deleting all or part of a gene can be achieved by inducing homologous recombination by introducing a nucleotide sequence or vector containing a nucleotide sequence homologous to the desired gene into a microorganism. The nucleotide sequence or vector to be introduced may contain a dominant selection marker, but is not limited to this.

[0108] 2) Modification of the expression regulatory region can be, for example, by deletion, addition, non-conserved or conserved substitution or a combination thereof, or by substituting the sequence with a sequence having weaker activity, resulting in mutations in the expression regulatory region (or expression regulatory sequence). The expression regulatory region may include, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences regulating transcription and translation termination.

[0109] Furthermore, modifications to amino acid sequences or polynucleotide sequences in 3) and 4) can be achieved by inducing mutations in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide through deletion, insertion, non-conservative or conserved substitution, or a combination thereof, thereby weakening the activity of the polypeptide, or by replacing the sequence with a modified amino acid sequence or polynucleotide sequence that has weaker activity or with a modified amino acid sequence or polynucleotide sequence that does not exhibit activity, but are not limited thereto. For example, gene expression can be suppressed or weakened by introducing mutations into the polynucleotide sequence and generating a stop codon, but are not limited thereto.

[0110] In addition, 5) modifying the start codon or the base sequence of the 5'-UTR of the gene encoding the polypeptide can be, for example, replacing the start codon with a codon whose polypeptide expression rate is lower than that of the endogenous start codon, but is not limited thereto.

[0111] 6) The introduction of an antisense oligonucleotide (e.g., antisense RNA) that is complementary to the transcript of the gene encoding the polypeptide can be performed with reference to [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0112] 7) Add a sequence complementary to the SD sequence upstream of the Shine-Dalgarno (SD) sequence of the gene encoding the polypeptide to form a secondary structure that prevents ribosome attachment, which may inhibit mRNA translation or slow down its translation rate.

[0113] 8) Reverse transcription engineering (RTE) involves adding a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the polynucleotide sequence encoding the polypeptide. This can be done by generating an antisense nucleotide that is complementary to the transcript of the gene encoding the polypeptide to reduce its activity.

[0114] 9) Regulating the cellular localization of the peptide can be achieved by targeting the peptide to specific organelles or specific spaces within the cell. For example, the peptide can be targeted to the periplasm or cytoplasm by adding or removing a leader sequence that plays a role in protein targeting, but is not limited to this.

[0115] As the microorganism according to any one of the foregoing specific embodiments, the microorganism disclosed herein may be a Corynebacterium genus microorganism, wherein a threonine dehydratase derived from Arabidopsis thaliana is introduced, and wherein endogenous threonine dehydratase activity is absent, but is not limited thereto.

[0116] Endogenous threonine dehydratases can originate from Corynebacterium species, specifically from Corynebacterium glutamicum. The amino acid and polynucleotide sequences of threonine dehydratases derived from Corynebacterium glutamicum can be obtained from publicly available databases, including, but not limited to, those in NCBI's GenBank.

[0117] In one example, a threonine dehydratase derived from *Corynebacterium glutamicum* may comprise, but is not limited to, the amino acid sequence of SEQ ID NO: 41 or an amino acid sequence having at least 60% homology or identity with it, as long as it retains the threonine dehydratase activity derived from *Corynebacterium glutamicum*. Specifically, a polypeptide exhibiting threonine dehydratase activity derived from *Corynebacterium glutamicum* may have, comprise, consist of, or substantially consist of the following sequence: the amino acid sequence of SEQ ID NO: 41 or 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: 41. In one example, a threonine dehydratase derived from *Corynebacterium glutamicum* may refer to a protein inherent in *Corynebacterium glutamicum*, but is not limited to it. Specifically, the threonine dehydratase derived from Corynebacterium glutamicum can be a threonine dehydratase derived from Corynebacterium glutamicum consisting of the amino acid sequence of SEQ ID NO:41, which is inherently present in Corynebacterium glutamicum, but is not limited thereto.

[0118] In addition, the base sequence encoding threonine dehydratase from Corynebacterium glutamicum can be the base sequence encoding a protein that displays the activity of threonine dehydratase from Corynebacterium glutamicum.

[0119] For example, a threonine dehydratase derived from Corynebacterium glutamicum having the amino acid sequence of SEQ ID NO: 41 can be encoded by a polynucleotide having, comprising, consisting of, or substantially consisting of the sequence of SEQ ID NO: 42 or 43, or a base 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%, or at least 98% and less than 100% homology or identity with the sequence of SEQ ID NO: 42 or 43, but not limited thereto. The base sequence of SEQ ID NO: 42 or SEQ ID NO: 43 can be obtained from known databases, examples of which include, but are not limited to, NCBI's GenBank.

[0120] Meanwhile, the Corynebacterium genus microorganisms with the ability to produce branched-chain amino acids disclosed herein include any naturally occurring wild-type microorganism, Corynebacterium genus microorganisms with enhanced ability to produce branched-chain amino acids by enhancing or weakening the activity of genes related to the branched-chain amino acid production mechanism, or Corynebacterium genus microorganisms with the ability to produce branched-chain amino acids by introducing or enhancing the activity of exogenous genes.

[0121] Another aspect of this disclosure provides a method for producing branched-chain amino acids, comprising culturing a Corynebacterium microorganism of the present disclosure in a culture medium, wherein the microorganism is inoculated with a threonine dehydratase derived from Arabidopsis thaliana.

[0122] Another aspect of this disclosure provides a method for improving the production of branched-chain amino acids, comprising culturing the Corynebacterium genus of this disclosure in a culture medium, wherein the microorganism is inoculated with a threonine dehydratase derived from Arabidopsis thaliana.

