Microorganism into which exogenous NAD-dependent isocitrate dehydrogenase is introduced, and method for producing l-amino acid using same

Abstract

The present application relates to: a Corynebacterium genus microorganism having an L-amino acid producing ability, into which an NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans or a polynucleotide encoding the same is introduced; a method for producing an L-amino acid, the method comprising a step of culturing the microorganism in a culture medium; a composition for producing an L-amino acid, the composition comprising the microorganism, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof; and to the use of said microorganism in the production of L-amino acids.

Description

Technical Field

[0001] This disclosure relates to a Corynebacterium microorganism capable of producing L-amino acids, wherein an NAD-dependent isocitrate dehydrogenase derived from Streptococcus mutans or a polynucleotide encoding therefrom is introduced; a method for producing L-amino acids, the method comprising the step of culturing the microorganism in a culture medium; a composition for producing L-amino acids, the composition comprising the microorganism, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof; and the use of the microorganism in the production of L-amino acids. Background Technology

[0002] The production of target substances (such as amino acids) in microorganisms has been extensively studied as an environmentally friendly and safe method, with ongoing research focusing on the production of large quantities of target substances in the genus *Corynebacterium*. *Corynebacterium* sp., particularly *Corynebacterium glutamicum*, are Gram-positive microorganisms frequently used for the production of L-amino acids and other useful substances.

[0003] L-amino acids are the basic structural units of proteins and are important materials used in pharmaceuticals, food additives, animal feed, nutritional supplements, pesticides, disinfectants, and more. Various studies have been conducted to develop efficient microorganisms and technologies for fermentation processes in order to produce L-amino acids and other useful substances. For example, target-specific methods are primarily used, where the expression of genes encoding enzymes involved in L-tryptophan biosynthesis is increased, or genes unnecessary for biosynthesis are removed (US 8945907 B2).

[0004] L-Tryptophan is an essential amino acid and has been widely used as a raw material in feed additives, pharmaceutical products (such as infusion solutions), and health food ingredients. It can be produced through chemical synthesis, enzymatic reactions, and fermentation. However, currently, direct fermentation using microorganisms is the primary method. Previous research indicates that among the 20 amino acids, tryptophan biosynthesis requires the highest level of energy, a fact confirmed by actual intracellular quantitative analysis (Proc. Natl. Acad. Sci. USA (2002) V99, pp3695-3700). Therefore, further research is needed to effectively increase L-Tryptophan production.

[0005] L-histidine is one of the 20 standard amino acids and is classified as an essential amino acid for children's growth. L-histidine participates in important physiological processes such as antioxidation and immune regulation, and is therefore used in the medical industry, for example, in drugs for treating gastric ulcers, as a raw material for cardiovascular drugs, and in amino acid infusion solutions. Histidine is mainly found in hemoglobin and is primarily produced through protein hydrolysis extraction using blood meal as a raw material. However, this method has drawbacks such as low efficiency and environmental pollution. On the other hand, it is possible to produce L-histidine through microbial fermentation, but large-scale industrialization has not yet been achieved. Therefore, research is still needed to effectively increase the production capacity of L-histidine. Summary of the Invention

[0006] Technical issues

[0007] The objective of this disclosure is to provide a microorganism incorporating an exogenous NAD-dependent isocitrate dehydrogenase, and a method for producing L-amino acids using the microorganism.

[0008] Technical solution

[0009] One aspect of this disclosure provides a Corynebacterium microorganism capable of producing L-amino acids, wherein an NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans or a polynucleotide encoding therethe is introduced.

[0010] In one specific embodiment, the L-amino acid may be any one or more selected from the group consisting of L-tryptophan and L-histidine.

[0011] In another specific embodiment, the NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans may contain the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80% or more sequence identity with it.

[0012] In another specific embodiment, the NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans may be encoded by an icd gene.

[0013] In another specific embodiment, the polynucleotide encoding the NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans may include the nucleotide sequence of SEQ ID NO: 2.

[0014] In another specific embodiment, the Corynebacterium genus microorganism may be Corynebacterium glutamicum.

[0015] With respect to any one of the aforementioned specific embodiments, the Corynebacterium genus microorganism may have an increased ability to produce L-amino acids compared to unmodified microorganisms.

[0016] Another aspect of this disclosure provides a method for producing L-amino acids, the method comprising the step of culturing a Corynebacterium microorganism capable of producing L-amino acids in a culture medium, the microorganism being introduced with an NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans or a polynucleotide encoding therethe.

[0017] In one specific embodiment, the L-amino acid may be any one or more selected from the group consisting of L-tryptophan and L-histidine.

[0018] In another specific embodiment, the method may further include the step of recovering L-amino acids from cultured microorganisms, cultures of said microorganisms, fermentation products of said microorganisms, or culture media.

[0019] Another aspect of this disclosure provides a composition for producing L-amino acids, the composition comprising a Corynebacterium spp. microorganism capable of producing L-tryptophan, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof, wherein the microorganism is introduced with an NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans or a polynucleotide encoding therefrom.

[0020] In one specific embodiment, the L-amino acid may be any one or more selected from the group consisting of L-tryptophan and L-histidine.

[0021] Beneficial effects

[0022] The Corynebacterium genus microorganisms disclosed herein capable of producing L-amino acids can produce L-amino acids in high yields, thereby being effectively applied to the industrial production of L-amino acids. The microorganisms are infused with NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans or the polynucleotide encoding it. Detailed Implementation

[0023] This disclosure will be described in detail below. Furthermore, each description and embodiment disclosed herein can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed herein fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited to the specific embodiments described below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to further clarify the level and scope of the subject matter to which this disclosure pertains.

[0024] definition

[0025] As used in this disclosure and the appended claims, the singular forms (“a”, “an”, and “the”) may include plural indicators unless otherwise stated. Unless otherwise stated, singular terms shall include plural terms, and plural terms shall include singular terms. As used in this disclosure and the appended claims, unless otherwise stated, the use of “or” may include “and / or”.

[0026] As used herein, the term "approximately" may appear before a specific numerical value. The term "approximately" as used herein includes not only the exact number listed after the term, but also the range that is close to or approximates that number. Whether any number is close to or approximates the specific number presented can be determined by considering the context in which the number is presented. For example, the term "approximately" may refer to the range of -10% to +10% of a value. In another instance, the term "approximately" may refer to the range of -5% to +5% of a given numerical value, but is not limited to this.

[0027] As used herein, terms such as “first, second, third…”, “i), ii), iii)…” or “(a), (b), (c), (d)…” are used to distinguish individual components, and when these terms are used to refer to steps of a method, use, or experiment, they are not limited to being performed sequentially or sequentially; for example, there may be no time interval between these steps, or they may be performed simultaneously, or they may be performed sequentially, in reverse order, or at random intervals of seconds, minutes, hours, days, or months.

[0028] As used herein, the term "composed of" means that the total percentage of the specific features, steps, ingredients, or other components listed after the term is 100%. The features, steps, ingredients, or other components listed after the term "composed of" may be necessary or mandatory. For example, any other features, steps, ingredients, or other components, or non-essential features, steps, ingredients, or other components, may be excluded in addition to those listed after the term "composed of".

[0029] As used herein, the term “substantially composed of” can mean that the one or more unspecified features, steps, ingredients or other components of the object claimed herein may be present when they are substantially unaffected by the presence of one or more unspecified features, steps, ingredients or other components.

[0030] As used herein, the term "comprising" means the presence of the features, steps, ingredients, or other components listed after the term, and does not exclude the presence of one or more additional features, steps, ingredients, or other components. The features, steps, ingredients, or other components listed after the term "comprising" herein may be essential or mandatory. However, in some embodiments, the term may also include any other or non-essential features, steps, ingredients, or other components.

[0031] Proteins, polypeptides

[0032] As used herein, the terms "protein" or "peptide" refer to a polymer or oligomer of consecutive amino acid residues. In this disclosure, the terms "peptide," "protein," and "peptide" are used interchangeably.

[0033] As used herein, the terms "mature polypeptide" or "mature protein" refer to a polypeptide or protein in the form that lacks a signal sequence or pro-peptide sequence. A mature polypeptide or mature protein can be the functional form of a polypeptide or protein. A mature polypeptide or mature protein can refer to the final form of a polypeptide after translation; and / or after post-translational modifications. Examples of post-translational modifications include, but are not limited to, N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, leader sequence removal, etc.

[0034] In this disclosure, amino acid sequences are described in the direction from N-terminus to C-terminus, unless otherwise stated.

[0035] Regarding the amino acid sequences in this disclosure, it is clear that a polypeptide or protein "comprising" the amino acid sequence described by a specific sequence number, a polypeptide or protein "composed of" the amino acid sequence described by a specific sequence number, or a polypeptide or protein "having" the amino acid sequence described by a specific sequence number may also include any polypeptide or protein in which some of these amino acids are deleted, modified, substituted, or added, provided that it has the same or corresponding activity as the polypeptide or protein composed of the amino acid sequence of the corresponding sequence number. For example, a polypeptide or protein may also include polypeptides or proteins in which amino acids that do not change the protein function are added or deleted inside or before / after (N-terminus or C-terminus), naturally occurring mutations, their silent mutations, or conserved substitutions, provided that they have the same or corresponding activity.

[0036] In addition, for example, peptides or proteins conjugated with an N-terminal signal (or leader) sequence that participates in protein (peptide) translocation in a co-translational or post-translational manner, or peptides or proteins conjugated with another sequence or adapter to enable the identification, purification or synthesis of peptides or proteins, may also be included in the scope of peptides or proteins having an amino acid sequence described by a specific sequence number.

