Microorganism for producing L-tryptophan and method for producing L-tryptophan using same

Abstract

The present disclosure relates to: a Corynebacterium genus microorganism having an L-tryptophan-producing ability, into which a pyruvate-phosphate dikinase derived from foal foal xylose or a polynucleotide encoding the same is introduced; a method for producing L-tryptophan, comprising a step of culturing the microorganism in a culture medium; a composition for producing L-tryptophan comprising the microorganism, a culture product of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof; and the use of said microorganism for the production of L-tryptophan.

Description

Technical Field

[0001] This disclosure relates to a Corynebacterium microorganism capable of producing L-tryptophan, wherein a pyruvate-phosphate dikinase derived from Komagataeibacter xylinus or a polynucleotide encoding therefrom is introduced; a method for producing L-tryptophan, comprising culturing the microorganism in a culture medium; a composition for producing L-tryptophan comprising the microorganism, a culture product of the microorganism, a fermentation product of the microorganism, or a combination of two or more thereof; and the use of the microorganism for producing L-tryptophan. Background Technology

[0002] Various studies on the production of target substances (e.g., amino acids) in microorganisms have involved eco-friendly and safe production methods, with continuous research focusing on the large-scale production of target substances in Corynebacterium sp. microorganisms. Corynebacterium sp., especially Corynebacterium glutamicum, are Gram-positive microorganisms commonly used for the production of L-amino acids and other useful substances. Various studies are underway to develop microorganisms and fermentation processes with high-efficiency production for the production of L-amino acids and other useful substances.

[0003] L-Tryptophan is an essential amino acid that has been widely used as a raw material in pharmaceutical products such as feed additives and infusion solutions, as well as a component of health foods. Therefore, L-Tryptophan can be produced through chemical synthesis, enzymatic reactions, and fermentation, but currently, the primary method used is direct fermentation using microorganisms.

[0004] Microorganisms possess an aromatic biosynthetic pathway, in which phosphoenolpyruvate (PEP), an intermediate product of glycolysis, and erythrose-4-phosphate (E4P), a product of the pentose phosphate pathway, are polymerized in the biosynthesis of L-tryptophan via 3-deoxy-D-arabinohepenolate 7-phosphate (DAHP) synthase (EC 2.5.1.54). Previous studies have shown that, based on intracellular quantitative analysis, tryptophan biosynthesis requires the highest energy level among 20 amino acids (Proc. Natl. Acad. Sci. USA, (2002) V 99, pp 3695-3700).

[0005] Therefore, to ensure a stable supply of E4P, methods to increase biosynthesis by enhancing the expression of the tktA gene (NCBI gene ID: 12931960) encoding transketolase (EC 2.2.1.1) have been investigated (Current Opinion in Biotechnology, (2009) V20, pp 651-658). Furthermore, to maintain intracellular energy levels, research is underway to reduce the use of the high-energy substance ATP (FEMS Microbiol Lett, (2009) V297, pp 217-224).

[0006] However, gluconeogenesis is generally known to have low activity in nutrient-rich culture environments (J Bacteriol. 2013 Sep, 195(18), 4283-4296.; Nature Communications Vol. 8, Article No.: 14316, 2017). Furthermore, there is a problem that the amount of tryptophan precursor phosphoenolpyruvate is reduced due to the production of byproducts such as acetic acid during L-tryptophan production, thereby decreasing L-tryptophan yield.

[0007] Therefore, research on effectively improving L-tryptophan production capacity remains necessary. Summary of the Invention

[0008] Technical issues

[0009] The inventors have demonstrated that L-tryptophan production capacity is increased in Corynebacterium microorganisms in which pyruvate-phosphokinase derived from Xylospora xylose or its polynucleotide is introduced, thereby completing this disclosure.

[0010] Technical solution

[0011] One object of this disclosure is to provide a Corynebacterium microorganism with L-tryptophan production capacity, wherein a pyruvate-phosphokinase derived from Xylose-derived Corynebacterium or a polynucleotide encoding the same is introduced.

[0012] In one embodiment, the pyruvate-phosphokinase derived from *Xylostella xylospora* may include the amino acid sequence of SEQ ID NO:1.

[0013] In another implementation, the pyruvate phosphate dual kinase derived from *Xylostella xylospora* can be encoded by the ppdK gene.

[0014] As a microorganism according to any of the above embodiments, the Corynebacterium genus microorganism may be Corynebacterium glutamicum.

[0015] As a microorganism according to any of the above embodiments, the Corynebacterium genus microorganism may have an increased L-tryptophan production capacity compared to unmodified microorganisms.

[0016] Another object of this disclosure is to provide a method for producing L-tryptophan, which includes culturing a Corynebacterium microorganism capable of producing L-tryptophan in a culture medium wherein a pyruvate-phosphokinase derived from Xylose-Coccobacillus or a polynucleotide encoding the same is introduced therein.

[0017] In one embodiment, the method may further include recovering L-tryptophan from cultured microorganisms, culture products of said microorganisms, fermentation products of said microorganisms, or culture media.

[0018] Another object of this disclosure is to provide a composition for producing L-tryptophan, comprising: a Corynebacterium microorganism capable of producing L-tryptophan, wherein a pyruvate-phosphokinase derived from *Xylostella spp.* or a polynucleotide encoding therefrom is introduced; a culture product of said microorganism; a fermentation product of said microorganism; or a combination of two or more thereof.

[0019] Another object of this disclosure is to provide the use of a composition for the production of L-tryptophan, said composition comprising a Corynebacterium genus microorganism capable of L-tryptophan production, wherein a pyruvate-phosphokinase derived from *Xylostella spp.* or a polynucleotide encoding therefrom is introduced; a culture product of said microorganism; a fermentation product of said microorganism; or a combination of two or more thereof.

[0020] Beneficial effects

[0021] The present disclosure provides a Corynebacterium genus microorganism capable of producing L-tryptophan, incorporating pyruvate-phosphokinase derived from Xylospora xylose or its encoded polynucleotide, which can produce L-tryptophan in high yield and is therefore suitable for industrial production of L-tryptophan. Detailed Implementation

[0022] This disclosure will now be described in detail. Furthermore, each description and embodiment disclosed herein can 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 by the specific descriptions below. In addition, numerous papers and patent documents are cited throughout this specification. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety and will more clearly describe the level of the technical field to which this disclosure pertains and the content of this disclosure.

[0023] definition

[0024] As used in the specification and appended claims, the singular forms (“a”, “an”, and “the”) include indicators of the plural, unless the context clearly indicates otherwise. Unless the context clearly indicates otherwise, singular terms shall include the plural, and plural terms shall include the singular. As used in the specification and appended claims, unless otherwise indicated, the use of “or” may be used to include “and / or”.

[0025] 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. In one instance, 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 value, but is not limited to these.

[0026] As used herein, descriptions such as “first, second, third…”, “i), ii), iii)…” or “(a), (b), (c), (d)…” can be used to distinguish similar compositions. When these terms are used to refer to steps of a method, use, or determination, these terms do not imply that the steps are 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, randomly, or in reverse order, with intervals of seconds, minutes, hours, days, or months.

[0027] 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".

[0028] 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.