[0123] As used herein, the term "culture" refers to the growth of the strains of this disclosure under appropriately controlled environmental conditions. The culture process of this disclosure can be carried out in suitable culture media and under suitable culture conditions known in the art. Those skilled in the art can readily adapt and use this culture method according to the selected strain. Specifically, the culture can be a batch culture, a continuous culture, or a fed-batch culture, but is not limited thereto.

[0124] As used herein, the term "culture medium" refers to a mixture containing nutrients as the main components required for culturing the microorganisms of this disclosure, and providing nutrients, growth factors, etc., including water essential for survival and development. Specifically, any culture medium and culture conditions can be used to culture the strains of this disclosure without particular limitation, as long as the medium is used for the general culture of microorganisms. The microorganisms of this disclosure can be cultured under aerobic conditions in a general culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while controlling temperature, pH, etc. For example, culture media for Corynebacterium spp. can be found in the literature ["Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981)].

[0125] In this disclosure, carbon sources include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; or amino acids such as glutamic acid, methionine, and lysine. Additionally, natural organic nutrients such as starch hydrolysate, molasses, molasses, rice bran, cassava, bagasse, and corn steep liquor can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and various other carbon sources can be used without limitation in appropriate amounts. These carbon sources can be used alone or in combination of at least two, but are not limited thereto.

[0126] As nitrogen sources, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate can be used, or organic nitrogen sources such as amino acids (e.g., glutamic acid, methionine, and glutamine), peptone, NZ-amines, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysate, fish or its decomposition products, and defatted soybean meal or its decomposition products can be used. These nitrogen sources can be used alone or in combination of at least two, but are not limited thereto.

[0127] Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or their corresponding sodium-containing salts can be used as phosphorus sources. Sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc., can be used as inorganic compounds. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors can be added to the culture medium in batches or continuously. However, the culture medium is not limited to these.

[0128] In the cultivation of the microorganisms disclosed herein, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture medium in a suitable manner to adjust the pH of the medium. During cultivation, antifoaming agents such as polyethylene glycol esters of fatty acids can be used to suppress foaming. To maintain an aerobic state in the culture medium, oxygen or oxygen-containing gas can be injected. To maintain an anaerobic or slightly aerobic state in the culture medium, no gas injection is necessary, or nitrogen, hydrogen, or carbon dioxide gas can be injected. However, the cultivation conditions are not limited to these.

[0129] In the cultivation of this disclosure, the cultivation temperature can be maintained between 27°C and 37°C, specifically between 30°C and 33°C, and the cultivation can be carried out for about 20 to 120 hours, but the cultivation conditions are not limited to these.

[0130] As used herein, the term "culture" refers to a culture medium, a concentrated culture medium, a dried product of a culture medium, a culture filtrate, a concentrated culture filtrate, or a dried product of a culture filtrate obtained by culturing a specific microorganism in a culture medium. A culture medium is a solution containing a specific microorganism, while a culture filtrate is a solution substantially free of the specific microorganism (where "substantially" means that the specific microorganism has been excluded by means of filtration, etc., but does not mean that the microorganism is completely absent from the filtrate). The form of a culture is not limited, and in one instance, it may be in the form of a liquid, an emulsion, or a solid. Specifically, for the purposes of this disclosure, the culture may contain branched-chain amino acids.

[0131] As used herein, the term "fermentation" refers to the process by which microorganisms use their enzymes to break down organic matter, excluding putrefaction. Fermentation and putrefaction proceed using similar processes. However, during decomposition, fermentation produces useful substances, while putrefaction produces foul odors or harmful substances.

[0132] In this disclosure, the method for obtaining fermentation products from the strain is not particularly limited and can be obtained according to methods conventionally used in the relevant or similar technical fields.

[0133] As used herein, the term "fermentation product" includes all types of materials containing fermentation products produced by a strain, including not only the fermentation material itself but also the culture medium in which the strain and the culture coexist; fermentation products obtained by filtering the strain from the culture medium; fermentation products obtained by sterilizing the strain in the culture medium and then filtering it; extracts obtained by extracting the fermentation product or the culture medium containing it; diluted solutions obtained by diluting the fermentation product or its extracts; concentrates; dried products obtained by drying the fermentation product or its extracts; and lysates obtained by collecting and breaking the cells of the strain.

[0134] In the methods disclosed herein, the microorganisms can be cultured using any culture conditions and methods known in the art. Those skilled in the art can readily adapt and use this culture method according to the selected strain.

[0135] Branched-chain amino acids produced by the culture of this disclosure can be secreted into the culture medium or retained in the cells.

[0136] In one specific embodiment, the method for producing branched-chain amino acids disclosed herein may further include preparing the microorganisms of the present disclosure, preparing a culture medium or a combination thereof (in any order) for culturing the microorganisms, for example, prior to culturing.

[0137] The method for producing branched-chain amino acids disclosed herein may further include recovering the desired substance, particularly branched-chain amino acids, from cultured microorganisms, cultures of the microorganisms, fermentation products of the microorganisms, or culture media after culture. A recovery step may be further included after culture.

[0138] Recovery can be achieved by collecting the desired branched-chain amino acids using suitable methods known in the art, depending on the method of culturing the microorganisms disclosed herein, such as batch, continuous, or fed-batch culture. For example, recovery may include centrifugation, filtration, treatment with a crystalline protein precipitant (salting out), extraction, sonication, ultrafiltration, dialysis, various chromatographic methods (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography), HPLC, or combinations thereof. The desired substances, particularly branched-chain amino acids, can be recovered from the culture medium or microorganisms using suitable methods known in the art.