[0037] As used herein, the term "conservative substitution" refers to the replacement of an 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. For another example, amino acids can be divided into amino acids with charged side chains (charged amino acids), such as arginine, lysine, histidine, glutamic acid, and aspartic acid, and amino acids with uncharged side chains (uncharged amino acids; also called neutral amino acids), such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. For another example, phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. For another example, valine, leucine, and isoleucine can be classified as branched-chain amino acids. For yet another example, classifying 20 amino acids by size, starting with the smaller groups, amino acids can be divided into five groups: glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, and tyrosine. However, they are not necessarily limited to this. Often, conserved substitutions may have little or no effect on the activity of peptides or proteins.

[0038] Genes, polynucleotides

[0039] As used herein, the term "gene" narrowly refers to a polynucleotide that encodes a functional molecule, and broadly refers to a polynucleotide that contains the polynucleotide encoding the functional molecule and its upstream and downstream regions. In some embodiments, the functional molecule may be RNA or protein, and the gene may have sequences (introns) inserted between the coding regions (exons).

[0040] As used herein, the terms “polynucleotide,” “nucleic acid,” or “nucleic acid molecule” refer to a chain of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA) of a certain length or longer, as a polymer of nucleotides in which nucleotide monomers are linked together by covalent bonds to form a long chain. In this disclosure, the terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably.

[0041] Identity, homology

[0042] As used herein, the term “identity” or “homology” refers to the degree of similarity between two given amino acid or nucleotide sequences and can be expressed as a percentage. The terms “homology” and “identity” are generally used interchangeably.

[0043] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard alignment algorithms and can be used together with the default gap penalty established by the program used.

[0044] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using known computer algorithms, such as the “FASTA” program with default parameters as described in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444. Alternatively, sequence information can be compared using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.Biol.48: 443-453), such as the Needleman program (version 5.0.0 or later) in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), or GAP computer programs such as the Smith-Waterman algorithm (Smith and Waterman, Adv. Appl. Math (1981) 2:482) (including the GCG package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403). (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARILLO et al.] (1988) SIAM J Applied Math 48: 1073)). Homology, similarity, or identity can be determined, for example, by using BLAST or ClustalW from the National Center for Biotechnology Information.

[0045] Furthermore, whether any two polynucleotide sequences are homologous, similar, or identical to each other can be determined by Southern hybridization experiments under suitable hybridization conditions, and suitable hybridization conditions can be determined by methods well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual; FMAusubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto. For example, homologous or identical polynucleotide sequences can typically hybridize to at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or full-length sequence under stringent conditions.

[0046] 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., above, 9.50-9.51, 11.7-11.8). Examples include: polynucleotides with high homology or identity, i.e., polynucleotides with 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity, hybridizing with each other, while polynucleotides with lower homology or identity do not hybridize with each other, or conditions of washing once, especially two or three times, at a salt concentration and temperature equivalent to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, more specifically 68°C, 0.1×SSC, 0.1% SDS, which are typical washing conditions for Southern hybridization.

[0047] Hybridization can occur between nucleotides with complementary bases, but depending on the strictness of the hybridization, hybridized polynucleotides can contain some mismatches between bases. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this disclosure can comprise isolated nucleotide fragments complementary to the whole sequence as well as nucleotide sequences substantially similar to them.

[0048] For example, polynucleotides homologous to or identical with the polynucleotides disclosed herein can be detected by hybridization at a Tm value of 55°C. Furthermore, the Tm value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art.

[0049] The appropriate stringency of hybrid polynucleotides depends on the length and complementarity of the polynucleotides, and these variables are well known in the art (e.g., Sambrook et al., above).

[0050] Nucleic acid constructs, vectors, and transformation

[0051] As used herein, the term "nucleic acid construct" refers to an artificially designed single- or double-stranded nucleic acid molecule contained in a vector that can be used to integrate target genetic material into a suitable host or host cell. For example, a nucleic acid construct may contain a transgene delivered via a transformation vector that allows the inserted sequence to replicate and / or be expressed in a host cell. For example, the transgene may be cloned from an existing sequence or synthesized artificially.

[0052] As used herein, the term "vector" refers to a DNA construct used to deliver a target polynucleotide into a suitable host or host cell.

[0053] For example, the vector can be a nucleotide sequence containing a polynucleotide of the target polypeptide, operatively linked to a suitable expression regulatory region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. The expression regulatory region may contain a promoter capable of initiating transcription, any operon sequence controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences controlling the termination of transcription and translation. The vector can be transformed into a suitable host cell (microorganism) and then replicate or function independently of the host genome, or it can be integrated into the genome itself.

[0054] Furthermore, for example, the vector disclosed herein may contain a sequence for inserting the target polynucleotide into a chromosome. Insertion of the polynucleotide into the chromosome using the vector can be performed by any method known in the art, such as, but not limited to, homologous recombination.

[0055] There are no particular limitations on the vectors used in this disclosure, but any vector known in the art may be used. Examples of commonly used vectors may include natural or recombinant plasmids, granules, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or granule vectors, and the pDZ system, pDC system, pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, and pET system can be used as plasmid vectors. For example, pDZ, pDC, pDC24, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors may be used.

[0056] The vector may also contain selection markers to confirm transformation into host cells, or further, insertion into the host cell's chromosome. Selection markers are used to select cells transformed by the vector or to confirm whether the target polynucleotide has been inserted into the chromosome, and may use markers that confer selectable phenotypes (such as drug resistance, auxotrophic phenotypes, cytotoxic agent resistance, or surface peptide expression). Only cells expressing the selection markers can survive or exhibit different phenotypes under conditions treated with the selection agent, thus allowing for the selection of transformed cells.

[0057] As used herein, the term "transformation" refers to the introduction of a target polynucleotide or a vector containing it into a host cell (microorganism), thereby altering the genetic traits of the host cell (microorganism). Transformed polynucleotides can be located by insertion into the chromosome of the host cell (microorganism) or by being located extrachromosomally. Furthermore, polynucleotides include DNA or RNA. Depending on the purpose of the introduction, the polynucleotide can be introduced in a suitable form. For example, a polynucleotide for expressing a target polypeptide can be introduced into the host cell (microorganism) in the form of an expression cassette, a gene construct containing all the elements required for its autonomous expression. Expression cassettes typically contain a promoter, transcription termination signal, ribosome binding site, and translation termination signal operably linked to the coding sequence of the target polypeptide. Expression cassettes can be in the form of a self-replicating expression vector. Furthermore, polynucleotides can be introduced into the host cell (microorganism) as is and operably linked to the sequence required for expression in the host cell (microorganism), but are not limited thereto.

[0058] As used herein, the term "operably linked" refers to a construction that places a regulatory sequence in the appropriate position to guide the expression of a coding sequence. Therefore, the term "operably linked" encompasses the attachment or connection between a regulatory region (such as a promoter, terminator, signal sequence, or enhancer) with a functional domain having known or desired activity and a target (gene or polypeptide), thereby regulating the expression, secretion, or function of the target (gene or polypeptide) according to known or desired activity. For example, the term can refer to the functional linking of a polynucleotide sequence to a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding a target polypeptide.

[0059] 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, and secretion.

[0060] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule comprising a target polynucleotide sequence and a operatively linked regulatory sequence for its expression. For example, an expression vector may contain a nucleotide sequence of a polynucleotide encoding a target polypeptide, which is operatively linked to a suitable expression regulatory region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host.

[0061] As used herein, the term "regulatory sequence" refers to the polynucleotide sequence required to regulate the expression of a target polynucleotide sequence. Each regulatory sequence can be natural (from the same source) or exogenous (from a different gene), or a mutant or other artificial sequence relative to the coding sequence. Instances of regulatory sequences can include leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, operon sequences, sequences encoding ribosome binding sites, and sequences terminating transcription and translation. The smallest unit of a regulatory sequence can include a promoter and sequences terminating transcription and translation.

[0062] As used in this article, the term “genetic recombination” refers to a natural or artificial process in which the original sequence of the elements that make up a gene (such as DNA or RNA) is altered during dissociation and reassembly.

[0063] As used herein, the term "recombinant gene" refers to a gene with a novel genomic composition resulting from genetic recombination (e.g., chemical synthesis or genetic engineering techniques). As used herein, the terms "recombinant gene," "recombinant DNA," and "recombinant polynucleotide" are used interchangeably. For example, a recombinant gene can be an artificial combination of nucleic acid fragments (such as regulatory sequences) that do not exist in nature.

[0064] As used in this article, the term "recombinant protein" refers to a protein produced due to genetic recombination.

[0065] microorganism

[0066] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms, or naturally or artificially genetically modified prokaryotic and eukaryotic microorganisms, and 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 can be a microorganism containing genetic modifications for the production of desired polypeptides, proteins, or products. As used herein, the terms "microorganism," "strain," "host," and "host cell" are used interchangeably.

[0067] As used herein, the term "recombinant microorganism" refers to a microorganism that has been genetically modified and exhibits a genotype and / or phenotype different from that of a naturally occurring microorganism (e.g., when the genetic modification affects the nucleic acid sequence encoding the microorganism), and may include the offspring or all potential offspring of that microorganism. As used herein, the terms "recombinant microorganism," "genetically modified microorganism," "recombinant host cell," "recombinant cell," and "recombinant strain" are used interchangeably. For example, a recombinant microorganism may express genes not present in its natural (non-recombinant) form, or may not express genes expressed in their natural form, or may express natural genes in a manner different from those expressed in their natural form.

[0068] For example, the microorganisms disclosed herein may be microorganisms in which NAD-dependent isocitrate dehydrogenase protein or polynucleotides encoding it are introduced (e.g., recombinant microorganisms), but are not limited thereto.

[0069] As used herein, the term "microorganism capable of producing L-amino acids" refers to a microorganism that is able to produce L-amino acids in an organism, and can include any microorganism prepared by providing the ability to produce L-amino acids to microorganisms that are not naturally capable of producing L-amino acids or microorganisms that are naturally capable of producing L-amino acids. The ability to produce L-amino acids can be conferred or enhanced through species modification.