[0029] As used herein, the term "comprising / including" means the presence of the features, steps, ingredients, or other components listed after the term, and does not exclude the presence or addition of one or more features, steps, ingredients, or other components. The features, steps, ingredients, or other components listed after the term "comprising / including" herein may be necessary or mandatory. However, in some embodiments, the term may also include any other or non-essential features, steps, ingredients, or other components.

[0030] Proteins, polypeptides

[0031] As used herein, the terms "protein" or "polypeptide" refer to a polymer or oligomer of consecutive amino acid residues. In this disclosure, "polypeptide," "protein," and "peptide" are used interchangeably.

[0032] In some cases, the term "active amino acid sequence" can refer to "polypeptide," "protein," or "peptide," and when "polypeptide," "protein," "peptide," or "active amino acid sequence" has catalytic activity, it can be called an "enzyme."

[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, although they are described as "comprising / including" polypeptides or proteins with amino acid sequences described by a specific sequence number, "consisting of" amino acid sequences described by a specific sequence number, or "having" amino acid sequences described by a specific sequence number, it is apparent that any polypeptide or protein having an amino acid sequence in which a portion of the sequence is deleted, modified, substituted, conservatively substituted, or added, may fall within the scope of this invention if 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, it may include polypeptides or proteins having sequence additions or deletions that do not alter protein function, naturally occurring mutations, their silencing mutations, or conservative substitutions within or upstream of the polypeptide or protein sequence (N-terminus or C-terminus), provided that they have the same or corresponding activity as the polypeptide or protein.

[0036] For example, a polypeptide or protein that can be co-translated or post-translated with an N-terminal signal (or leader) sequence involved in protein (peptide) translocation, or that can be co-translated or co-translated with another sequence or linker to identify, purify or synthesize a polypeptide or protein, or that falls within the range of polypeptides or proteins whose amino acid sequences are described by a specific 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. Amino acids can be classified into the following groups:

[0038] In one example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In another example, amino acids with charged side chains (charged amino acids) include arginine, lysine, histidine, glutamic acid, and aspartic acid; and amino acids with uncharged side chains (uncharged amino acids; also called neutral amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In another example, aromatic amino acids include phenylalanine, tryptophan, and tyrosine. In another example, branched-chain amino acids include valine, leucine, and isoleucine. In yet another example, the 20 amino acids can be divided into five groups based on their size, starting with the relatively small group: 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, the classification of amino acids is not limited to these examples. Generally, conserved substitutions have little or no effect on the activity of polypeptides or proteins.

[0039] Genes, polynucleotides

[0040] As used herein, the term "gene" refers to a polynucleotide encoding a functional molecule and a polynucleotide including upstream and downstream regions of the polynucleotide, or functional RNA. In some embodiments, a gene may have sequences (introns) inserted between the various coding regions (exons).

[0041] As used herein, the terms "polynucleotide," "nucleic acid," or "nucleic acid molecule" refer to a polymer of nucleotide monomers linked together by covalent bonds to form a long chain, which is a DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA) chain having at least a certain length. In this disclosure, "polynucleotide," "nucleic acid," or "nucleic acid molecule" are used interchangeably.

[0042] Identity, homology

[0043] As used herein, the term “identity” or “homology” refers to the degree of correlation between two given amino acid sequences or nucleotide sequences and may be expressed as a percentage. The terms “homology” and “identity” are generally used interchangeably.

[0044] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard alignment algorithms, and can be used together with default gap penalties established by the program used.

[0045] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined by known computer algorithms (such as the “FASTA” program) using the default parameters in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444. Alternatively, it can be determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), which uses the following execution: Needleman program (version 5.0.0 or later) in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (GCG package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF et al., J MOLECBIOL 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). For example, homology, similarity, or identity can be determined by using BLAST or ClustalW from the National Center for Biotechnology Information.

[0046] Furthermore, whether any two polynucleotide sequences are homologous, similar, or identical can be determined by comparing the sequences under defined stringent conditions using Southern hybridization experiments, and the appropriate hybridization conditions to be defined can be determined by methods within the scope of this disclosure, which are known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York), but are not limited thereto. Essentially, homologous or identical polynucleotide sequences are generally expected to hybridize under stringent conditions with all or at least about 50%, 60%, 70%, 80%, or 90% of the full sequence length.

[0047] As used herein, “strict conditions” refer to conditions that enable specific hybridization between polynucleotides. Such conditions are described in detail in the literature (see Sambrook et al., above, 9.50-9.51, 11.7-11.8). For example, stringent conditions may 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 may include typical Southern hybridization washing conditions, i.e., washing once, especially twice or three times, at a salt concentration and temperature corresponding 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.

[0048] Hybridization requires that the two nucleotides have complementary sequences, although mismatches between bases are possible due to the strictness of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, this disclosure may also include isolated nucleic acid fragments complementary to the whole sequence, as well as base sequences substantially similar to them.

[0049] For example, T can be used m The hybridization conditions, with a value of 55°C, were used to detect polynucleotides homologous or identical to those disclosed herein under the conditions described above. Furthermore, T... m The value can be 60°C, 63°C or 65°C, but is not limited to these, and can be appropriately adjusted by those skilled in the art for their purposes.

[0050] 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., J. Sambrook et al., above).

[0051] Nucleic acid constructs, vectors, and transformation

[0052] As used herein, the term “nucleic acid construct” refers to a single-stranded or double-stranded nucleic acid molecule that includes one or more regulatory sequences and is artificially synthesized or modified to contain specific sequences in a manner not found in nature or isolated from nature.

[0053] As used herein, the term "vector" refers to a DNA construct used to deliver a target polynucleotide into a suitable host or host cell. In one instance, a vector may contain a nucleotide sequence of a polynucleotide encoding a target polypeptide that is operatively linked to a suitable expression regulatory region (expression regulatory sequence) to enable expression of the target polypeptide in a suitable host cell, but is not limited thereto.

[0054] The expression regulatory region can contain a promoter capable of initiating transcription, any operon sequence regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences regulating transcription and translation termination. Once transformed into a suitable host cell (microorganism), the vector can replicate or function independently of the host genome, or it can integrate into its genome.

[0055] There are no particular limitations on the vectors used in this disclosure, and any vector known in the art may be used. Examples of commonly used vectors 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; those based on pDZ, pDC, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. In one example, pDZ, pDC, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, and other vectors may be used.

[0056] In one example, the target polynucleotide can be inserted into a chromosome using a vector for intracellular chromosome insertion. Insertion of the polynucleotide into the chromosome can be performed by any method known in the art, such as homologous recombination, but not limited to this. The vector may also contain a selection marker to confirm insertion into the chromosome. The selection marker is used to select cells transformed by the vector, i.e., to confirm the insertion of the target polynucleotide, and may use a marker that provides a selectable phenotype (such as drug resistance, auxotrophic phenotype, cytotoxic agent resistance, or surface peptide expression). Only cells expressing the selection marker are able to survive or exhibit a different phenotype under conditions treated with a selection agent, thus allowing for the selection of transformed cells.