[0139] Furthermore, the method for producing branched-chain amino acids disclosed herein may further include purification. Purification can be performed using suitable methods known in the art. In one instance, when the method for producing branched-chain amino acids disclosed herein includes both recovery and purification, these steps may be performed sequentially or discontinuously, simultaneously, or as an integrated step in any order, but are not limited thereto.

[0140] In the methods disclosed herein, threonine dehydratase, introduction, branched-chain amino acids, etc., are as described in other aspects above.

[0141] Another aspect of this disclosure provides a composition for producing branched-chain amino acids, comprising: a Corynebacterium microorganism of this disclosure, wherein the microorganism is inoculated with a threonine dehydratase derived from Arabidopsis thaliana; a culture of the microorganism; a fermentation product of the microorganism; or a combination of at least two of the above.

[0142] The compositions disclosed herein may further comprise suitable excipients commonly used in compositions for the production of branched-chain amino acids. Examples of such excipients include preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, or isotonic agents, but are not limited thereto.

[0143] In one specific embodiment, the compositions disclosed herein may comprise microbially effective amounts or amounts suitable for being present in the compositions used in production.

[0144] In the compositions disclosed herein, threonine dehydratase, induction, branched-chain amino acids, etc., are as described in other aspects above.

[0145] Another aspect of this disclosure provides the use of Corynebacterium microorganisms of this disclosure for the production of branched-chain amino acids, wherein the microorganisms are infused with a threonine dehydratase derived from Arabidopsis thaliana.

[0146] In the uses disclosed herein, threonine dehydratase, induction, branched-chain amino acids, etc., are as described in other aspects above.

[0147] [Modes for Implementing the Invention] This disclosure will be described in detail by way of examples. However, these examples are given for illustrative purposes only, and the scope of this disclosure is not intended to be limited by these examples. Furthermore, any technical descriptions lacking in this disclosure can be fully understood and readily practiced by those skilled in the art to which this disclosure pertains or in related fields.

[0148] Example 1: Construction of an L-valine-producing strain infused with exogenous threonine dehydratase The exogenous threonine dehydratase gene was inserted into the chromosome of the valine-producing strain Corynebacterium glutamicum KCCM11201P (US8,465,962 B).

[0149] Example 1-1: Construction of an L-valine-producing strain lacking endogenous ilvA (Cgl) To construct a vector (SEQ ID NO: 42) for the deletion of the gene encoding *Corynebacterium glutamicum* autothreonine dehydratase (SEQ ID NO: 41), chromosome KCCM11201P was used as a template for PCR. The PCR conditions were as follows: initial denaturation at 95°C for 10 minutes; 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 40 seconds, and extension at 72°C for 40 seconds; followed by a final extension at 72°C for 5 minutes. As a result, autothreonine dehydratase was obtained using primers SEQ ID NO: 1 and 2. ilvA The 643 bp DNA fragment upstream of the 5' region was used to obtain autologous DNA using primers SEQ ID NO: 3 and 4. ilvA A 777 bp DNA fragment downstream of the 3' region.

[0150] According to the provided manual, two amplified DNA fragments and the pDC24 vector treated with BamHI and XbaI (New England Biolabs, Beverly, MA) were cloned using the In-Fusion Cloning Kit (TaKaRa) to construct *Corynebacterium glutamicum* autologous DNA. ilvA The vector pDC24_ΔilvA is missing.

[0151] [Table 1] The constructed pDC24_ΔilvA was transformed into *Corynebacterium glutamicum* KCCM11201P via homologous recombination on the chromosome (van der Rest et al., *Appl Microbiol Biotechnol* 52:541-545, 1999). Strains that had inserted the vector into the chromosome via homologous sequence recombination were selected on a medium containing 25 mg / L kanamycin. Subsequently, PCR was performed on the *Corynebacterium glutamicum* transformants that had undergone the second recombination using primers of SEQ ID NO: 5 and 6 to confirm autologous recombination. ilvA The missing part. ilvA The strain whose ORF gene was deleted from the chromosome of the parent strain KCCM11201P was named Corynebacterium glutamicum KCCM11201P_ΔilvA.

[0152] Examples 1-2: Construction of strains infused with threonine dehydratase (ilvA(Ath)) derived from Arabidopsis thaliana In order to insert a foreign protein encoding a threonine dehydratase protein ilvA Autologous Corynebacterium glutamicum ilvA The gene was used as the insertion site. For this purpose, chromosome KCCM11201P was used as a template for PCR. The PCR conditions were as follows: initial denaturation at 95°C for 10 minutes; 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 50 seconds; followed by a final extension at 72°C for 5 minutes. As a result, *Corynebacterium glutamicum* autogenous cells were obtained using primers SEQ ID NO: 7 and 8. ilvA A 638 bp DNA fragment upstream of the 5' region (hereinafter referred to as "fragment A") was used with primers of SEQ ID NO: 9 and 10 to obtain *Corynebacterium glutamicum* autogenous DNA. ilvA The 776 bp DNA fragment downstream of the 3' region (hereinafter referred to as "fragment B").