[0070] As used herein, the term "unmodified microorganism (strain)" does not exclude the inclusion of microorganisms (strains) that may have naturally occurring mutations, and can be a wild-type microorganism (strain) or a natural microorganism (strain) itself, or a microorganism (strain) before its traits are altered by genetic variation due to natural or artificial factors. In this disclosure, the term "unmodified microorganism (strain)" may be used interchangeably with "pre-modified microorganism (strain)," "unmutated microorganism (strain)," "parental microorganism," "parental strain," "wild-type microorganism (strain)," "reference microorganism (strain)," or "standard microorganism (strain)." In this disclosure, unmodified microorganism may refer to a microorganism (strain) in which the NAD-dependent isocitrate dehydrogenase protein or the polynucleotide encoding it of this disclosure has not been introduced, or a microorganism (strain) before its introduction, but is not limited thereto. Furthermore, unmodified microorganisms in this disclosure may be microorganisms that do not contain the polypeptide composed of SEQ ID NO: 1 or the polynucleotide composed of SEQ ID NO: 2, but are not limited thereto.

[0071] Increased protein (peptide) activity

[0072] As used herein, the term “increased protein (peptide) activity” refers to an enhanced activity of a protein (peptide) in a host cell (microorganism) compared to its endogenous activity. This increase can be used interchangeably with terms such as activation, upregulation, overexpression, and enhancement. The host cell (microorganism) can be a prokaryotic or eukaryotic microorganism.

[0073] Increased protein (peptide) activity can include two scenarios: exhibiting protein (peptide) activity that is not endogenously present in the host cell (microorganism), or exhibiting enhanced protein (peptide) activity compared to endogenous activity or activity before modification.

[0074] For example, “exhibiting protein (peptide) activity not present in endogenous proteins” or “exhibiting enhanced protein (peptide) activity” can be caused by “the introduction of peptides (proteins),” but is not limited to this.

[0075] As used herein, the term “introduction” of a protein (peptide) refers to the expression in a microorganism of a gene not originally present in the microorganism, compared to the endogenous or unmodified activity of the corresponding protein, to exhibit the activity of the specific protein or to exhibit enhanced, increased, or elevated peptide activity. For example, a polynucleotide encoding a specific protein (peptide) can be introduced into the chromosome of a host cell (microorganism), or a vector containing a polynucleotide encoding a specific protein (peptide) can be introduced into a host cell (microorganism) to exhibit its activity.

[0076] "Endogenous activity" refers to the activity of a specific protein (peptide) that was originally present in the host cell (microorganism) or the unmodified host cell (microorganism) before the trait change due to genetic variation caused by natural or artificial factors. This can be used interchangeably with "activity before modification".

[0077] An increase in protein (peptide) activity compared to endogenous activity means that the activity and / or concentration (expression level) of the protein (peptide) in the host cell (microorganism) is higher than the activity and / or concentration (expression level) of the protein (peptide) originally present in the untransformed host cell (microorganism) or the unmodified host cell (microorganism).

[0078] For example, an increase indicates the presence of the activity of a corresponding protein (peptide) that was not originally present, or its activity or concentration based on the activity or concentration of the host cell (microbe) before transformation or the unmodified host cell (microbe), typically by about 1% or more, about 10% or more, about 25% or more, about 50% or more, about 75% or more, about 100% or more, about 150% or more, about 200% or more, about 300% or more, about 400% or more, or about 500% or more, up to about 1000% or about 2000% or more, but not limited to these.

[0079] Increased protein (peptide) activity can be achieved by introducing exogenous proteins (peptides) or by increasing the activity of existing proteins (peptides). This increase can be confirmed by the degree of increase in the activity and expression level of the corresponding protein (peptide), or by the increase in the amount of product resulting from the activity of the corresponding protein (peptide).

[0080] Increased protein (peptide) activity can be achieved by a variety of methods well known in the art, and such methods are not limited, as long as the activity of the target protein (peptide) can be increased compared to the host cell (microbe) before modification. Specifically, genetic engineering and / or protein engineering, which are well known to those skilled in the art, can be used; these are routine methods in molecular biology, but the method is not limited thereto (e.g., Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012, etc.).

[0081] Specifically, the increase in protein (peptide) activity disclosed herein can be:

[0082] 1) Increase the copy number of polynucleotides encoding proteins (peptides) within the cell;

[0083] 2) Modify the expression regulatory regions of genes encoding proteins (peptides) on chromosomes (e.g., introduce modifications into the expression regulatory regions, replace them with sequences that have stronger activity, or insert sequences that have stronger activity).

[0084] 3) Modify the nucleotide sequence of the start codon or 5'-UTR of the gene transcript that encodes a protein (peptide);

[0085] 4) Modifying the amino acid sequence of proteins (peptides) to enhance their activity;

[0086] 5) Modify the polynucleotide sequence encoding a protein (peptide) to enhance the activity of the protein (peptide) (e.g., modify the polynucleotide sequence of a gene encoding a protein (peptide) to encode a protein (peptide) that has been modified to enhance the activity of the protein (peptide)).

[0087] 6) Introduce a foreign protein (peptide) that exhibits protein (peptide) activity or a foreign polynucleotide encoding that protein (peptide);

[0088] 7) Codon optimization for polynucleotides encoding proteins (peptides);

[0089] 8) Analyze the tertiary structure of proteins (peptides) to select and modify exposed sites, or to chemically modify them; or

[0090] 9) Controlling the intracellular localization of proteins (peptides); or

[0091] 10) A combination of two or more of 1) through 9), but not limited thereto.

[0092] For example,

[0093] 1) Increasing the copy number of polynucleotides encoding proteins (peptides) within cells can be achieved by introducing a vector containing the polynucleotide encoding the protein (peptide) into a host cell (microorganism), the polynucleotide being operatively linked to a suitable regulatory sequence. Alternatively, this can be achieved by introducing one or two or more copies of the polynucleotide encoding the protein (peptide) into the chromosome of the host cell (microorganism), the polynucleotide being operatively linked to a suitable regulatory sequence. Introduction into the chromosome can be done by, but is not limited to, introducing a vector capable of inserting the polynucleotide into the chromosome of the host cell (microorganism). The vector is as described above. The regulatory sequence for the polynucleotide encoding sequence can be natural (from the same source) or exogenous (from a different gene), or it can be a mutant sequence or other artificial sequence, and can induce the expression of the polynucleotide in the host cell (microorganism).

[0094] 2) Replacing the expression regulatory region (or expression regulatory sequence) of a gene encoding a protein (peptide) on a chromosome with a highly active sequence can be achieved, for example, by introducing modifications into the sequence through deletion, insertion, substitution, or a combination thereof to further enhance the activity of the expression regulatory region, or by replacing the replacement sequence with a sequence of stronger activity. Expression regulatory regions may include, but are not limited to, promoters, operon sequences, sequences encoding ribosome binding sites, and sequences regulating transcription and translation termination. For example, a strong promoter can replace the original promoter, but this is not the only possibility.

[0095] Examples of known strong promoters may include, but are not limited to, the cj1 to cj7 promoters (US 7662943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the λ phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13(sm3) promoter (US 10584338 B2), the O2 promoter (US 10273491 B2), the tkt promoter, and the yccA promoter.

[0096] 3) Modifying the coding start codon or 5'-UTR nucleotide sequence of a gene that encodes a protein (peptide) can be achieved, for example, by modifying the nucleotide sequence to encode another start codon that has a higher protein (peptide) expression rate compared to the endogenous start codon, or an RBS sequence that has a higher protein (peptide) expression rate compared to the endogenous RBS (ribosome binding site) sequence, but is not limited thereto.

[0097] 4) and 5) Modification of the amino acid sequence or polynucleotide sequence of a protein (peptide) can be achieved by introducing modifications into the sequence to enhance the activity of the protein (peptide) through deletion, insertion, or substitution of the amino acid sequence of the protein (peptide) or the polynucleotide sequence encoding the protein (peptide), or by replacing it with a modified amino acid sequence or polynucleotide sequence to enhance its activity, but is not limited thereto. For example, substitution can be carried out by inserting polynucleotides into the chromosome through homologous recombination, but is not limited thereto.

[0098] 6) The introduction of exogenous polynucleotides exhibiting protein (peptide) activity can be achieved by introducing an exogenous polynucleotide encoding a protein (peptide) exhibiting the same / similar activity as the protein (peptide) into a host cell (microorganism). Exogenous polynucleotides can be used without restriction, regardless of their origin or sequence, as long as they exhibit the same / similar activity as the protein (peptide). Those skilled in the art can implement the methods used in the introduction by appropriately selecting known transformation methods, and the expression of the introduced polynucleotide in the host cell can produce a protein (peptide), thereby increasing its activity.

[0099] 7) Codon optimization of polynucleotides encoding proteins (peptides) can be achieved by optimizing the codons of endogenous polynucleotides to increase transcription or translation in the host cell (microorganism), or by optimizing the codons of exogenous polynucleotides so that the exogenous polynucleotides can achieve optimized transcription and translation in the host cell (microorganism).

[0100] 8) Analyzing the tertiary structure of proteins (peptides) to select and modify exposed sites, or to chemically modify them, can be achieved, for example, by comparing the sequence information of the protein (peptide) to be analyzed with a database (which stores the sequence information of known proteins) to determine template protein candidates based on sequence similarity, and thus confirming the structure based on this information, thereby selecting and transforming or modifying the exposed sites to be modified or chemically modified.

[0101] 9) Controlling the intracellular localization of proteins (peptides) can mean targeting proteins (peptides) to specific organelles or specific intracellular spaces within the cell, for example, by adding or removing a leader sequence that functions in the target protein (peptide) to target the periplasm or cytoplasm, but is not limited to this.