[0057] As used herein, the term "transformation" refers to the introduction of a vector containing a target polynucleotide into a host cell (microorganism) to alter the genetic characteristics of the host cell (microorganism). The transformed polynucleotide may be integrated into and located within or outside the chromosome of the host cell (microorganism). Furthermore, the polynucleotide may include DNA and / or RNA. Depending on the purpose of the introduction, the polynucleotide may be introduced in a suitable form. For example, a polynucleotide for expressing a target polypeptide may 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 may typically contain a promoter, transcription terminator, ribosome binding site, or translation terminator operatively linked to the polynucleotide. Expression cassettes may be in the form of a self-replicating expression vector. Furthermore, the polynucleotide may be introduced into the host cell (microorganism) as is and operatively linked to the sequence required for expression in the host cell, but is not limited thereto.

[0058] As used herein, the term "operably linked" refers to a construct that places a regulatory sequence in the appropriate position to regulate the expression of a coding sequence. Therefore, the term "operably linked" includes attachment or connection between a regulatory region (such as a promoter, stop codon, 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 according to known or desired activity. For example, it 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, it may contain a nucleotide sequence of a polynucleotide encoding a target polypeptide, which is operatively linked to a suitable expression regulatory region (expression regulatory sequence) to enable expression of the target polypeptide in a suitable host cell.

[0061] As used herein, the term "regulatory sequence" refers to a polynucleotide sequence essential for the expression of a target polynucleotide sequence. Each regulatory sequence can be native (from the same source) or exogenous (from a different gene) to the coding sequence, or it can be a mutant sequence or other artificial sequence. Examples of regulatory sequences can include leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, operon sequences, sequences encoding ribosome binding sites, and sequences regulating the termination of 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 elements that make up a gene, such as DNA or RNA, are altered from their original sequence during disassembly and recombination.

[0063] As used herein, the term "recombinant gene" refers to a gene with a novel genomic structure 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. In one instance, a recombinant gene may include an artificial combination of nucleic acid fragments not found in nature, such as regulatory sequences.

[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 prokaryotic or eukaryotic microorganisms that have undergone natural or artificial genetic modifications, 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 include genetically modified microorganisms that produce desired polypeptides, proteins, or products. In this disclosure, "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 pyruvate phosphate dual kinase or a polynucleotide encoding therethe is introduced (e.g., recombinant microorganisms), but are not limited thereto.

[0069] As used herein, the term "microorganism with L-tryptophan production capacity" refers to a microorganism capable of producing L-tryptophan in an organism, and may include all microorganisms conferred with L-tryptophan production capacity that do not possess endogenous L-tryptophan production capacity, or microorganisms with endogenous L-tryptophan production capacity. L-tryptophan production capacity can be conferred or enhanced through species modification.

[0070] As used herein, the term "unmodified microorganism (strain)" does not exclude microorganisms (strains) that may contain naturally occurring mutations, and may refer to wild-type microorganisms (strains) or naturally occurring microorganisms (strains) themselves, or microorganisms (strains) before their traits are altered due to genetic modifications caused by natural or artificial factors. As used herein, "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, the term may refer to microorganisms (strains) in which pyruvate phosphate dual kinase or the polynucleotide encoding it has not been introduced, or microorganisms (strains) before such 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 increase in the activity of a protein (peptide) in a host cell (microorganism) compared to its endogenous activity. Increase may 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: either the protein (peptide) activity is not endogenously present in the host cell (microorganism), or the protein (peptide) activity is enhanced compared to its endogenous or unmodified activity.

[0074] For example, a description of "exhibiting protein (peptide) activity that is not endogenously present" or exhibiting enhanced protein (peptide) activity may be caused by "the introduction of protein (peptide)," but is not limited to this.

[0075] As used herein, the term "introduction" of a protein (peptide) refers to the expression of a gene not originally present in a microorganism, thereby causing the microorganism to exhibit the activity of that specific protein, or the activity of the peptide to be enhanced, increased, or elevated compared to the endogenous activity or unmodified activity of the corresponding protein. This can, for example, be caused by introducing a gene encoding a protein (peptide) into a host cell (microorganism). For instance, this could involve introducing a polynucleotide encoding a specific protein (peptide) into the chromosome of a host cell (microorganism), or introducing a vector containing a polynucleotide encoding a specific protein (peptide) into a host cell (microorganism), thereby exhibiting or enhancing that activity.

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

[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 peptide originally present in the untransformed host cell (microorganism) or the unmodified host cell (microorganism).

[0078] In one instance, an increase can mean exhibiting the activity of a corresponding protein (peptide) that was not originally expressed, or its activity or concentration is typically based on an increase of 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] Enhanced protein (peptide) activity can be achieved by introducing exogenous proteins (peptides) or increasing the activity of endogenous proteins (peptides). Whether protein (peptide) activity is enhanced can be confirmed by the activity level of the corresponding protein (peptide), its expression level, or by an increase in the amount of product produced by the corresponding protein (peptide).

[0080] Enhancing protein (peptide) activity can be achieved through various methods well known in the art, provided that it enhances the activity of the target protein (peptide) compared to the host cell (microbe) before modification. Specifically, genetic engineering and / or protein engineering, well known to those skilled in the art, are conventional methods in molecular biology, but are not limited to these methods (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 enhancement of the protein (peptide) of this disclosure can be achieved through the following:

[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., induce modifications within the expression regulatory regions, replace with more active sequences, or insert more active sequences).

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

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

[0086] 5) Modifying the polynucleotide sequence encoding a protein (peptide) enhances the protein (peptide) activity (e.g., modifying the polynucleotide sequence encoding a gene to encode the modified protein (peptide) to enhance the protein (peptide) activity);

[0087] 6) Introduce exogenous proteins (peptides) that exhibit protein (peptide) activity or exogenous polynucleotides encoding them;

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

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

[0090] 9) Selected from two or more of the above 1) to 8), but not particularly limited thereto.

[0091] For example,

[0092] 1) Increasing the intracellular copy number of polynucleotides encoding proteins (peptides) can be achieved by introducing a vector containing a polynucleotide encoding a 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 a polynucleotide encoding a 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 performed by introducing a vector capable of inserting the polynucleotide into the host cell (microorganism) chromosome, but is not limited to this. 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).

[0093] 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 to the sequence through deletion, insertion, substitution, or a combination thereof to further enhance the activity of the expression regulatory region, or by replacing the sequence with a more active sequence. The expression regulatory region may include, but is not particularly limited to, promoters, operon sequences, sequences encoding ribosome binding sites, and sequences regulating transcription and translation termination. In one example, the method may include replacing the original promoter with a strong promoter, but is not limited to this.

[0094] Examples of known strong promoters include the cj1 to cj7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, λ phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13(sm3) promoter (US10584338 B2), O2 promoter (US10273491 B2), tkt promoter, yccA promoter, etc., but strong promoters are not limited to these.

[0095] 3) Modifying the nucleotide sequence or 5'-UTR of a gene encoding a protein (peptide) can be achieved, for example, by modifying the nucleotide sequence to encode another start codon (which has a higher protein (peptide) expression rate compared to the endogenous start codon) or an RBS (ribosome binding site) sequence (which has a higher protein (peptide) expression rate compared to the endogenous RBS sequence), but is not limited thereto.

[0096] 4) and 5) Modification of the amino acid sequence or polynucleotide sequence of a protein (peptide) can be achieved by introducing modifications to the sequence through deletion, insertion, substitution, or a combination thereof of the amino acid sequence of the protein (peptide) or the polynucleotide sequence encoding the protein (peptide), to enhance the activity of the protein (peptide), or by replacing the sequence with a modified amino acid sequence or polynucleotide sequence to increase activity, but is not limited thereto. For example, substitution can be achieved by inserting polynucleotides into the chromosome through homologous recombination, but is not limited thereto.