[0153] [Table 2] Meanwhile, in order to obtain a gene fragment with threonine dehydratase activity, based on the ilvA amino acid sequence (SEQ ID NO: 13) from Arabidopsis thaliana published in the NCBI database, codon optimization was performed to suit the codons used by Corynebacterium glutamicum, resulting in the synthesis of a 1779 bp gene fragment from Arabidopsis thaliana. ilvA Gene (SEQ ID NO: 14) (Bionics). Subsequently, synthetically derived from Arabidopsis thaliana was used. ilvA Using the gene as a template, PCR was performed with primers SEQ ID NO: 11 and 12 to obtain a 1681 bp DNA fragment (hereinafter referred to as "fragment C") containing ilvA(Ath, Δ1–45, T46M), which is the gene (SEQ ID NO: 16) encoding the amino acid sequence of ilvA(Ath) with the transport peptide removed (SEQ ID NO: 15). The PCR conditions were as follows: initial denaturation at 95°C for 10 minutes; 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute and 30 seconds; followed by a final extension at 72°C for 5 minutes.

[0154] According to the provided manual, the previously obtained fragments A, B, and C, as well as the pDC24 vector treated with BamHI and XbaI (New England Biolabs, Beverly, MA), were cloned using the In-Fusion Cloning Kit (TaKaRa) to construct an autologous strain capable of inserting the ilvA (Ath, Δ1-45, T46M) gene into Corynebacterium glutamicum. ilvA The carrier of position, namely pDC24_ΔilvA::ilvA(Ath).

[0155] The constructed pDC24_ΔilvA::ilvA(Ath) was transformed into *Corynebacterium glutamicum* KCCM11201P via homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains that inserted the vector into the chromosome via homologous sequence recombination were selected on a medium containing 25 mg / L kanamycin. Subsequently, PCR was performed on the *Corynebacterium glutamicum* transformants that had undergone the second recombination using primers of SEQ ID NO: 5 and 6, followed by sequence analysis to confirm autologous recombination. ilvA Whether ORF is missing and whether it originates from Arabidopsis thaliana. ilvA Whether it has been imported into the corresponding location. The autologous strain of parental strain KCCM11201P... ilvA ORF is a chromosome derived from Arabidopsis thaliana. ilvAThe strain with the (Ath, Δ1–45, T46M) gene substitution was named Corynebacterium glutamicum KCCM11201P_ΔilvA::ilvA(Ath).

[0156] Examples 1-3: Construction of strains infused with threonine dehydratase (ilvA(Eco)) derived from Escherichia coli To verify the L-valine production capacity of strains infused with exogenous threonine dehydratases other than those from Arabidopsis thaliana, an L-valine-producing strain infused with the ilvA(Eco) gene from Escherichia coli was constructed. As in Examples 1-2 above, to utilize the autologous... ilvA Using the gene as the insertion site, autologous genes were obtained using primers of SEQ ID NO: 7 and 8. ilvA The 638 bp DNA fragment A upstream of the 5' region was obtained autologously using primers of SEQ ID NO: 9 and 10. ilvA The 776 bp DNA fragment B downstream of the 3' region.

[0157] [Table 3] Meanwhile, to obtain the gene fragment (SEQ ID NO: 20) encoding threonine dehydratase (SEQ ID NO: 19) from *E. coli*, PCR was performed using chromosomal DNA from *E. coli* MG1655 as a template, with primers from SEQ ID NO: 17 and 18, yielding a 1578 bp DNA fragment containing ilvA(Eco) (hereinafter referred to as "fragment D"). The PCR conditions were as follows: initial denaturation at 95°C for 10 minutes; 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute and 30 seconds; followed by a final extension at 72°C for 5 minutes.

[0158] According to the provided manual, the previously obtained fragments A, B, and D, as well as the pDC24 vector treated with BamHI and XbaI (New England Biolabs, Beverly, MA), were cloned using the In-Fusion Cloning Kit (TaKaRa) to construct an autologous strain capable of inserting the ilvA(Eco) gene into Corynebacterium glutamicum. ilvA The carrier of the location, namely pDC24_ΔilvA::ilvA(Eco).

[0159] The constructed pDC24_ΔilvA::ilvA(Eco) was transformed into *Corynebacterium glutamicum* KCCM11201P via homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains that had inserted the vector into the chromosome via homologous sequence recombination were selected on a medium containing 25 mg / L kanamycin. Subsequently, PCR was performed on the *Corynebacterium glutamicum* transformants that had undergone the second recombination using primers of SEQ ID NO: 5 and 6, followed by sequence analysis to confirm autologous transformation. ilvA Whether ORF is missing and whether it originates from E. coli. ilvA Whether it has been imported into the corresponding location. The autologous strain of parental strain KCCM11201P... ilvA The strain whose ORF was replaced by ilvA(Eco) derived from Escherichia coli on the chromosome was named Corynebacterium glutamicum KCCM11201P_ΔilvA::ilvA(Eco).

[0160] Examples 1-4: Constructing imports from Tomato ( Solanum lycopersicum strains of threonine dehydratase (ilvA(Sly)) To verify the L-valine production capacity of strains infused with exogenous threonine dehydrating enzymes other than those from Arabidopsis thaliana, an L-valine-producing strain infused with the ilvA(Sly) gene from tomato was constructed. As in Examples 1-2 above, to utilize the autologous... ilvA Using the gene as the insertion site, autologous genes were obtained using primers of SEQ ID NO: 7 and 8. ilvA The 638 bp DNA fragment A upstream of the 5' region was obtained autologously using primers of SEQ ID NO: 9 and 10. ilvA The 776 bp DNA fragment B downstream of the 3' region.