[0102] This increase in protein (peptide) activity may mean an increase in the activity or concentration of the corresponding protein (peptide) relative to the activity or concentration of the protein (peptide) expressed in the wild-type or unmodified host cells (microorganisms), or an increase in the amount of product produced by the corresponding protein (peptide), but is not limited thereto.

[0103] The modification of some or all of the polynucleotides in the host cell (microorganism) disclosed herein can be achieved, but is not limited to, by (a) homologous recombination using an engineered nuclease (e.g., CRISPR-Cas9) via a vector for chromosome insertion or genome editing, and / or (b) by light (e.g., ultraviolet light and radiation, etc.) induction and / or chemical treatment.

[0104] nourish

[0105] As used herein, the term "culture" refers to the growth of microorganisms under appropriately controlled environmental conditions. The culture process can be carried out in suitable culture media and under culture conditions known in the art. Depending on the selected microorganisms, those skilled in the art can readily adapt such a culture process for use. Specifically, the culture can be a batch culture, a continuous culture, and / or a fed-batch culture, but is not limited thereto.

[0106] As used herein, the term "culture medium" refers to a mixture of substances containing nutrients required for the cultivation of microorganisms as its main components, providing nutrients and growth factors, as well as water necessary for survival and growth. Specifically, the culture medium and other culture conditions used to cultivate the microorganisms of this disclosure can be any culture medium used for the general cultivation of microorganisms without any particular limitation. For example, 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 simultaneously adjusting temperature, pH, etc. For example, culture media for Corynebacterium spp. can be found in the literature [Manual of Methods for General Bacteriology, American Bacteriological Society (Washington, D.C., USA, 1981)].

[0107] In this disclosure, carbon sources may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients such as starch hydrolysate, molasses, molasses, rice bran, cassava, cane molasses, and corn steep liquor may be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) may be used, and various other carbon sources may be used in unlimited quantities. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0108] Nitrogen sources can include inorganic nitrogen sources, such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; amino acids, such as glutamic acid, methionine, and glutamine; and organic nitrogen sources, such as peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysate, fish or its degradation products, and defatted soybean meal or its degradation products. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0109] Phosphorus sources may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. Additionally, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the culture medium in batches or continuously, but are not limited to this.

[0110] Furthermore, during the cultivation of the microorganisms disclosed herein, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc., in an appropriate manner. Additionally, the use of antifoaming agents such as fatty acid polyethylene glycol esters during cultivation can prevent bubble formation. Furthermore, oxygen or oxygen-containing gas can be injected into the culture medium to maintain aerobic conditions; or nitrogen, hydrogen, or carbon dioxide can be injected to maintain anaerobic or slightly aerobic conditions, but gas injection is not required, and the method is not limited to these methods.

[0111] In the cultivation process disclosed herein, the cultivation temperature can be maintained between 20°C and 45°C, specifically between 25°C and 40°C, and the cultivation can be carried out for approximately 10 hours to approximately 160 hours, but is not limited thereto.

[0112] As used herein, the term "culture product" means a culture solution, a concentrated culture solution, a dried product of a culture solution, 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, and means that the culture solution may contain the specific microorganism, while the culture filtrate substantially does not contain the specific microorganism (in this context, it substantially means excluding the specific microorganism separated by filtration, etc., but does not mean that the microorganism is completely excluded from the filtrate). The dosage form of the culture product is not limited and can be, for example, a liquid, an emulsion, or a solid.

[0113] As used in this article, the term "fermentation" refers to the process by which microorganisms use their enzymes to break down organic matter, excluding putrefaction. Fermentation and putrefaction occur through similar processes. However, when the result of decomposition is the production of useful substances, it is called fermentation; when it produces foul odors or harmful substances, it is called putrefaction.

[0114] In this disclosure, the method for obtaining fermentation products from microorganisms is not particularly limited and can be carried out according to methods commonly used in the art or similar fields.

[0115] As used herein, the term "fermentation product" can include not only the fermentation material itself, but also all kinds of materials containing fermentation products produced by microorganisms, such as materials containing fermenting microorganisms, fermentation products produced by fermenting microorganisms, fermentation products of cultured products, concentrated fermentation products; dried fermentation products, filtrates of fermentation products, filtrates of concentrated fermentation products, dried filtrates of fermentation products, extracts of fermentation products, or diluted solutions of fermentation products, etc.

[0116] Detailed description of this disclosure

[0117] The specific implementation schemes of this disclosure will be described in more detail below.

[0118] One aspect of this disclosure provides a Corynebacterium microorganism capable of producing L-amino acids, wherein an NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans or a polynucleotide encoding the same is introduced.

[0119] As used herein, the term "NAD-dependent isocitrate dehydrogenase (ICD)" refers to the NAD-dependent enzyme among isocitrate dehydrogenases that catalyzes oxidative decarboxylation to produce α-ketoglutarate and CO2. The NAD-dependent isocitrate dehydrogenase of this disclosure is interchangeable with ICD.

[0120] Specifically, the NAD-dependent isocitrate dehydrogenase protein disclosed herein may be a protein having NAD-dependent isocitrate dehydrogenase activity encoded by an icd gene, but its type is not particularly limited, as long as the protein has activity corresponding to NAD-dependent isocitrate dehydrogenase activity. NAD-dependent isocitrate dehydrogenase proteins encoded by icd genes are known in the art, and the amino acid and polynucleotide sequences of NAD-dependent isocitrate dehydrogenase proteins can be obtained from known databases, such as NCBI's GenBank, but are not limited thereto.

[0121] For example, an NAD-dependent isocitrate dehydrogenase protein derived from *Streptococcus mutans* may contain the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having 60% or more homology or identity with it, but is not limited thereto, as long as it possesses NAD-dependent isocitrate dehydrogenase activity. Specifically, any protein having a portion of the amino acid sequence of SEQ ID NO: 1 that has been deleted, modified, substituted, or added may be included in an NAD-dependent isocitrate dehydrogenase protein, provided that it exhibits efficacy corresponding to that of an NAD-dependent isocitrate dehydrogenase protein. Furthermore, any protein having or containing an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with the amino acid sequence of SEQ ID NO: 1, consisting of or substantially consisting of that amino acid sequence, and exhibiting efficacy corresponding to that of an NAD-dependent isocitrate dehydrogenase may be included in an NAD-dependent isocitrate dehydrogenase protein.

[0122] Furthermore, the sequence of the polynucleotide encoding the NAD-dependent isocitrate dehydrogenase protein derived from *Streptococcus mutans* can be obtained, for example, based on codon information known in the art, wherein the protein has the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60% or more homology or identity with it. For example, the NAD-dependent isocitrate dehydrogenase protein can be encoded by a polynucleotide having or containing the sequence of SEQ ID NO: 2, or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity with the sequence of SEQ ID NO: 2, a nucleotide sequence composed of such nucleotide sequence, or a nucleotide sequence substantially composed of such nucleotide sequence, but is not limited thereto. Furthermore, the nucleotide sequence of SEQ ID NO: 2 can be obtained from known databases, such as NCBI's GenBank, but is not limited thereto.

[0123] In this disclosure, a polynucleotide (gene) containing the nucleotide sequence of SEQ ID NO: 2 may be used interchangeably with a polynucleotide (gene) having the nucleotide sequence of SEQ ID NO: 2, a polynucleotide (gene) composed of the nucleotide sequence of SEQ ID NO: 2, or an icd gene.

[0124] Due to codon degeneracy or considering preferred codons in organisms expressing the NAD-dependent isocitrate dehydrogenase protein of this disclosure, the polynucleotides of this disclosure can undergo various modifications in their coding regions without altering the amino acid sequence of the NAD-dependent isocitrate dehydrogenase protein of this disclosure. Therefore, based on codon degeneracy, it is apparent that polynucleotides that can be translated into polypeptides consisting of the amino acid sequence of the NAD-dependent isocitrate dehydrogenase protein of this disclosure, or polypeptides homologous to or identical with it, may also be included in the polynucleotides of this disclosure. For example, the polynucleotides of this disclosure may be SEQ ID NO: 2, or its degenerate sequence.

[0125] For example, the polynucleotides disclosed herein may have or contain nucleotide sequences that are 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity with SEQ ID NO: 2, or may be composed of or substantially composed of nucleotide sequences that are 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity with SEQ ID NO: 2, but are not limited thereto.

[0126] Furthermore, the polynucleotides of this disclosure may contain probes that can be prepared from known gene sequences, such as any sequence that can be hybridized under stringent conditions with a sequence that is fully or partially complementary to the polynucleotide sequence of this disclosure to encode the NAD-dependent isocitrate dehydrogenase protein of this disclosure, without limitation.

[0127] The Corynebacterium species disclosed herein have the ability to produce L-amino acids.

[0128] In this disclosure, specifically, the L-amino acid may be L-tryptophan or L-histidine.

[0129] Regarding the purposes of this disclosure, the microorganisms of this disclosure may comprise all microorganisms capable of producing desired L-amino acids, wherein an NAD-dependent isocitrate dehydrogenase protein or a polynucleotide encoding the same is introduced. For example, the microorganisms of this disclosure may be genetically modified or recombinant microorganisms wherein an NAD-dependent isocitrate dehydrogenase protein or a polynucleotide encoding the same is introduced, thereby increasing the ability to produce L-amino acids. Specifically, recombinant microorganisms with increased L-amino acid production capacity may be, but are not limited to, microorganisms with increased L-amino acid production capacity compared to natural wild-type microorganisms or unmodified microorganisms with or without endogenous NAD-dependent isocitrate dehydrogenase activity.