[0097] 6) The method of introducing exogenous polynucleotides exhibiting protein (peptide) activity can be achieved by introducing exogenous polynucleotides encoding proteins (peptides) that exhibit the same / similar activity as the protein (peptide) into host cells (microorganisms). Exogenous polynucleotides can be used without restriction, regardless of their source or sequence, as long as they exhibit the same / similar activity as the protein (peptide). The introduction can be carried out by a transformation method known in the art, appropriately selected by a person skilled in the art, and the expression of the introduced polynucleotide in the host cells can produce the protein (peptide), thereby enhancing its activity.

[0098] 7) Codon optimization of polynucleotides encoding proteins (peptides) can be achieved by: optimizing the codons of endogenous polynucleotides to increase transcription or translation within the host cell (microorganism), or by optimizing the codons to enable optimized transcription and translation of exogenous polynucleotides within the host cell (microorganism).

[0099] 8) Methods for analyzing the tertiary structure of proteins (peptides) and thereby selecting and modifying exposed sites or chemically modifying them can be achieved by, for example, comparing the sequence information of the protein (peptide) to be analyzed with a database storing known protein sequence information to determine template protein candidates based on the degree of 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.

[0100] 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 cell (microorganism), or an increase in the amount of product produced by the corresponding protein (peptide), but is not limited thereto.

[0101] The modification of some or all polynucleotides in microorganisms disclosed herein can be achieved by: (a) homologous recombination of vectors used for chromosome insertion in microorganisms or genome editing using engineered nucleases (e.g., CRISPR-Cas9), and / or (b) induction by light such as ultraviolet light and irradiation and / or chemical treatment, but not limited thereto.

[0102] nourish

[0103] 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. Such a culture process can be readily adapted for use by those skilled in the art based on the strain to be selected. Specifically, the culture can be a batch culture, a continuous culture, and / or a fed-batch culture, but is not limited thereto.

[0104] As used herein, the term "culture medium" refers to a mixture of substances containing nutrients required for the cultivation of microorganisms as its main component, 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 routine microbial culture, without any particular limitation. For example, the microorganisms of this disclosure can be cultured under aerobic conditions in a conventional culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while adjusting temperature, pH, etc. For example, culture media for Corynebacterium spp. can be found in the literature ["Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981)].

[0105] 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. Furthermore, carbon sources may include natural organic nutrients such as starch hydrolysate, molasses, saccharin paste, rice bran, cassava, cane molasses, and corn steep liquor. Specifically, carbohydrates such as glucose and aseptically pretreated molasses (i.e., molasses converted to reducing sugars) may be used. In addition, various other carbon sources may be used in appropriate amounts without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0106] 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 decomposition products, and defatted soybean meal or its decomposition products. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0107] Phosphorus sources may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or their corresponding sodium-containing salts. Examples of inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, and calcium carbonate. 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 these phosphorus sources are not limited to these methods.

[0108] Furthermore, during the cultivation of the microorganisms disclosed herein in an appropriate manner, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid. Additionally, antifoaming agents such as fatty acid polyethylene glycol esters can be used during cultivation to prevent bubble formation. Furthermore, oxygen or oxygen-containing gases can be injected into the culture medium to maintain aerobic conditions; or nitrogen, hydrogen, or carbon dioxide can be injected, or no gas can be injected, to maintain anaerobic or microaerophilic conditions, but the gases are not limited to these.

[0109] The temperature during the cultivation process disclosed herein can be in the range of 20°C to 45°C, specifically 25°C to 40°C, and the cultivation can last from 10 hours to 160 hours, but is not limited thereto.

[0110] As used herein, the term "culture product" means a culture medium, concentrated culture medium, dried culture medium, culture filtrate, concentrated culture filtrate, or dried culture filtrate obtained by culturing a specific microorganism in a culture medium, and means that the culture medium may contain the specific microorganism, while the culture filtrate substantially does not contain the specific microorganism (in particular, 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, emulsion, or solid.

[0111] As used in this article, the term "fermentation" refers to the process by which microorganisms break down organic matter using their own enzymes without spoiling it. Fermentation and putrefaction occur through similar processes, but when the decomposition produces useful substances, it is called fermentation, while when it produces odorous or harmful substances, it is called putrefaction.

[0112] In this disclosure, there are no particular limitations on the methods for obtaining fermentation products from microorganisms, and the products can be obtained according to methods commonly used in the art or similar fields.

[0113] As used herein, the term "fermentation product" can include not only the fermented 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 products of fermentation products, filtrate of fermentation products, filtrate of concentrated fermentation products, dried products of filtrate of fermentation products, extracts of fermentation products, or diluted solutions of fermentation products.

[0114] Detailed description of this disclosure

[0115] The embodiments of this disclosure will be described in detail below:

[0116] One aspect of this disclosure provides a Corynebacterium microorganism capable of producing L-tryptophan, wherein a pyruvate-phosphokinase or a polynucleotide encoding it is introduced from Xylose-derived Corynebacterium.

[0117] As used herein, the term "pyruvate phosphate dual kinase (PPDK)" refers to a catalyst... This is an enzyme in the family of reaction transferases. The pyruvate-phosphokinase disclosed herein can be used interchangeably with PPDK. Specifically, the pyruvate-phosphokinase disclosed herein can be a protein having pyruvate-phosphokinase activity encoded by the ppdk gene, but its type is not particularly limited, as long as the protein has the corresponding pyruvate-phosphokinase activity. The pyruvate-phosphokinase encoded by the ppdk gene is known in the art, and the amino acid and polynucleotide sequences of pyruvate-phosphokinase can be obtained from known databases such as NCBI's GenBank, but are not limited thereto.

[0118] In one instance, a pyruvate-phosphokinase derived from *Xylostella xylostella* may include, but is not limited to, the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having 60% or more homology or identity with it, as long as it possesses pyruvate-phosphokinase activity. Specifically, any protein having a partially deleted, modified, substituted, or added amino acid sequence from SEQ ID NO:1, as long as it exhibits the corresponding activity of pyruvate-phosphokinase, falls within the scope of pyruvate-phosphokinase. Furthermore, any protein having or including an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with SEQ ID NO:1, composed of or substantially composed of the aforementioned amino acid sequence, and exhibiting the corresponding activity of pyruvate-phosphokinase, falls within the scope of pyruvate-phosphokinase.

[0119] Furthermore, the polynucleotide sequence encoding pyruvate-phosphokinase derived from *Xylostella spp.* having the amino acid sequence of SEQ ID NO:1 or having 60% or more homology or identity with the amino acid sequence, can be obtained, for example, based on codon information known in the art. In one example, pyruvate-phosphokinase can be encoded by a polynucleotide that may have or include 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, composed of, or substantially composed of, the aforementioned nucleotide sequences, but 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.

[0120] In this disclosure, a polynucleotide (gene) containing the nucleotide sequence of SEQ ID NO:2 can 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 ppdk.