[0161] [Table 4] Meanwhile, in order to obtain a gene fragment with threonine dehydratase activity, based on the ilvA amino acid sequence (SEQ ID NO: 23) from tomato published in the NCBI database, codon optimization was performed to suit the codons used by Corynebacterium glutamicum, resulting in the synthesis of a 1821 bp gene fragment from tomato. ilvA Gene (SEQ ID NO: 24) (Bionics). Subsequently, synthetic tomato-derived genes were used. ilvAUsing the gene as a template, PCR was performed with primers SEQ ID NO: 21 and 22 to obtain a 1696 bp DNA fragment (hereinafter referred to as "fragment E") containing ilvA(Sly, Δ1-53, I54M). This fragment encodes the gene (SEQ ID NO: 26) encoding the amino acid sequence of ilvA(Sly) with the transport peptide removed (SEQ ID NO: 25). The PCR conditions were as follows: initial denaturation at 95°C for 10 minutes; 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute and 30 seconds; followed by a final extension at 72°C for 5 minutes.

[0162] According to the provided manual, the previously obtained fragments A, B, and E, as well as the pDC24 vector treated with BamHI and XbaI (New England Biolabs, Beverly, MA), were cloned using the In-Fusion Cloning Kit (TaKaRa) to construct an autologous strain capable of inserting the ilvA (Sly, Δ1-53, I54M) gene into Corynebacterium glutamicum. ilvA The carrier of position, namely pDC24_ΔilvA::ilvA(Sly).

[0163] The constructed pDC24_ΔilvA::ilvA(Sly) was transformed into *Corynebacterium glutamicum* KCCM11201P via homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains that inserted the vector into the chromosome via homologous sequence recombination were selected on a medium containing 25 mg / L kanamycin. Subsequently, PCR was performed on the *Corynebacterium glutamicum* transformants that had undergone the second recombination using primers of SEQ ID NO: 5 and 6, followed by sequence analysis to confirm autologous recombination. ilvA Is ORF missing and does it originate from tomatoes? ilvA Whether it has been imported into the corresponding location. The autologous strain of parental strain KCCM11201P... ilvA The strain whose ORF was replaced by the tomato-derived ilvA (Sly, Δ1-53, I54M) on the chromosome was named Corynebacterium glutamicum KCCM11201P_ΔilvA::ilvA(Sly).

[0164] Examples 1-5: Constructing imports from spinach ( Spinach strains of threonine dehydratase (ilvA(Sol)) To verify the L-valine production capacity of strains infused with exogenous threonine dehydrating enzymes other than those from Arabidopsis thaliana, an L-valine-producing strain infused with the ilvA(Sol) gene from spinach was constructed. As in Examples 1-2 above, to utilize the autologous... ilvA Using the gene as the insertion site, autologous genes were obtained using primers of SEQ ID NO: 7 and 8. ilvA The 638 bp DNA fragment A upstream of the 5' region was obtained autologously using primers of SEQ ID NO: 9 and 10. ilvA The 776 bp DNA fragment B downstream of the 3' region.

[0165] [Table 5] Meanwhile, in order to obtain a gene fragment with threonine dehydratase activity, based on the ilvA amino acid sequence (SEQ ID NO: 29) from spinach published in the NCBI database, codon optimization was performed to suit the codons used by Corynebacterium glutamicum, resulting in the synthesis of a 1842 bp gene fragment from spinach. ilvA Gene (SEQ ID NO: 30) (Bionics). Subsequently, synthetic spinach-derived genes were used. ilvA Using the gene as a template, PCR was performed with primers SEQ ID NO: 27 and 28 to obtain a 1714 bp DNA fragment containing ilvA(Sol, Δ1-54, V55M) (hereinafter referred to as "fragment F"). This fragment encodes the gene (SEQ ID NO: 32) encoding the amino acid sequence of ilvA(Sol) with the transport peptide removed (SEQ ID NO: 31). The PCR conditions were as follows: initial denaturation at 95°C for 10 minutes; 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute and 30 seconds; followed by a final extension at 72°C for 5 minutes.

[0166] According to the provided manual, the previously obtained fragments A, B, and F, as well as the pDC24 vector treated with BamHI and XbaI (New England Biolabs, Beverly, MA), were cloned using the In-Fusion Cloning Kit (TaKaRa) to construct an autologous strain capable of inserting the ilvA(Sol, Δ1-54, V55M) gene into Corynebacterium glutamicum. ilvA The carrier of position, namely pDC24_ΔilvA::ilvA(Sol).

[0167] The constructed pDC24_ΔilvA::ilvA(Sol) was transformed into *Corynebacterium glutamicum* KCCM11201P via homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains that inserted the vector into the chromosome via homologous sequence recombination were selected on a medium containing 25 mg / L kanamycin. Subsequently, PCR was performed on the *Corynebacterium glutamicum* transformants that had undergone the second recombination using primers of SEQ ID NO: 5 and 6, followed by sequence analysis to confirm autologous recombination. ilvA Is ORF missing and does it originate from spinach? ilvA Whether it has been imported into the corresponding location. The autologous strain of parental strain KCCM11201P... ilvA The strain whose ORF was replaced by ilvA(Sol, Δ1-54, V55M) derived from spinach on the chromosome was named Corynebacterium glutamicum KCCM11201P_ΔilvA::ilvA(Sol).

[0168] Examples 1-6: Constructing and importing materials derived from *Bruguiera divaricata* (… Brachypodium distachyon strains of threonine dehydratase (ilvA(Bdi)) To verify the L-valine production capacity of strains infused with exogenous threonine dehydrating enzymes other than those from Arabidopsis thaliana, an L-valine-producing strain infused with the ilvA(Bdi) gene from Brachypodium distichum was constructed. As in Examples 1-2 above, to utilize the autologous... ilvA Using the gene as the insertion site, autologous genes were obtained using primers of SEQ ID NO: 7 and 8. ilvA The 638 bp DNA fragment A upstream of the 5' region was obtained autologously using primers of SEQ ID NO: 9 and 10. ilvA The 776 bp DNA fragment B downstream of the 3' region.