[0130] For example, microorganisms capable of producing L-amino acids, i.e., prokaryotic or eukaryotic microorganisms capable of producing L-amino acids in organisms, can include all microorganisms endogenously capable of producing L-amino acids, or microorganisms in which the ability to produce L-amino acids has been conferred by the introduction of the NAD-dependent isocitrate dehydrogenase protein of this disclosure into strains that do not possess the ability to produce L-amino acids. The ability to produce L-amino acids can be conferred or enhanced through species modification.

[0131] The microorganisms disclosed herein may include all microorganisms in which NAD-dependent isocitrate dehydrogenase protein or polynucleotides encoding it are introduced by various known methods.

[0132] For example, the recombinant microorganisms of this disclosure capable of producing L-amino acids can be microorganisms in which a foreign gene encoding the NAD-dependent isocitrate dehydrogenase protein of this disclosure, particularly a foreign gene encoding the NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans, is introduced via vector transformation to produce L-amino acids.

[0133] For example, the microorganisms that produce L-amino acids can be microorganisms that have been introduced with a polynucleotide sequence that encodes a protein containing the amino acid sequence of SEQ ID NO: 1, or a protein containing an amino acid sequence that has at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity with the amino acid sequence of SEQ ID NO: 1.

[0134] For example, the microorganisms that produce L-amino acids can be microorganisms that introduce polynucleotides capable of encoding a protein containing an amino acid sequence having at least 80% homology with the amino acid sequence of SEQ ID NO: 1; or polynucleotides containing the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity with the nucleotide sequence of SEQ ID NO: 2.

[0135] For example, the microorganisms with increased L-amino acid production capacity disclosed herein may be microorganisms with increased L-amino acid production capacity compared to unmodified microorganisms, but are not limited thereto. For example, the unmodified microorganisms used as target strains for comparing increased L-amino acid production capacity may be strains CM05-9157 or CA14-0114, but are not limited thereto.

[0136] For example, a microorganism with increased ability to produce L-amino acids may have an increased ability to produce L-amino acids by about 1% or more, specifically about 1% or more, about 2.5% or more, about 5% or more, about 6% or more, about 7% or more, about 8% or more, about 9% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, or about 26% or more (there is no particular upper limit, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 45% or less, about 40% or less, or about 35% or less), but is not limited thereto, as long as it has an increased + value compared to the production capacity of the parent microorganism (parent strain) or the unmodified microorganism. In another example, a recombinant strain with increased L-amino acid production capacity may have an increased L-amino acid production capacity of about 1.1 times or more, about 1.15 times or more, about 1.2 times or more, about 1.25 times or more, or about 1.26 times or more (with no particular upper limit, e.g., about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less, about 1.5 times or less, about 1.4 times or less, about 1.35 times or less) compared to the unmodified parental microorganism (parental strain) or the unmodified microorganism, but is not limited thereto.

[0137] For example, the microorganisms disclosed herein capable of producing L-amino acids can be prokaryotic or eukaryotic cells, but specifically prokaryotic cells. Prokaryotic cells may include, but are not limited to, microorganisms belonging to, genus such as *Escherichia* sp., *Erwinia* sp., *Serratia* sp., *Providencia* sp., *Corynebacterium* sp., *Pseudomonas* sp., *Leptospira* sp., *Salmonella* sp., *Brevibacteria* sp., *Hypomononas* sp., *Chromobacterium* sp., and *Norcardia* sp. Specifically, the microorganisms may be *Escherichia* or *Corynebacterium*. More specifically, the microorganisms may be *Corynebacterium*.

[0138] Regarding the microorganisms according to any one of the foregoing specific embodiments, the microorganisms disclosed herein may be Corynebacterium genus microorganisms.

[0139] The *Corynebacterium* species mentioned may be *Corynebacterium glutamicum*, *Corynebacterium crudilactis*, *Corynebacterium deserti*, *Corynebacterium efficiens*, *Corynebacterium callunae*, *Corynebacterium stationis*, *Corynebacterium singulare*, *Corynebacterium halotolerans*, *Corynebacterium striatum*, *Corynebacterium ammoniagenes*, *Corynebacterium pollutisoli*, *Corynebacterium imitans*, *Corynebacterium testudinoris*, or *Corynebacterium flavescens*. Specifically, the microorganisms disclosed herein may be *Corynebacterium glutamicum*, but are not limited thereto.

[0140] Meanwhile, the Corynebacterium genus microorganisms with the ability to produce L-amino acids disclosed herein may include all naturally occurring wild-type microorganisms themselves, Corynebacterium genus microorganisms in which the activity of genes related to the L-amino acid production mechanism is increased or decreased, thereby having an enhanced ability to produce L-amino acids, or Corynebacterium genus microorganisms in which the activity of exogenous genes is introduced or increased, thereby having an enhanced ability to produce L-amino acids.

[0141] Another aspect of this disclosure provides a method for producing L-amino acids, the method comprising the step of culturing a Corynebacterium microorganism capable of producing L-amino acids in a culture medium, wherein the microorganism is introduced with an NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans of this disclosure or a polynucleotide encoding therethe.

[0142] In the methods disclosed herein, the microorganisms can be cultured using any culture conditions and methods known in the art. Depending on the selected microorganisms, those skilled in the art can readily adapt this culture method for use.

[0143] The L-amino acids produced by the culture of this disclosure can be released into the culture medium or retained in the cells.

[0144] In one specific embodiment, the method for producing L-amino acids of this disclosure may further include the steps of preparing the microorganisms of this disclosure, preparing a culture medium for culturing the strain, or a combination thereof (in any order, regardless of the sequence), for example, prior to the culturing step.

[0145] The method for producing L-amino acids disclosed herein may further include a step of recovering the desired substance, particularly L-amino acids, from cultured microorganisms, the culture product of said microorganisms, the fermentation product of said microorganisms, or the culture medium. A recovery step may be further included after the culturing step.

[0146] Recovery can be achieved by collecting the desired L-amino acids using the methods of culturing microorganisms disclosed herein, for example, using suitable methods known in the art according to batch culture, continuous culture, or fed-batch culture methods. For example, centrifugation, filtration, treatment with a protein crystallizing precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatography methods (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC, or combinations thereof can be used to recover the desired substance, particularly L-amino acids, from the culture medium or microorganisms using suitable methods known in the art.

[0147] Furthermore, the method for producing L-amino acids disclosed herein may further include a purification step, which may be performed using suitable methods known in the art. In one embodiment, when the method for producing L-amino acids disclosed herein includes both a recovery step and a purification step, the recovery step and the purification step may be performed sequentially or intermittently, or simultaneously, or may be integrated into a single step, but the method is not limited thereto.

[0148] In the methods disclosed herein, the NAD-dependent isocitrate dehydrogenase, the introduction, and the L-amino acids are as described in other aspects above.

[0149] Another aspect of this disclosure provides a composition for producing L-amino acids, the composition comprising a Corynebacterium spp. microorganism capable of producing L-amino acids, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof, wherein the microorganism is introduced with the NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans of this disclosure or a polynucleotide encoding therethe.

[0150] The compositions disclosed herein may further comprise any suitable excipients commonly used in compositions for the production of L-amino acids, and such excipients may comprise, for example, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or isotonic agents, etc., but are not limited thereto.

[0151] In one specific embodiment, each component present in the compositions of this disclosure may be included in a microbially effective amount or in an amount that may be suitably present in the compositions used for production.

[0152] In the compositions disclosed herein, the NAD-dependent isocitrate dehydrogenase, the delivery system, and L-amino acids are as described in other aspects above.

[0153] Another aspect of this disclosure provides the use of Corynebacterium microorganisms capable of producing L-amino acids in the production of L-amino acids, wherein the microorganisms are introduced with the NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans of this disclosure or the polynucleotide encoding therethe.

[0154] In the purposes of this disclosure, the NAD-dependent isocitrate dehydrogenase, delivery, and L-amino acids are as described in other aspects above.

[0155] Methods of implementing the present invention

[0156] The present disclosure will be described in more detail below by way of exemplary embodiments. However, the following exemplary embodiments are merely preferred embodiments for illustrating the present disclosure and are therefore not intended to limit the scope of the disclosure thereto. Meanwhile, technical matters not described in this specification can be fully understood and readily implemented by those skilled in the art or similar art.

[0157] Example 1. Exploration and selection of NAD-dependent isocitrate dehydrogenase (icd) genes

[0158] To select organisms possessing NAD-dependent isocitrate dehydrogenase (icd), a BLAST search was performed using the amino acid sequence of endogenous isocitrate dehydrogenase from *Corynebacterium glutamicum* as the query sequence, based on the NCBI and Kegg databases. As a result, considering the biosafety level and availability suitable for production strains, three types of microorganisms expected to possess NAD-dependent isocitrate dehydrogenase were selected, as shown in Table 1 below.

[0159] [Table 1]

[0160]

[0161] Example 2. Preparation of L-tryptophan-producing microorganisms infused with exogenous NAD-dependent isocitrate dehydrogenase

[0162] Example 2-1. Preparation of plasmids for gene replacement

[0163] To replace the icd gene in the chromosome of Corynebacterium glutamicum, plasmid pDC24 (SEQ ID NO: 86) was used as the parent vector to construct a plasmid that could replace the coding sequence of the endogenous icd gene with the coding sequence of the exogenous icd gene.

[0164] Specifically, chromosomal DNA of Corynebacterium glutamicum ATCC13869 was used as a template. Primer pairs SEQ ID NO: 7 and SEQ ID NO: 8 were used to amplify the upstream region on the chromosome where homologous recombination occurred, and primer pairs SEQ ID NO: 9 and SEQ ID NO: 10 were used to amplify the downstream region. Then, PCR was performed to obtain the respective gene fragments.

[0165] Use Solg TM PCR was performed using Pfu-X DNA polymerase (SolGent Co.) under the following conditions: denaturation at 95°C for 4 minutes, followed by 27 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 50 seconds, and then polymerization at 72°C for 5 minutes. The primer sequences used are shown in Table 2 below.