[0121] Due to codon degeneracy or considering preferred codons in organisms expressing the pyruvate-phosphokinase of this disclosure, the polynucleotides of this disclosure can be modified in various ways in the coding region without altering the amino acid sequence of the pyruvate-phosphokinase of this disclosure. Therefore, based on codon degeneracy, it is also evident that polynucleotides may include polypeptides that can be translated into a polypeptide consisting of the amino acid sequence of the pyruvate-phosphokinase of this disclosure, or polypeptides that are homologous to or identical with it. For example, the polynucleotides of this disclosure may be SEQ ID NO:2, or its degenerated sequence.

[0122] In another instance, the polynucleotide of this disclosure may have or include 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 SEQ ID NO:2, or may consist of or substantially consist of 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 SEQ ID NO:2, but is not limited thereto.

[0123] Furthermore, the polynucleotides disclosed herein may include probes that can be prepared from known gene sequences, such as any sequence that can hybridize under stringent conditions with all or part of the complementary sequence of the polynucleotide sequence disclosed herein to encode the pyruvate phosphate dual kinase of the present disclosure, without limitation.

[0124] For the purposes of this disclosure, the microorganisms of this disclosure may include all microorganisms capable of producing desired L-tryptophan by introducing pyruvate-phosphokinase or a polynucleotide encoding it. For example, the microorganisms of this disclosure are characterized by the introduction of pyruvate-phosphokinase activity, thereby increasing L-tryptophan production capacity, and may be genetically modified microorganisms or recombinant microorganisms, but are not limited thereto. Specifically, recombinant strains with increased L-tryptophan production capacity may be microorganisms with increased L-tryptophan production capacity compared to natural wild-type microorganisms, or unmodified microorganisms with or without endogenous pyruvate-phosphokinase activity, but are not limited thereto.

[0125] In one instance, a microorganism capable of producing L-tryptophan (which is a prokaryotic or eukaryotic microbial strain capable of producing L-tryptophan in an organism) may include all endogenously L-tryptophan-producing microorganisms, or microorganisms in which the L-tryptophan-producing ability has been conferred upon a parent strain that is not capable of L-tryptophan production by the activity of pyruvate-phosphokinase introduced in this disclosure. L-tryptophan production capacity may be conferred or enhanced through species modification.

[0126] The microorganisms disclosed herein may include all microorganisms that have been introduced with pyruvate phosphate dual kinase or the polynucleotide encoding it by various known methods.

[0127] In one instance, recombinant microorganisms capable of producing L-tryptophan may include all microorganisms that can be transformed by a vector and are therefore capable of producing L-tryptophan by introducing a foreign gene encoding the pyruvate-phosphate dual kinase disclosed herein (particularly a foreign gene encoding the pyruvate-phosphate dual kinase derived from Xylose-1,000).

[0128] For example, the microorganisms used to produce L-tryptophan can be microorganisms that have introduced a protein encoding an amino acid sequence containing SEQ ID NO:1, or a polynucleotide sequence 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 SEQ ID NO:1.

[0129] For example, the microorganisms used to produce L-tryptophan can be microorganisms that have been introduced with a polynucleotide 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 a polynucleotide 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.

[0130] In one instance, the microorganism with increased L-tryptophan production capacity disclosed herein may be a microorganism with increased L-tryptophan production capacity compared to an unmodified microorganism, but is not limited thereto. In one instance, the unmodified microorganism (which is the target strain used to compare the increased L-tryptophan production capacity) may be strain CM05-9157, but is not limited thereto.

[0131] In one instance, a microorganism with increased L-tryptophan production capacity may have an increase of 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 16% or more, about 17% or more, or about 18% 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, about 35% or less, about 30% or less, about 25% or less, or about 20% or less) of L-tryptophan production capacity compared to the production capacity of the parent microorganism (parent strain) or the unmodified microorganism, but is not limited thereto, as long as there is a positive increase compared to the production capacity of the parent microorganism (parent strain) or the unmodified microorganism. In another instance, a microorganism with increased L-tryptophan production capacity may have an increased L-tryptophan production capacity of approximately 1.1 times or more, approximately 1.15 times or more, approximately 1.16 times or more, approximately 1.17 times or more, or approximately 1.18 times or more (with no particular upper limit, e.g., approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, approximately 2 times or less, approximately 1.5 times or less, approximately 1.4 times or less, approximately 1.3 times or less, or approximately 1.2 times or less) compared to the L-tryptophan production capacity of the parent microorganism before modification (parental strain) or the unmodified microorganism, but is not limited thereto.

[0132] In one instance, the microorganism capable of producing L-tryptophan can be a prokaryotic or eukaryotic cell, but particularly a prokaryotic cell. Prokaryotic cells can include, for example, microorganisms belonging to the genera *Escherichia*, *Erwinia*, *Serratia*, *Providencia*, *Corynebacterium*, *Pseudomonas*, *Leptospira*, *Salmonella*, *Brevibacteria*, *Hypomononas*, *Chromobacterium*, and *Norcardia*, or fungi or yeasts, but are not limited thereto. Specifically, it can be a microorganism belonging to the genera *Escherichia*, *Corynebacterium*, *Leptospira*, and yeasts. More specifically, it can be a microorganism belonging to the genus *Corynebacterium*.

[0133] As a microorganism according to any of the above embodiments, the microorganism disclosed herein may be a Corynebacterium genus microorganism.

[0134] In one instance, the microorganisms disclosed herein 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.

[0135] Specifically, the microorganisms disclosed herein may be Corynebacterium genus, more specifically, Corynebacterium glutamicum, but are not limited thereto.

[0136] Meanwhile, the Corynebacterium genus microorganisms with L-tryptophan production capacity disclosed herein may include all of the following: naturally wild-type microorganisms themselves; Corynebacterium genus microorganisms in which the activity of genes related to the L-tryptophan production mechanism is increased or decreased, thereby having enhanced L-tryptophan production capacity; or Corynebacterium genus microorganisms in which the activity of exogenous genes is introduced or increased, thereby having enhanced L-tryptophan production capacity.

[0137] Another aspect of this disclosure provides a method for producing L-tryptophan, comprising culturing a Corynebacterium genus microorganism capable of producing L-tryptophan in a culture medium wherein a pyruvate-phosphokinase derived from *Xylostella spp.* or a polynucleotide encoding the pyruvate-phosphokinase is introduced therein.

[0138] In the methods disclosed herein, the microorganisms can be cultured using any culture conditions and methods known in the art. This culture process can be readily adapted by those skilled in the art to suit the specific microorganisms to be selected.

[0139] L-tryptophan produced by the culture method disclosed herein can be released into the culture medium or retained in the cells.

[0140] In one embodiment, the method for producing L-tryptophan disclosed herein may further include the steps of preparing the microorganisms of the present disclosure, preparing a culture medium for culturing the strains, or a combination thereof (in any order, regardless of the sequence), for example prior to the culturing step.

[0141] The method for producing L-tryptophan disclosed herein may further include a step of recovering the desired substance, particularly L-tryptophan, from cultured microorganisms, microbial culture products, microbial fermentation products, or culture media. A recovery step may also be included after the culturing step.

[0142] In the recovery step, the methods for culturing the microorganisms disclosed herein can be used, for example, according to batch culture, continuous culture, or fed-batch culture methods, using suitable methods known in the art to collect the desired L-tryptophan. For example, methods such as centrifugation, filtration, treatment with a protein crystallizing precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC, or combinations thereof, can be used, and suitable methods known in the art can be used to recover the desired substance, particularly L-tryptophan, from the culture medium or microorganisms.