[0169] [Table 6] Simultaneously, to obtain a gene fragment with threonine dehydratase activity, a 1392 bp ilvA(Bdi) gene (SEQ ID NO: 36) (Bionics) was synthesized based on the ilvA amino acid sequence (SEQ ID NO: 35) from *Brachys glutamicum* publicly available in the NCBI database. Codon optimization was performed to suit the codons used by *Corynebacterium glutamicum*. Using the synthesized ilvA(Bdi) gene as a template, PCR was performed using primers of SEQ ID NO: 33 and 34 to obtain a 1426 bp DNA fragment containing ilvA(Bdi) (hereinafter referred to as "fragment G"). The PCR conditions were as follows: initial denaturation at 95°C for 10 minutes; 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute and 20 seconds; followed by a final extension at 72°C for 5 minutes.

[0170] According to the provided manual, the previously obtained fragments A, B, and G, as well as the pDC24 vector treated with BamHI and XbaI (New England Biolabs, Beverly, MA), were cloned using the In-Fusion Cloning Kit (TaKaRa) to construct an autologous vector capable of inserting the ilvA(Bdi) gene into Corynebacterium glutamicum. ilvA The carrier of position, namely pDC24_ΔilvA::ilvA(Bdi).

[0171] The constructed pDC24_ΔilvA::ilvA(Bdi) was transformed into *Corynebacterium glutamicum* KCCM11201P via homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains that inserted the vector into the chromosome via homologous sequence recombination were selected on a medium containing 25 mg / L kanamycin. Subsequently, PCR was performed on the *Corynebacterium glutamicum* transformants that had undergone the second recombination using primers of SEQ ID NO: 5 and 6, followed by sequence analysis to confirm autologous transformation. ilvA Whether ORF is missing and whether it originates from *Bruguiera gymnorhiza*. ilvA Whether it has been imported into the corresponding location. The autologous strain of parental strain KCCM11201P... ilvA The strain whose ORF was replaced by ilvA(Bdi) derived from *B. bipinnatifida* on the chromosome was named *Corynebacterium glutamicum* KCCM11201P_ΔilvA::ilvA(Bdi).

[0172] Example 2: Evaluation of L-valine production capacity of strains introduced with exogenous threonine dehydratase Flask evaluations were performed to compare the valine production capacity of *Corynebacterium glutamicum* KCCM11201P with that of the six strains constructed in Examples 1-1 to 1-6 (i.e., KCCM11201P_ΔilvA, KCCM11201P_ΔilvA::ilvA(Ath), KCCM11201P_ΔilvA::ilvA(Eco), KCCM11201P_ΔilvA::ilvA(Sly), KCCM11201P_ΔilvA::ilvA(Sol), and KCCM11201P_ΔilvA::ilvA(Bdi)) (all of which are valine-producing strains). Each strain was continuously subcultured in nutrient medium, inoculated into 250 mL corner-baffle flasks containing 25 mL of production medium, and cultured with shaking at 30 °C and 200 rpm for 72 hours. The valine production capacity of each strain was measured and is shown in Table 7 below.

[0173] [Nutritional medium (pH 7.2)] 10 g glucose, 5 g meat broth, 10 g peptone, 2.5 g sodium chloride, 5 g yeast extract, 20 g agar, and 2 g urea (based on 1 L of distilled water) [Production medium (pH 7.0)] 100 g glucose, 40 g ammonium sulfate, 2.5 g soybean protein, 5 g corn steep liquor solids, 3 g urea, 1 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 100 μg biotin, 1 mg thiamine hydrochloride, 2 mg calcium pantothenate, 3 mg nicotinamide, and 30 g calcium carbonate (based on 1 L of distilled water). [Table 7] As shown in Table 7 above, KCCM11201P_ΔilvA::ilvA(Ath) exhibited the highest valine production capacity among the evaluated strains, with a value of 0.040 g / L / h. Specifically, compared with... ilvA Compared to the KCCM11201P strain without deletion, the KCCM11201P_Δ strain with ilvA deletion... ilvA The strain exhibited very low valine production capacity, while KCCM11201P_ΔilvA::ilvA(Ath) showed valine production capacity equivalent to 114% of that of KCCM11201P. Furthermore, it was confirmed that relative to... ilvA The missing KCCM11201P_ΔilvA strain originated from Arabidopsis thaliana. ilvA The introduction of this technology will increase valine production capacity to 333%.

[0174] Example 3: Construction of Corynebacterium glutamicum CJ7V strain infused with ilvA (Ath) and evaluation of L-valine production capacity. To verify whether the introduction of ilvA (Ath) could also increase the L-valine production capacity of other Corynebacterium glutamicum strains that produce L-valine, a single mutant [ilvN (A42V); Biotechnology and Bioprocess Engineering, June 2014, Vol. 19, No. 3, pp. 456–467] was introduced into wild-type Corynebacterium glutamicum ATCC14067 to construct strain CJ7V (US 2020-0362374 A1) with enhanced L-valine production capacity.

[0175] Specifically, genomic DNA from wild-type Corynebacterium glutamicum strain ATCC14067 was extracted using the G-spin Total DNA Extraction Mini Kit (Intron, catalog number 17045) according to the kit's instructions. Genomic DNA was used as a template for PCR. This was to construct a model for introducing the A42V mutation. ilvN Gene vectors were obtained using primer pairs of SEQ ID NO: 37 and 38, and primer pairs of SEQ ID NO: 39 and 40, to obtain gene fragments (H and I). PCR conditions were as follows: initial denaturation at 94°C for 5 minutes; 25 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 60 seconds; followed by a final extension at 72°C for 7 minutes.