[0166] [Table 2]

[0167]

[0168] Using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY2009, NEBuilder HiFi DNA Assembly Master Mix), the upstream and downstream fragments of the region on the chromosome undergoing homologous recombination, which were amplified by the above PCR, and the chromosome integration vector pDC24 digested with the smaI restriction enzyme, were cloned to obtain the recombinant plasmid, which was named pDC24Δicd(13869).

[0169] Example 2-2. Preparation of tryptophan-producing Corynebacterium spp. microorganisms infused with NAD-dependent isocitrate dehydrogenase derived from Streptococcus mutans.

[0170] The NAD-dependent isocitrate dehydrogenase selected in Example 1 from *Streptococcus mutans* has the amino acid sequence of SEQ ID NO: 1. Information about the gene encoding NAD-dependent isocitrate dehydrogenase and its surrounding base sequence was obtained from US NIH GenBank, and based on this, a codon-optimized icd gene (SEQ ID NO: 2) from *Streptococcus mutans* was synthesized using the gene synthesis service of Bionics Co., Ltd. To insert this gene into the genomic DNA of *Corynebacterium glutamicum*, the synthesized gene was used as a template and amplified by PCR using the primer pair of SEQ ID NO: 11 and SEQ ID NO: 12. Solg... TM Pfu-X DNA polymerase was used as the polymerase, and PCR was performed under the following conditions: 27 cycles of denaturation at 95°C for 2 minutes, followed by denaturation at 95°C for 20 seconds, annealing at 60°C for 40 seconds, and polymerization at 72°C for 1 minute, and then polymerization at 72°C for 5 minutes. The primer sequences used are shown in Table 3 below.

[0171] [Table 3]

[0172]

[0173] Subsequently, the gene fragment of NAD-dependent isocitrate dehydrogenase from *Streptococcus mutans* and the chromosome integration vector pDC24Δicd(13869) digested with smaI restriction enzyme prepared in Example 2-1 were cloned and amplified using the Gibson assembly method (DG Gibson et al., *NATURE METHODS*, VOL.6 NO.5, MAY 2009, NEBuilder high-fidelity DNA assembly premix) to obtain the recombinant plasmid, which was named pDC24Δicd(13869)-icd(S.mu). Cloning was performed by mixing the Gibson assembly reagent, the gene fragment, and the digested vector at an appropriate molar ratio, followed by incubation at 50°C for 1 hour. The pDC24Δicd(13869)-icd(S.mu) vector, thus prepared, was transformed into tryptophan-producing strain CM05-9157 (US Patent Publication No. US 2023-0134555 A1) via electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). A double crossover was then performed to obtain strains in which the coding sequence of the endogenous icd gene on the chromosome was replaced with the coding sequence of the icd gene derived from *Streptococcus mutans*. The obtained strains were identified by PCR and genome sequencing using primers SEQ ID NO: 13 and SEQ ID NO: 14, which amplify the outer regions of the upstream and downstream homologous recombination regions at the site of gene substitution, respectively. The primer sequences used are shown in Table 4 below.

[0174] [Table 4]

[0175]

[0176] The strain obtained by the above method was named CM05-9157Δicd::icd(S.mu).

[0177] Examples 2-3. Preparation of tryptophan-producing Corynebacterium spp. microorganisms infused with NAD-dependent isocitrate dehydrogenase derived from Streptococcus guinea pig.

[0178] The NAD-dependent isocitrate dehydrogenase selected in Example 1 from *Streptococcus guinea pig* has the amino acid sequence SEQ ID NO: 3. Information about the corresponding gene and its surrounding base sequence was obtained from US NIH GenBank, and based on this, a codon-optimized icd gene (SEQ ID NO: 4) from *Streptococcus guinea pig* was synthesized using the gene synthesis service of Bionics Co., Ltd. To insert this gene into the genomic DNA of *Corynebacterium glutamicum*, the synthesized gene was used as a template, and PCR was performed in the same manner as in Examples 2-2 using primer pairs SEQ ID NO: 15 and SEQ ID NO: 16. The primer sequences used are shown in Table 5 below.

[0179] [Table 5]

[0180]

[0181] Subsequently, the gene fragment of NAD-dependent isocitrate dehydrogenase from *Streptococcus guinea pig*, amplified using the Gibson assembly method, and the chromosome integration vector pDC24Δicd(13869) prepared in Example 2-1 and digested with the smaI restriction enzyme were cloned to obtain a recombinant plasmid, named pDC24Δicd(13869)-icd(S.ca). Cloning was performed by mixing the Gibson assembly reagent, gene fragment, and digested vector in an appropriate molar ratio and then incubating at 50°C for 1 hour. The pDC24Δicd(13869)-icd(S.ca) vector thus prepared was transformed into tryptophan-producing strain CM05-9157 by electroporation, followed by double crossover to obtain a strain in which the coding sequence of the icd gene from *Streptococcus guinea pig* replaced the coding sequence of the endogenous icd gene on the chromosome. The strain was identified by PCR and genome sequencing using primers SEQ ID NO: 13 and SEQ ID NO: 14 from Table 4, which can amplify the outer regions of the upstream and downstream regions of homologous recombination at the site where the gene was replaced, respectively.

[0182] The strain obtained by the above method was named CM05-9157Δicd::icd(S.ca).

[0183] Examples 2-4. Preparation of tryptophan-producing Corynebacterium spp. microorganisms infused with NAD-dependent isocitrate dehydrogenase derived from Streptococcus monitori.

[0184] The NAD-dependent isocitrate dehydrogenase selected from *Streptococcus monitori* in Example 1 has the amino acid sequence SEQ ID NO: 5. Information about the corresponding gene and its surrounding base sequence was obtained from US NIH GenBank, and based on this, a codon-optimized icd gene (SEQ ID NO: 6) from *Streptococcus monitori* was synthesized using the gene synthesis service of Bionics Co., Ltd. To insert this gene into the genomic DNA of *Corynebacterium glutamicum*, the synthesized gene was used as a template, and PCR was performed in the same manner as in Examples 2-2 using primer pairs SEQ ID NO: 17 and SEQ ID NO: 18. The primer sequences used are shown in Table 6 below.

[0185] [Table 6]

[0186]

[0187] Subsequently, the gene fragment of NAD-dependent isocitrate dehydrogenase from *Streptococcus monitori*, amplified using the Gibson assembly method, and the chromosome integration vector pDC24Δicd(13869) prepared in Example 2-1 and digested with the smaI restriction enzyme were cloned to obtain a recombinant plasmid, named pDC24Δicd(13869)-icd(S.va). Cloning was performed by mixing the Gibson assembly reagent, gene fragment, and digested vector in an appropriate molar ratio and then incubating at 50°C for 1 hour. The pDC24Δicd(13869)-icd(S.va) vector thus prepared was transformed into tryptophan-producing strain CM05-9157 by electroporation, followed by double crossover to obtain a strain in which the coding sequence of the icd gene from *Streptococcus monitori* replaced the coding sequence of the endogenous icd gene on the chromosome. The strain was identified by PCR and genome sequencing using primers SEQ ID NO: 13 and SEQ ID NO: 14, which can respectively amplify the outer regions of the upstream and downstream regions of homologous recombination at the site where the gene was replaced.

[0188] The strain obtained by the above method was named CM05-9157Δicd::icd(S.va).

[0189] Example 3. Evaluation of L-tryptophan production capacity of tryptophan-producing Corynebacterium spp. microorganisms with introduced exogenous NAD-dependent isocitrate dehydrogenase gene.

[0190] To test the L-tryptophan production capacity of the strains CM05-9157Δicd::icd(S.mu), CM05-9157Δicd::icd(S.ca), and CM05-9157Δicd::icd(S.va) prepared in Examples 2-2, 2-3, and 2-4, respectively, as well as the parental strain CM05-9157 without the introduction of exogenous genes, these strains were cultured in the following manner.

[0191] Each strain was inoculated into a 250-mL corner-baffled flask containing 25 mL of seed culture medium and incubated at 30°C with shaking at 200 rpm for 20 hours. Next, 1 mL of the seed culture was inoculated into a 250-mL corner-baffled flask containing 25 mL of production culture medium and incubated at 30°C with shaking at 200 rpm for 24 hours. After incubation, the L-tryptophan yield was measured by HPLC. The compositions of the seed and production media are shown below, and the L-tryptophan concentration in the media for each test strain is shown in Table 7.

[0192] Seed culture medium (pH 7.0)

[0193] 20 g glucose, 10 g peptone, 5 g yeast extract, 1.5 g urea, 4 g KH2PO4, 8 g K2HPO4, 0.5 g MgSO4 7H2O, 100 µg biotin, 1000 µg thiamine hydrochloride, 2000 µg calcium pantothenate, 2000 µg nicotinamide (based on 1 liter of distilled water).

[0194] <Production medium (pH 7.0)>

[0195] 30 g glucose, 15 g (NH4)2SO4, 1.2 g MgSO4 7H2O, 1 g KH2PO4, 5 g yeast extract, 900 µg biotin, 4500 µg thiamine hydrochloride, 4500 µg calcium pantothenate, 30 g CaCO3 (based on 1 liter of distilled water).

[0196] [Table 7]

[0197]

[0198] As shown in Table 7, the CM05-9157Δicd::icd(S.mu) strain, which incorporated a gene from *Streptococcus mutans*, produced a final L-tryptophan yield of 2.32 g / L in shake-flask culture, demonstrating a 26.6% increase in fermentation yield compared to the parental strain CM05-9157, which produced 1.83 g / L. In contrast, the CM05-9157Δicd::icd(S.ca) and CM05-9157Δicd::icd(S.va) strains, which incorporated genes from *Streptococcus guinea pig* and *Streptococcus monitor lizard*, respectively, produced 1.77 g / L and 1.82 g / L of L-tryptophan, respectively, representing a decrease in yield of approximately 3.4% and 0.9% compared to the parental strain CM05-9157.