[0143] Furthermore, the method for producing L-tryptophan disclosed herein may also include a purification step, which may be performed using suitable methods known in the art. In one example, when the method for producing L-tryptophan disclosed herein includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or intermittently without regard to the order, or they may be performed simultaneously, or they may be integrated into a single step, but the method is not limited thereto.

[0144] In the methods disclosed herein, the pyruvate phosphate dual kinase, delivery, and L-tryptophan, etc., are as described in other aspects above.

[0145] Another aspect of this disclosure provides a composition for producing L-tryptophan, comprising: a Corynebacterium microorganism capable of producing L-tryptophan, wherein a pyruvate-phosphokinase derived from *Xylostella spp.* or a polynucleotide encoding therefrom is introduced; a culture product of said microorganism; a fermentation product of said microorganism; or a combination of two or more thereof.

[0146] The compositions disclosed herein may also contain any suitable excipients commonly used in compositions for the production of L-tryptophan, and such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or isotonic agents.

[0147] In one embodiment, the various components present in the compositions of this disclosure may be included in a microbiologically effective amount or in an amount that may be suitably present in the compositions used for production.

[0148] In the compositions disclosed herein, the pyruvate phosphate dual kinase, delivery, and L-tryptophan, etc., are as described in other aspects above.

[0149] Another aspect of this disclosure provides the use of a Corynebacterium microorganism capable of producing L-tryptophan, wherein a pyruvate-phosphokinase derived from Xylospora xylose or a polynucleotide encoding the same is introduced.

[0150] In the purposes of this disclosure, the pyruvate phosphate dual kinase, delivery, and L-tryptophan, etc., are as described in other aspects above.

[0151] Mode of implementing the present invention

[0152] The present disclosure will now be described in detail by way of embodiments. However, these embodiments are merely preferred embodiments given for illustrative purposes, and therefore, the scope of the present disclosure is not intended to be limited to or restricted by these embodiments. Furthermore, those skilled in the art or similar fields will fully understand and readily implement technical features not described herein.

[0153] Example 1: Screening and selection of phosphoenolpyruvate synthase (ppsA) and pyruvate phosphate dual kinase (ppdK) genes

[0154] To select phosphoenolpyruvate synthase or pyruvate and pyruvate phosphate dual kinase with high sugar heteroactivity, an acetic acid bacteria and methanogens were searched based on literature review results, using carbon sources consisting of 1 to 3 carbons. Considering the biosafety level and availability suitable for production strains, four types of microorganisms expected to possess phosphoenolpyruvate synthase or pyruvate and pyruvate phosphate dual kinase were selected, as shown in Table 1 below.

[0155] [Table 1]

[0156]

[0157] Example 2. Preparation of L-tryptophan-producing microorganisms infused with exogenous pyruvate-phosphokinase dual kinase

[0158] Example 2-1. Preparation of plasmids for inserting genes

[0159] Plasmids were prepared to introduce the genes for pyruvate-phosphokinase dual kinase or phosphoenolpyruvate synthase into the transposon gene region of the chromosome of Corynebacterium glutamicum via homologous recombination.

[0160] Specifically, using Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template, PCR was performed using primer pairs SEQ ID NO:11 and 12 and primer pairs SEQ ID NO:13 and 14 to obtain the corresponding fragments. Solg... TM Pfu-X DNA polymerase was used for PCR, and PCR was performed 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.

[0161] [Table 2]

[0162]

[0163] The recombinant plasmid was obtained by cloning two fragments amplified by PCR and the pDCM2 vector (Korean Patent No. 10-2278000) digested by SmaI restriction enzyme using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, May 2009, NEBuilder HiFi DNA Assembly Master Mix), and named pDCM2-△Tn.

[0164] Example 2-2. Preparation of Corynebacterium microorganisms infused with pyruvate-phosphokinase derived from Xylospora xylose

[0165] To introduce the ppdK gene (NZ_CP024644.1, SEQ ID NO:2) encoding pyruvate-phosphokinase derived from *K. xylinus* into *Corynebacterium glutamicum*, firstly, based on the *K. xylinus* ppdK gene (SEQ ID NO:2) synthesized using the gene synthesis service of Bionix Co., Ltd., PCR was performed using the primer pair of SEQ ID NO:15 and 16 to amplify the ppdK gene.

[0166] Use Solg TM Pfu-X DNA polymerase was used as the polymerase, and PCR was performed under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of 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.

[0167] [Table 3]

[0168]

[0169] Furthermore, to obtain the Pcj7 promoter, PCR was performed using primers SEQ ID NO:17 and SEQ ID NO:18, based on p117-cj7-gfp (US 7662943 B2) as a template. Solg... TM Pfu-X DNA polymerase (SolGentco.) was used as the polymerase, and PCR was performed under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of denaturation at 95°C for 20 seconds, annealing at 60°C for 40 seconds, and polymerization at 72°C for 30 seconds, and then polymerization at 72°C for 5 minutes.

[0170] [Table 4]

[0171]

[0172] Subsequently, the amplified *Xylostella xylostella* ppdK gene, Pcj7 promoter region, and pDCM2-ΔTn prepared in Example 2-1 by ScaI restriction enzyme digestion were cloned using the Gibson assembly method (DG Gibson et al., *NATURE METHODS*, VOL. 6 NO. 5, May 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid, named pDCM2-ΔTn::Pcj7-ppdK(K.xy). Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in calculated molar amounts and then incubating at 50°C for 1 hour.

[0173] The pDCM2-△Tn::Pcj7-ppdK(K.xy) vector prepared therefrom was transformed into tryptophan-producing strain CM05-9157 (Korean Patent No. 10-2278000) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), followed by a secondary crossover to obtain a strain in which a single copy of the Pcj7-ppdK(K.xy) gene was inserted between transposon genes on the chromosome. The obtained strain was identified by genome sequencing and PCR using primers SEQ ID NO:19 and SEQ ID NO:20, which amplify the outer regions of the upstream and downstream regions of the gene insertion site, respectively.

[0174] [Table 5]

[0175]

[0176]

[0177] The strain obtained was named CM05-9157::Pcj7-ppdK(K.xy).

[0178] Examples 2-3. Preparation of Corynebacterium microorganisms infused with pyruvate-phosphokinase derived from Acetobacter pasteurellii subsp.

[0179] To introduce the gene (NZ_CP021922.1, SEQ ID NO:4) encoding pyruvate-phosphokinase (SEQ ID NO:3) from *Acetobacter pasteurellii* subsp. *pasteurella* into *Corynebacterium glutamicum*, firstly, based on the ppdK gene (SEQ ID NO:4) of *Acetobacter pasteurellii* subsp. *pasteurella* synthesized using the gene synthesis service of Bionix Co., Ltd. as a template, PCR was performed using the primer pair of SEQ ID NO:21 and 22 to amplify the ppdK gene.

[0180] Use Solg TM Pfu-X DNA polymerase was used as the polymerase, and PCR was performed under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of 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.