[0176] As a result, 528 bp and 509 bp polynucleotide fragments H and I were obtained, respectively. Using these two fragments as templates, overlap PCR was performed with primers of SEQ ID NO: 37 and 40 to obtain 1010 bp PCR products.

[0177] The 10¹⁰ bp PCR product obtained was treated with the restriction enzyme SmaI (New England Biolabs, Beverly, MA), and then ligated to the pDC24 vector treated with the same restriction enzyme using T4 ligase (New England Biolabs, Beverly, MA). The constructed vector was transformed into *E. coli* DH5α, selected on LB medium containing kanamycin, and the DNA was obtained using a DNA-spin plasmid purification kit (iNtRON). This will be used for... ilvN The vector that introduced the A42V mutation was named pDC24-ilvN(A42V).

[0178] [Table 8] Subsequently, pDC24-ilvN(A42V) was transformed into wild-type *Corynebacterium glutamicum* strain ATCC14067 via homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains that inserted the vector into the chromosome via homologous sequence recombination were selected on a medium containing 25 mg / L kanamycin. Then, PCR was performed on the *Corynebacterium glutamicum* transformants that had undergone the second recombination using SEQ ID NOs: 37 and 40 to amplify the gene fragment, followed by sequence analysis to confirm the introduction of the mutant strain. The recombinant strain was named *Corynebacterium glutamicum* CJ7V.

[0179] In the same manner as in Example 1-1, by using for missing autologous tissue ilvA The vector (pDC24_ΔilvA) was transformed into Corynebacterium glutamicum CJ7V to construct a strain, which was named Corynebacterium glutamicum CJ7V_ΔilvA.

[0180] Meanwhile, in the same manner as in Examples 1-2, the method for introducing Arabidopsis thaliana was used. ilvA The vector (pDC24_ΔilvA::ilvA(Ath)) was transformed into Corynebacterium glutamicum CJ7V to construct a strain, which was named Corynebacterium glutamicum CJ7V_ΔilvA::ilvA(Ath). To compare the L-valine production capacity of the constructed strains, the strains were cultured in the same manner as in Example 2, and the valine production capacity of each strain was measured and shown in Table 9 below.

[0181] [Table 9] As shown in Table 9 above, when ilvA When absent, L-valine production capacity is only 0.006 g / L / h, while the production of L-valine from Arabidopsis thaliana has been confirmed. ilvA The introduction of [a specific ingredient] increased L-valine production capacity. Furthermore, it was confirmed that [the effect was] relative to [other factors]. ilvA The deletion strain CJ7V_ΔilvA is derived from Arabidopsis thaliana. ilvA The introduction of this technology will increase L-valine production capacity to 400%.

[0182] Example 4: Construction of a leucine-producing strain with introduced exogenous ilvA (Ath) and evaluation of leucine production capacity. Example 4-1: Construction of an L-leucine-producing strain with autologous ilvA(Cgl) deletion In order to construct a gene for the deletion of autothreonine dehydratase encoding Corynebacterium glutamicum ( ilvA The vector (SEQ ID NO: 43, NCBI accession number "NCgl2046") was used, and PCR was performed using chromosome ATCC13032 as a template. The PCR conditions were as follows: initial denaturation at 95°C for 10 minutes; 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 40 seconds, and extension at 72°C for 40 seconds; followed by a final extension at 72°C for 5 minutes. As a result, autologous chromosomes were obtained using primers of SEQ ID NO: 44 and 45. ilvA A 1043 bp DNA fragment upstream of the 5' region was obtained using primers of SEQ ID NO: 46 and 47. ilvA A 1043 bp DNA fragment downstream of the 3' region was extracted. Two amplified DNA fragments were purified using a PCR Purification Kit (QIAGEN), and the purified amplification products were treated with the restriction enzyme SmaI. The treated products were then cloned into the pDC24 vector (SEQ ID NO: 51) after heat treatment at 65°C for 20 minutes using an In-Fusion Cloning Kit (TaKaRa) according to the provided manual, thereby constructing the pDC24_ΔilvA13032 vector for the deletion of autologous Corynebacterium glutamicum ilvA.

[0183] [Table 10] The constructed pDC24_ΔilvA13032 was transformed into the leucine-producing strain Corynebacterium glutamicum CJL8109 (WO 2022 / 163981 A1) via electroporation to construct its autologous... ilvA Missing strain. This is how a strain with missing strains is constructed. ilvA The strain CJL8109 was named CJL8126.

[0184] Example 4-2: Construction of an L-leucine-producing strain infused with threonine dehydratase (ilvA(Ath)) derived from Arabidopsis thaliana In order to insert a foreign protein encoding a threonine dehydratase protein ilvA Autologous Corynebacterium glutamicum ilvAThe gene was used as the insertion site. PCR was performed using the pDC24_ΔilvA::ilvA(Ath) vector prepared in Examples 1-2 as a template. The PCR conditions were as follows: initial denaturation at 95°C for 10 minutes; 28 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 50 seconds; followed by a final extension at 72°C for 5 minutes. As a result, primers of SEQ ID NO: 48 and 49 were used to obtain a gene containing *Arabidopsis thaliana*. ilvA A 1681 bp DNA fragment of the gene (SEQ ID NO: 16) was used. The amplification product was purified using a PCR purification kit (QIAGEN) and used as the insert DNA fragment for vector construction. The purified amplification product was treated with the restriction enzyme SmaI and cloned using the In-Fusion HD cloning kit (Clontech) according to the provided manual, wherein the molar ratio of the pDC24_ΔilvA13032 vector (heat-treated at 65°C for 20 min) to the insert DNA fragment was adjusted to 1:2 to construct the vector pDC24_ΔilvA13032::ilvA(Ath) for introducing ilvA(Ath) into the chromosome. Table 11 describes the sequences and details of the primers used in this example.