[0199] These results indicate that L-tryptophan production capacity only increases significantly when the exogenous NAD-dependent isocitrate dehydrogenase gene from Streptococcus mutans is introduced into Corynebacterium microorganisms.

[0200] Example 4. Preparation of L-histidine-producing microorganisms infused with exogenous NAD-dependent isocitrate dehydrogenase

[0201] Example 4-1. Preparation of histidine-producing Corynebacterium microorganisms

[0202] To evaluate L-histidine production capacity, wild-type Corynebacterium glutamicum ATCC13032 was used as the starting microorganism to prepare strain CA14-0114 (in which genes in the histidine biosynthesis pathway were enhanced).

[0203] Specifically, to relieve feedback inhibition on the HisG protein (the first enzyme in the L-histidine biosynthesis pathway), the codon of the hisG gene was modified to produce a protein in which amino acids 233 and 235 from the N-terminus of HisG are simultaneously replaced by histidine (glycine) and glutamine (threonine), respectively (SEQ ID NO: 19) (ACS Synth.Biol., 2014, 3 (1), pp 21-29). Furthermore, to enhance the activity of the hisE gene, which resides in the same operon as hisG, the start codon was replaced by ATG. Additionally, to enhance the L-histidine biosynthesis pathway, the promoters of the biosynthetic genes hisN, hisH, hisD, hisA, and hisB were replaced with strong promoters, and the pathway was further enhanced by introducing additional copies of the hisE(g1a)G(G233H / T235Q) operon and the hisD gene.

[0204] Example 4-1-1. Preparation of histidine-producing microorganisms that relieve feedback inhibition

[0205] To prepare a histidine-producing strain in which feedback inhibition was relieved, PCR was performed using chromosomal DNA of *Corynebacterium glutamicum* ATCC13032 as a template and primer pairs of SEQ ID NO: 20 and SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23. Using the two amplified DNA fragments as templates and primers of SEQ ID NO: 20 and SEQ ID NO: 23, PCR was performed in the same manner as in Example 2 to obtain the 'hisE(g1a)G(G233H / T235Q)' gene fragment. Furthermore, PCR was performed using chromosomal DNA of ATCC13032 as a template and primers of SEQ ID NO: 71 and 72 to obtain the upstream region of the hisE gene.

[0206] [Table 8]

[0207]

[0208] To replace the strong promoter, PCR was performed using the synthetic promoter Pspl13 (SEQ ID NO: 24, Korean Patent No. 10-1783170) as a template and primers SEQ ID NO: 25 and SEQ ID NO: 26, in the same manner as in Example 1. After treating the pDC24 vector with the restriction endonuclease Sma1, the upstream DNA fragment of the hisE gene, the Pspl13 promoter, and the 'hisE(g1a)G(G233H / T235Q)' gene fragment were cloned using the Gibson assembly method to obtain a recombinant plasmid, named pDC24ΔPn_hisEG::Pspl13_hisE(g1a)G(G233H / T235Q). Gibson cloning was performed in the same manner as in Example 1.

[0209] The constructed pDC24ΔPn_hisEG::Pspl13_hisE(g1a)G(G233H / T235Q) vector was transformed into *Corynebacterium glutamicum* ATCC13032 via electroporation, followed by a two-stage exchange process to obtain the strain (where feedback inhibition was relieved by introducing a mutation into the existing hisG gene, and hisE activity was enhanced by replacing the start codon of the hisE gene). The corresponding genetic modifications were confirmed by PCR using primers SEQ ID NO: 27 and SEQ ID NO: 28 and genome sequencing. The strain thus obtained was named CJ-HIS1.

[0210] Example 4-1-2. Preparation of histidine-producing microorganisms with enhanced biosynthetic pathways via promoter substitution

[0211] Next, in order to enhance the activity of the biosynthetic genes hisN, hisH, hisD, hisA, and hisB, plasmids were prepared that replaced the wild-type promoters of each gene with stronger promoters.

[0212] Specifically, chromosomal DNA from Corynebacterium glutamicum ATCC13032 was used as a template, along with primers SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, and SEQ ID NO:37 and SEQ ID NO:38, and PCR was performed in the same manner as in Example 2. The upstream regions of the hisN, hisH, hisD, hisA, and hisB genes were obtained.

[0213] Furthermore, using chromosomal DNA from Corynebacterium glutamicum ATCC13032 as a template and primers from SEQ ID NO: 73 and SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78, SEQ ID NO: 79 and SEQ ID NO: 80, and SEQ ID NO: 81 and SEQ ID NO: 82, the downstream regions of the hisN, hisH, hisD, hisA, and hisB genes were obtained.

[0214] [Table 9]

[0215]

[0216] To replace the endogenous promoters of the hisN, hisH, and hisD genes with the strong promoter Pcj7 (SEQ ID NO: 39, patent number 10-0620092), genomic DNA of *Corynebacterium ammoniagenes* was used as a template, along with primers SEQ ID NO: 40 and SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 43, and SEQ ID NO: 83 and SEQ ID NO: 84, and PCR was performed in the same manner as in Example 2.

[0217] [Table 10]

[0218]

[0219] In addition, in order to replace the endogenous promoters of the hisA and hisB genes with the strong promoter Pspl13 promoter (SEQ ID NO: 24, Korean Patent No. 10-1783170), PCR was performed in the same manner as in Example 2 using Pspl13 promoter as a template and primers of SEQ ID NO: 44 and SEQ ID NO: 45, SEQ ID NO: 46 and SEQ ID NO: 85.

[0220] [Table 11]

[0221]

[0222] After treating the pDC24 vector with the restriction endonuclease Sma1, the upstream DNA fragments of the amplified hisN, hisH, and hisD genes, the Pcj7 promoter fragment, and the downstream DNA fragments of the hisN, hisH, and hisD genes were cloned using the Gibson assembly method to obtain recombinant plasmids, which were named pDC24ΔPn::Pcj7_hisN, pDC24ΔPn::Pcj7_hisH, and pDC24ΔPn::Pcj7_hisD, respectively. Similarly, after treating the pDC24 vector with the restriction endonuclease Sma1, the upstream DNA fragments of the amplified hisA and hisB genes, the Pspl13 promoter fragment, and the downstream DNA fragments of the hisA and hisB genes were cloned using the Gibson assembly method to obtain recombinant plasmids, which were named pDC24ΔPn::Pspl13_hisA and pDC24ΔPn::Pspl13_hisB, respectively. Gibson cloning was performed in the same manner as in Example 2.

[0223] The constructed pDC24ΔPn::Pcj7_hisN vector was transformed into CJ-HIS1 constructed in Example 4-1-1 via electroporation. A second exchange was performed to replace the promoter in the existing hisN gene, resulting in a strain with enhanced corresponding genes. The corresponding genetic modifications were confirmed by PCR using primers SEQ ID NO: 47 and SEQ ID NO: 48 and genome sequencing. The strain thus obtained was named CJ-HIS2.

[0224] Next, the constructed pDC24ΔPn::Pcj7_hisH vector was transformed into the CJ-HIS2 strain prepared above via electroporation. Through a second exchange, the promoter was replaced in the existing hisH gene, resulting in a strain with enhanced corresponding genes. The corresponding genetic modifications were confirmed by PCR using primers SEQ ID NO: 49 and SEQ ID NO: 50 and genome sequencing. The strain thus obtained was named CJ-HIS3.

[0225] Next, the constructed pDC24ΔPn::Pcj7_hisD vector was transformed into the CJ-HIS3 strain prepared above via electroporation. Through a second exchange, the promoter was replaced in the existing hisD gene, resulting in a strain with enhanced corresponding genes. The corresponding genetic modifications were confirmed by PCR using primers SEQ ID NO: 51 and SEQ ID NO: 52 and genome sequencing. The strain thus obtained was named CJ-HIS4.

[0226] Next, the constructed pDC24ΔPn::Pspl13_hisA vector was transformed into the CJ-HIS4 strain prepared above via electroporation. Through a second exchange, the promoter in the existing hisA gene was replaced, resulting in a strain with enhanced corresponding genes. The corresponding genetic modifications were confirmed by PCR using primers SEQ ID NO: 53 and SEQ ID NO: 54 and genome sequencing. The strain thus obtained was named CJ-HIS5.

[0227] Next, the constructed pDC24ΔPn::Pspl13_hisB vector was transformed into the CJ-HIS5 strain prepared above via electroporation. Through a second exchange, the promoter was replaced in the existing hisB gene, resulting in a strain with enhanced corresponding genes. The corresponding genetic modifications were confirmed by PCR using primers SEQ ID NO: 55 and SEQ ID NO: 56 and genome sequencing. The strain thus obtained was named CJ-HIS6.

[0228] [Table 12]

[0229]

[0230] Example 4-1-3. Preparation of histidine-producing microorganisms with enhanced biosynthetic pathways via additional gene insertion.

[0231] Next, the gene NCgl1021, known to encode transposons in Corynebacterium glutamicum, was used as the insertion site to additionally insert the hisE(g1a)G(G233H / T235Q) operon and the hisD gene. Specifically, to construct a vector for NCgl1021 deletion and target gene insertion, PCR was performed using the chromosome of ATCC13032 as a template and primer pairs SEQ ID NO: 57 and SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60, in the same manner as in Example 2, to amplify the left homologous arm region and the right homologous arm region of NCgl1021.