[0181] [Table 6]

[0182]

[0183] Furthermore, to obtain the Pcj7 promoter, PCR was performed using primers from SEQ ID NO:17 and SEQ ID NO:23, based on p117-cj7-gfp (US 7662943 B2) as a template. Solg... TM Pfu-X DNA polymerase (SolGentco.) was used as the polymerase, and PCR was performed under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of denaturation at 95°C for 20 seconds, annealing at 60°C for 40 seconds, and polymerization at 72°C for 30 seconds, and then polymerization at 72°C for 5 minutes.

[0184] [Table 7]

[0185]

[0186] Subsequently, the amplified ppdK gene, Pcj7 promoter region, and pDCM2-ΔTn prepared in Example 2-1 by ScaI restriction enzyme digestion were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, May 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid, named pDCM2-ΔTn::Pcj7-ppdK(A.pa). Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in calculated molar amounts and then incubating at 50°C for 1 hour.

[0187] Subsequently, the pDCM2-△Tn::Pcj7-ppdK(A.pa) vector prepared therefrom was transformed into tryptophan-producing strain CM05-9157 (Korean Patent No. 10-2278000) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), followed by a second exchange to obtain a strain in which a single copy of the Pcj7-ppdK(A.pa) gene was inserted between transposon genes on the chromosome. The obtained strain was identified by genome sequencing and PCR using primers SEQ ID NO:19 and SEQ ID NO:20, which amplify the outer regions of the upstream and downstream regions of the gene insertion site, respectively.

[0188] The strain obtained was named CM05-9157::Pcj7-ppdK(A.pa).

[0189] Examples 2-4. Preparation of Corynebacterium spp. microorganisms infused with pyruvate-phosphokinase derived from *Dystrophococcus acetophilus* C2A.

[0190] To introduce the gene (NZ_AE010299.1, SEQ ID NO:6) encoding pyruvate-phosphokinase (SEQ ID NO:5) derived from *Dystrophus acetophilus* C2A into *Corynebacterium glutamicum*, firstly, based on the ppdK gene of *Dystrophus acetophilus* C2A (SEQ ID NO:6) synthesized using the gene synthesis service of Bionix Co., Ltd., PCR was performed using primer pairs SEQ ID NO:24 and 25 to amplify the ppdK gene.

[0191] Use Solg TM Pfu-X DNA polymerase was used as the polymerase, and PCR was performed under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of 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.

[0192] [Table 8]

[0193]

[0194] Furthermore, to obtain the Pcj7 promoter, PCR was performed using primers from SEQ ID NO:17 and SEQ ID NO:26, based on p117-cj7-gfp (US 7662943 B2) as a template. Solg... TMPfu-X DNA polymerase (SolGentco.) was used as the polymerase, and PCR was performed under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of denaturation at 95°C for 20 seconds, annealing at 60°C for 40 seconds, and polymerization at 72°C for 30 seconds, and then polymerization at 72°C for 5 minutes.

[0195] [Table 9]

[0196]

[0197] Subsequently, the amplified ppdK gene, Pcj7 promoter region, and pDCM2-ΔTn prepared in Example 2-1 by ScaI restriction enzyme digestion were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, May 2009, NEBuilder HiFi DNAAssembly Master Mix). This yielded a recombinant plasmid named pDCM2-ΔTn::Pcj7-ppdK(M.ac). Cloning was performed by mixing the Gibson assembly reagent and the gene fragments in calculated molar amounts and then incubating at 50°C for 1 hour.

[0198] Subsequently, the pDCM2-△Tn::Pcj7-ppdK(M.ac) vector prepared therefrom was transformed into tryptophan-producing strain CM05-9157 (Korean Patent No. 10-2278000) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), followed by a second exchange to obtain a strain in which a single copy of the Pcj7-ppdK(M.ac) gene was inserted between transposon genes on the chromosome. The obtained strain was identified by genome sequencing and PCR using primers SEQ ID NO:19 and SEQ ID NO:20, which amplify the outer regions of the upstream and downstream regions of the gene insertion site, respectively.

[0199] The strain obtained was named CM05-9157::Pcj7-ppdK(M.ac).

[0200] Examples 2-5. Preparation of Corynebacterium spp. microorganisms infused with pyruvate-phosphokinase derived from Escherichia coli

[0201] To introduce ppsA (NZ_CP084899.1, SEQ ID NO:8), which encodes phosphoenolpyruvate synthase (SEQ ID NO:7) from Escherichia coli (an ortholog of the ppdk gene), into Corynebacterium glutamicum, PCR was first performed using primers SEQ ID NO:27 and 28 as a template based on E. coli W3110 chromosomal DNA to amplify the ppsA gene.

[0202] Use Solg TM Pfu-X DNA polymerase was used as the polymerase, and PCR was performed under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of 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.

[0203] [Table 10]

[0204]

[0205] Furthermore, to obtain the Pcj7 promoter, PCR was performed using primers from SEQ ID NO:17 and SEQ ID NO:29, based on p117-cj7-gfp (US 7662943 B2) as a template. Solg... TM Pfu-X DNA polymerase (SolGentco.) was used as the polymerase, and PCR was performed under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of denaturation at 95°C for 20 seconds, annealing at 60°C for 40 seconds, and polymerization at 72°C for 30 seconds, and then polymerization at 72°C for 5 minutes.

[0206] [Table 11]

[0207]

[0208] Subsequently, the amplified *E. coli* ppsA gene, Pcj7 promoter region, and pDCM2-ΔTn prepared in Example 2-1 by ScaI restriction enzyme were cloned using the Gibson assembly method (DG Gibson et al., *NATURE METHODS*, VOL. 6 NO. 5, May 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid, named pDCM2-ΔTn::Pcj7-ppsA(E.co). Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in calculated molar amounts and then incubating at 50°C for 1 hour.

[0209] Subsequently, the pDCM2-△Tn::Pcj7-ppsA(E.co) vector prepared therefrom was transformed into tryptophan-producing strain CM05-9157 (Korean Patent No. 10-2278000) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), followed by a second exchange to obtain a strain in which a single copy of the Pcj7-ppsA(E.co) gene was inserted between transposon genes on the chromosome. The obtained strain was identified by genome sequencing and PCR using primers SEQ ID NO:19 and SEQ ID NO:20, which amplify the outer regions of the upstream and downstream regions of the gene insertion site, respectively.

[0210] The strain obtained was named CM05-9157::Pcj7-ppsA(E.co).

[0211] Examples 2-6. Preparation of Corynebacterium microorganisms infused with pyruvate-phosphokinase derived from Corynebacterium glutamicum ATCC13869

[0212] To introduce ppsA (NZ_CP016335.1, SEQ ID NO:10), which encodes phosphoenolpyruvate synthase (SEQ ID NO:9) derived from Corynebacterium glutamicum ATCC13869 (and is an ortholog of the ppdk gene), into Corynebacterium glutamicum, PCR was first performed using primers SEQ ID NO:30 and 31, based on Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template, to amplify the ppsA gene.

[0213] Use Solg TM Pfu-X DNA polymerase was used as the polymerase, and PCR was performed under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of 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.

[0214] [Table 12]

[0215]

[0216] Furthermore, to obtain the Pcj7 promoter, PCR was performed using primers SEQ ID NO:17 and SEQ ID NO:32, based on p117-cj7-gfp (US 7662943 B2) as a template. Solg... TMPfu-X DNA polymerase (SolGentco.) was used as the polymerase, and PCR was performed under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of denaturation at 95°C for 20 seconds, annealing at 60°C for 40 seconds, and polymerization at 72°C for 30 seconds, and then polymerization at 72°C for 5 minutes.