[0185] [Table 11] The constructed pDC24_ΔilvA13032::ilvA(Ath) was transformed into the leucine-producing strain *Corynebacterium glutamicum* CJL8109 (WO 2022 / 163981 A1) via homologous recombination on the chromosome (van der Rest et al., Appl Microbiol Biotechnol 52:541-545, 1999). Strains that inserted the vector into the chromosome via homologous sequence recombination were selected on a medium containing 25 mg / L kanamycin. Subsequently, PCR was performed on the *Corynebacterium glutamicum* transformants that had undergone the second recombination using primers of SEQ ID NO: 50 and 6, followed by sequence analysis to confirm autologous recombination. ilvA Whether ORF is missing and whether it originates from Arabidopsis thaliana. ilvA Whether it has been imported into the corresponding location. The autologous parent strain CJL8109 is included. ilvA The strain whose ORF was replaced by ilvA (Ath, Δ1–45, T46M) derived from Arabidopsis thaliana on its chromosome was named Corynebacterium glutamicum CJL8127.

[0186] To evaluate the leucine production capacity of the above-mentioned strains, a shake-flask fermentation titer assessment was conducted.

[0187] - Production culture medium: 100 g glucose, 40 g (NH4)2SO4, 2.5 g soybean protein, 5 g corn steep liquor solids, 3 g urea, 1 g KH2PO4, 0.5 g MgSO4∙7H2O, 100 μg biotin, 1000 μg thiamine hydrochloride, 2000 μg calcium pantothenate, 3000 μg nicotinamide, and 30 g calcium carbonate (based on 1 liter of distilled water), pH 7.0 Each parental strain of *Corynebacterium glutamicum* ATCC13032 and CJL8109, as well as the constructed strains CJL8126 and CJL8127, was inoculated into a 250 mL baffled Erlenmeyer flask containing 25 mL of production medium using a platinum ring. The flasks were then incubated at 30 °C and 200 rpm for 60 hours with shaking to produce leucine. After cultivation, the amount of leucine produced was measured by HPLC. The leucine concentration in the culture medium of each test strain is shown in Table 12 below.

[0188] [Table 12] As shown in Table 12 above, this confirms... ilvA The leucine production capacity of strain CJL8126 was reduced. In contrast, strain CJL8127 (which was introduced with leucine from Arabidopsis thaliana) showed reduced leucine production. ilvA In the case of a leucine-producing strain, the leucine production capacity was increased by 217% compared to the parent strain CJL8109. Furthermore, it was confirmed that compared to... ilvA The deletion strain CJL8126 was introduced with a strain derived from Arabidopsis thaliana. ilvA The leucine production capacity of strain CJL8127 was increased to 241%.

[0189] As described above, those skilled in the art will understand that this disclosure can be implemented in other specific forms without departing from the technical spirit or essential characteristics of this disclosure. In this regard, it should be understood that the foregoing embodiments are illustrative in all respects and should not be construed as restrictive. The scope of this disclosure should be interpreted to include the meaning and scope of the appended claims, but not the specific embodiments, and all changes or variations derived from equivalent concepts fall within the scope of this disclosure.

Claims

1. A Corynebacterium genus microorganism, wherein the microorganism is infused with a threonine dehydratase derived from Arabidopsis thaliana.

2. The microorganism according to claim 1, wherein the Arabidopsis-derived threonine dehydratase comprises an amino acid sequence having at least 75% identity with SEQ ID NO:

15.

3. The microorganism according to claim 1, wherein the threonine dehydratase derived from Arabidopsis thaliana is composed of... ilvA Gene encoding.

4. The microorganism according to claim 3, wherein it is derived from Arabidopsis thaliana. ilvA The gene contains a base sequence that is at least 75% identical to SEQ ID NO:

16.

5. The microorganism according to claim 1, wherein the microorganism is a microorganism in which endogenous threonine dehydratase activity is further lacking.

6. The microorganism according to claim 1, wherein the microorganism is Corynebacterium glutamicum.

7. The microorganism according to claim 1, wherein the microorganism has the ability to produce branched-chain amino acids.

8. The microorganism according to claim 7, wherein the branched-chain amino acid is L-valine or L-leucine.

9. The microorganism of claim 1, wherein the microorganism has an increased ability to produce branched-chain amino acids compared to unmodified microorganisms.

10. A method for producing branched-chain amino acids, comprising culturing Corynebacterium microorganisms in a culture medium, wherein the microorganisms are inoculated with a threonine dehydratase derived from Arabidopsis thaliana.

11. The method of claim 10, further comprising recovering branched-chain amino acids from cultured microorganisms, cultures of said microorganisms, fermentation products of said microorganisms, or culture media after culture.

12. A method for increasing the production of branched-chain amino acids, comprising culturing Corynebacterium microorganisms in a culture medium, wherein the microorganisms are inoculated with a threonine dehydratase derived from Arabidopsis thaliana.

13. A composition for producing branched-chain amino acids, comprising: a Corynebacterium microorganism, wherein the microorganism is inoculated with a threonine dehydratase derived from Arabidopsis thaliana; a culture of the microorganism; a fermentation product of the microorganism; or a combination of at least two of the above.

14. The use of Corynebacterium microorganisms for the production of branched-chain amino acids, wherein the microorganisms are infused with threonine dehydratase derived from Arabidopsis thaliana.

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