[0232] [Table 13]

[0233]

[0234] In the same manner as in Example 2, PCR was performed using the vector pDC24ΔPn ::Pspl13_hisE(g1a)G(G233H / T235Q) constructed in Example 4-1-1 as a template and primers of SEQ ID NO: 61 and SEQ ID NO: 62 to obtain the 'Pspl13_hisE(g1a)G(G233H / T235Q)' gene fragment. Furthermore, in the same manner as in Example 2, PCR was performed using the vector pDC24ΔPn ::Pcj7_hisD constructed in Example 4-1-2 as a template and primers of SEQ ID NO: 63 and SEQ ID NO: 64 to obtain the 'Pcj7_hisD' gene fragment.

[0235] [Table 14]

[0236]

[0237] After treating the pDC24 vector with the restriction endonuclease Sma1, the amplified left and right homologous arm regions of NCgl1021, as well as the gene fragments 'Pspl13_hisE(g1a)G(G233H / T235Q)' and 'Pcj7_hisD', were cloned using the Gibson assembly method to obtain a recombinant plasmid, which was named 'pDC24ΔNCgl1021::Pspl13_hisE(g1a)G(G233H / T235Q)-Pcj7_hisD'. Gibson cloning was performed in the same manner as in Example 2. The constructed vector 'pDC24-Pspl13_hisE(g1a)G(G233H / T235Q)-Pcj7_hisD' was transformed into CJ-HIS6 prepared in Example 4-1-2 via electroporation, followed by double crossover to obtain a strain in which the histidine biosynthesis pathway was enhanced by additional gene insertion. The corresponding genetic modifications were confirmed by PCR and genome sequencing using primers of SEQ ID NO: 65 and SEQ ID NO: 66. The strain thus obtained was named CA10-0114.

[0238] [Table 15]

[0239]

[0240] Example 4-2. Preparation of plasmids for gene replacement

[0241] A plasmid was constructed to introduce the NAD-dependent isocitrate dehydrogenase from *Streptococcus mutans*, which exhibited excellent tryptophan production capabilities as described in Example 3, into the chromosome of a histidine-producing *Corynebacterium glutamate* strain. Plasmid pDC24 was used as the parental vector to construct a plasmid capable of replacing the coding sequence of the endogenous icd gene with the coding sequence of the exogenous icd gene under the natural promoter.

[0242] Specifically, chromosomal DNA from *Corynebacterium glutamicum* ATCC13032 was used as a template. Primer pairs SEQ ID NO: 67 and SEQ ID NO: 68 were used to amplify the upstream region of the chromosome where homologous recombination occurred, and primer pairs SEQ ID NO: 69 and SEQ ID NO: 70 were used to amplify the downstream region. PCR was then performed to obtain the respective gene fragments. Solg... TM PCR was performed using Pfu-X DNA polymerase (SolGent co.) under the following conditions: denaturation at 95°C for 4 minutes, followed by 27 cycles of denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 50 seconds, and then polymerization at 72°C for 5 minutes. The primer sequences used are shown in Table 16 below.

[0243] [Table 16]

[0244]

[0245] The upstream and downstream segments of the region on the chromosome where homologous recombination occurred were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL.6 NO.5, MAY2009, NEBuilder high-fidelity DNA assembly premix) and the chromosome integration vector pDC24 digested with the smaI restriction enzyme to obtain the recombinant plasmid, which was named pDC24Δicd(13032).

[0246] Example 4-3. Preparation of histidine-producing Corynebacterium spp. microorganisms infused with NAD-dependent isocitrate dehydrogenase derived from Streptococcus mutans.

[0247] To introduce the gene encoding NAD-dependent isocitrate dehydrogenase (SEQ ID NO: 1) from *Streptococcus mutans* selected in Example 1 into a histidine-producing *Corynebacterium glutamicum* strain, PCR was performed using the *Streptococcus mutans* icd gene (SEQ ID NO: 2) as a template and primers SEQ ID NO: 11 and SEQ ID NO: 12 from Table 3, as described in Examples 2-2. Solg... TM Pfu-X DNA polymerase was used as the polymerase, and PCR was performed under the following PCR amplification conditions: 27 cycles of denaturation at 95°C for 2 minutes, followed by denaturation at 95°C for 20 seconds, annealing at 60°C for 40 seconds, and polymerization at 72°C for 1 minute, and then polymerization at 72°C for 5 minutes.

[0248] Subsequently, the NAD-dependent isocitrate dehydrogenase gene fragment from *Streptococcus mutans* and the chromosome integration vector pDC24Δicd(13032) digested with SmaI restriction enzyme constructed in Example 2-1 were cloned and amplified using the Gibson assembly method (DG Gibson et al., *NATURE METHODS*, VOL.6 NO.5, MAY 2009, NEBuilder high-fidelity DNA assembly premix) to obtain the recombinant plasmid, which was named pDC24Δicd(13032)-icd(S.mu). Cloning was performed by mixing the Gibson assembly reagent, gene fragment, and digested vector at an appropriate molar ratio and then incubating at 50°C for 1 hour.

[0249] The pDC24Δicd(13032)-icd(S.mu) vector, thus prepared, was transformed into the histidine-producing strain CA14-0114 prepared in Example 4-1 by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), followed by double crossover to obtain a strain in which the coding sequence of the endogenous icd gene on the chromosome was replaced by the coding sequence of the icd gene derived from Streptococcus mutans. The obtained strain was identified by PCR and genome sequencing using primers of SEQ ID NO: 13 and SEQ ID NO: 14 in Table 4, which can amplify the outer regions of the homologous recombination upstream and downstream regions at the site of gene replacement, respectively.

[0250] The strain obtained by the above method was named CA14-0114Δicd::icd(S.mu).

[0251] Example 5. Evaluation of L-histidine production capacity of Corynebacterium spp. microorganisms with introduced exogenous NAD-dependent isocitrate dehydrogenase gene.

[0252] To test the L-histidine production capacity of the CA14-0114Δicd::icd (S.mu) strain prepared in Example 4 and the parental strain CA14-0114 without the introduction of exogenous genes, these strains were cultured in the following manner.

[0253] Each strain was inoculated into a 250-mL baffled Erlenmeyer flask containing 25 mL of seed culture medium and incubated at 30°C with shaking at 200 rpm for 48 hours. Next, 1 mL of the seed culture was inoculated into a 250-mL baffled Erlenmeyer flask containing 25 mL of production culture medium and incubated at 30°C with shaking at 200 rpm for 24 hours. After incubation, the yield of L-histidine was measured by HPLC. The compositions of the seed and production media are shown below, and the L-histidine concentration in the culture medium for each test strain is shown in Table 17.

[0254] Seed culture medium (pH 7.0)

[0255] 5% glucose, 1% bacterial peptone, 0.25% sodium chloride, 1% yeast extract, 0.4% urea (based on 1 liter of distilled water)

[0256] <Production medium (pH 7.0)>

[0257] 6% raw sugar, 2% ammonium sulfate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 2.0% corn steep liquor (CSL), 200 μg / L biotin, 30 g / L calcium carbonate (based on 1 liter of distilled water)

[0258] [Table 17]

[0259]

[0260] As shown in Table 17, the CA14-0114Δicd::icd(S.mu) strain, which was infused with the NAD-dependent isocitrate dehydrogenase gene from Streptococcus mutans, produced a final 6.2 g / L of L-histidine in shake flask culture, demonstrating a fermentation yield increase of approximately 34.8% compared to the parental strain CA14-0114, which produced 4.6 g / L.

[0261] These results indicate that introducing the NAD-dependent isocitrate dehydrogenase gene from Streptococcus mutans into Corynebacterium microorganisms not only increased L-tryptophan production capacity as demonstrated in Example 3, but also increased L-histidine production capacity.

[0262] Based on the foregoing description, those skilled in the art will understand that this disclosure can be implemented in different specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the above embodiments are not restrictive but illustrative in all respects. The scope of this disclosure is defined by the appended claims and not by the description that follows them; therefore, all variations and modifications falling within the boundaries and scope of the claims, or equivalents of such boundaries and scope, are included in the claims.

Claims

1. A Corynebacterium genus microorganism capable of producing L-amino acids, wherein an NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans or a polynucleotide encoding the same is introduced.

2. The Corynebacterium genus microorganism according to claim 1, wherein the L-amino acid is any one or more selected from the group consisting of L-tryptophan and L-histidine.

3. The Corynebacterium genus microorganism according to claim 1, wherein the NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80% or more sequence identity with it.

4. The Corynebacterium genus microorganism according to claim 1, wherein the NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans is encoded by the icd gene.

5. The Corynebacterium microorganism according to claim 1, wherein the polynucleotide encoding the NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans comprises the nucleotide sequence of SEQ ID NO:

2.

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

7. The Corynebacterium genus microorganism according to any one of claims 1 to 6, wherein the Corynebacterium genus microorganism has an increased ability to produce L-amino acids compared with unmodified microorganisms.

8. A method for producing L-amino acids, the method comprising the step of culturing a Corynebacterium microorganism capable of producing L-amino acids in a culture medium, wherein the microorganism is introduced with an NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans or a polynucleotide encoding therethe.

9. The method according to claim 8, wherein the L-amino acid is any one or more selected from the group consisting of L-tryptophan and L-histidine.

10. The method of claim 8, further comprising the step of recovering L-amino acids from cultured microorganisms, cultures of said microorganisms, fermentation products of said microorganisms, or culture media.

11. A composition for producing L-amino acids, the composition comprising a Corynebacterium spp. microorganism capable of producing L-amino acids, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof, wherein the microorganism is introduced with an NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans or a polynucleotide encoding thereas.

12. The composition according to claim 11, wherein the L-amino acid is any one or more selected from the group consisting of L-tryptophan and L-histidine.

13. Use of a Corynebacterium genus microorganism capable of producing L-amino acids in the production of L-amino acids, wherein the microorganism is introduced with an NAD-dependent isocitrate dehydrogenase protein derived from Streptococcus mutans or a polynucleotide encoding therethe.

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