[0217] [Table 13]

[0218]

[0219]

[0220] Subsequently, the ppsA gene, Pcj7 promoter region, and pDCM2-ΔTn prepared in Example 2-1 and digested with ScaI restriction enzyme from Corynebacterium glutamicum ATCC13869, which were amplified thereby, were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, May 2009, NEBuilder HiFi DNA Assembly Master Mix). This yielded a recombinant plasmid named pDCM2-ΔTn::Pcj7-ppsA(C.gl). Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in calculated molar amounts and then incubating at 50°C for 1 hour.

[0221] Subsequently, the pDCM2-△Tn::Pcj7-ppsA(C.gl) vector prepared therefrom was transformed into tryptophan-producing strain CM05-9157 (Korean Patent No. 10-2278000) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), followed by a second exchange to obtain a strain in which a single copy of the Pcj7-ppsA(C.gl) gene was inserted between transposon genes on the chromosome. The obtained strain was identified by genome sequencing and PCR using primers SEQ ID NO:19 and SEQ ID NO:20, which amplify the outer regions of the upstream and downstream regions of the gene insertion site, respectively.

[0222] The strain obtained was named CM05-9157::Pcj7-ppsA(C.gl).

[0223] Example 3. Evaluation of the L-tryptophan production capacity of L-tryptophan-producing microorganisms introduced with exogenous pyruvate phosphate dual kinase or its ortholog.

[0224] To verify the L-tryptophan production capacity of the strains CM05-9157::Pcj7-ppdK(K.xy), CM05-9157::Pcj7-ppdK(A.pa), CM05-9157::Pcj7-ppdK(M.ac), and CM05-9157::Pcj7-ppsA(E.co) prepared in Examples 2-2, 2-3, 2-4, and 2-5, respectively, the parental strain CM05-9157 without exogenous gene introduction, and CM05-9157::Pcj7-ppsA(C.gl) prepared in Examples 2-6, these strains were cultured and evaluated in the following manner.

[0225] Each strain was inoculated into a 250 mL corner-baffled flask containing less than 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 less than 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 culture medium for each test strain is shown in Table 14.

[0226] Seed culture medium (pH 7.0)

[0227] 20g glucose, 10g peptone, 5g yeast extract, 1.5g urea, 4g KH2PO4, 8g K2HPO4, 0.5g MgSO47H2O, 100μg biotin, 1000μg thiamine hydrochloride, 2000μg calcium pantothenate, 2000μg nicotinamide (per liter of distilled water).

[0228] <Production medium (pH 7.0)>

[0229] 30g glucose, 15g (NH4)2SO4, 1.2g MgSO4 7H2O, 1g KH2PO4, 5g yeast extract, 900μg biotin, 4500μg thiamine hydrochloride, 4500μg calcium pantothenate, 30g CaCO3 (per liter of distilled water).

[0230] [Table 14]

[0231] OD562 Tryptophan production (g / L) Tryptophan yield (*100g / g, %) CM05-9157 56.5 1.88 6.37 CM05-9157::Pcj7-ppsA(C.gl) 56.3 1.89 6.38 CM05-9157::Pcj7ppsA(E.co) 56.1 1.9 6.40 CM05-9157::Pcj7-ppdK(A.pa) 56.8 1.85 6.34 CM05-9157::Pcj7-ppdK(K.xy) 52.1 2.35 7.52 CM05-9157::Pcj7-ppdK(M.ac) 57.8 1.64 5.51

[0232] As shown in Table 14 above, the results confirmed that the tryptophan production of the CM05-9157::Pcj7-ppsA(C.gl) strain, which was introduced with the phosphoenolpyruvate synthase gene from Corynebacterium glutamicum, was 1.89 g / L, which was almost the same as that of the parent strain CM05-9157.

[0233] Furthermore, among pyruvate-phosphokinase genes or phosphoenolpyruvate synthase genes derived from various microorganisms (which are orthologs of the pyruvate-phosphokinase gene), only the CM05-9157::Pcj7-ppdK(K.xy) strain, which incorporated the pyruvate-phosphokinase gene from *Xylostella spp.*, ultimately produced 2.35 g / L of L-tryptophan in shake-flask culture. This L-tryptophan production capacity increased by approximately 18% compared to the parental strains CM05-9157 and / or CM05-9157::Pcj7-ppsA(C.gl).

[0234] In contrast, three strains (CM05-9157::Pcj7-ppdK(A.pa), CM05-9157::Pcj7-ppdK(M.ac), and CM05-9157::Pcj7-ppsA(E.co)) introduced with exogenous phosphoenolpyruvate synthase or pyruvate phosphokinase other than *C. xylose* showed a slight decrease in tryptophan production compared to the control group CM05-9157, or a relatively insignificant increase in tryptophan production.

[0235] These results indicate that L-tryptophan production can only be specifically increased when a specific pyruvate-phosphokinase gene derived from *Xylostella xylospora* is introduced into *Corynebacterium* microorganisms.

[0236] Based on the foregoing, those skilled in the art to which this disclosure pertains will understand that this disclosure may be implemented in other specific forms without modifying the technical concept or essential features of this disclosure. In this regard, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of this disclosure. Rather, this disclosure is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalent substitutions, and other embodiments that may be included within the spirit and scope of this disclosure as defined by the appended claims.

Claims

1. A Corynebacterium microorganism capable of producing L-tryptophan, wherein a pyruvate-phosphokinase or a polynucleotide encoding it is introduced from Xylose-derived Corynebacterium.

2. The microorganism according to claim 1, wherein the pyruvate-phosphokinase derived from *Xylostella xylostella* comprises the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 90% identity with SEQ ID NO:

1.

3. The microorganism according to claim 1, wherein the pyruvate phosphate dual kinase derived from *Xylostella xylostella* is encoded by the ppdK gene.

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

5. The microorganism according to any one of claims 1 to 4, wherein the Corynebacterium genus microorganism has an increased L-tryptophan production capacity compared with unmodified microorganisms.

6. A method for producing L-tryptophan, comprising culturing a Corynebacterium microorganism capable of producing L-tryptophan in a culture medium wherein a pyruvate-phosphokinase derived from *Xylostella spp.* or a polynucleotide encoding the same is introduced therein.

7. The method of claim 6 further comprises recovering L-tryptophan from the cultured microorganism, the culture product of the microorganism, the fermentation product of the microorganism, or the culture medium.

8. A composition for producing L-tryptophan, comprising: a Corynebacterium microorganism capable of producing L-tryptophan, wherein a pyruvate-phosphokinase derived from *Xylostella spp.* or a polynucleotide encoding therefrom is introduced; a culture product of said microorganism; a fermentation product of said microorganism; or a combination of two or more thereof.

9. Use of the composition for the production of L-tryptophan, the composition comprising: a Corynebacterium microorganism capable of producing L-tryptophan, wherein a pyruvate-phosphokinase derived from *Xylostella spp.* or a polynucleotide encoding therefrom is introduced; a culture product of said microorganism; a fermentation product of said microorganism; or a combination of two or more thereof.

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