L-tryptophan-producing microorganism and method for producing L-tryptophan using the same

Abstract

The present application relates to a Corynebacterium microorganism capable of producing L-tryptophan, into which a pyruvate phosphate dikinase protein derived from Komagataibacter xylinus or a polynucleotide encoding the same has been introduced; a method for producing L-tryptophan, which comprises culturing the microorganism in a medium; a composition for producing L-tryptophan, which comprises the microorganism, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more of these; and use of the microorganism for producing L-tryptophan.

Description

[Technical Field]

[0001] The present application relates to a Corynebacterium microorganism capable of producing L-tryptophan, into which a pyruvate phosphate dikinase protein derived from Komagataibacter xylinus or a polynucleotide encoding the same has been introduced; a method for producing L-tryptophan, which comprises culturing the microorganism in a medium; a composition for producing L-tryptophan, which comprises the microorganism, a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more of these; and use of the microorganism for producing L-tryptophan. [Background technology]

[0002] The production of target substances (e.g., amino acids) using microorganisms has been the subject of extensive research as an environmentally friendly and safe production method, and continuous research has been conducted into the mass production of target substances from microorganisms of the genus Corynebacterium. Microorganisms of the genus Corynebacterium, particularly Corynebacterium glutamicum, are Gram-positive microorganisms that are widely used to produce L-amino acids and other useful substances. To produce these L-amino acids and other useful substances, extensive research is being conducted to develop highly efficient production microorganisms and fermentation process technologies.

[0003] L-tryptophan is an essential amino acid and has been widely used as a feed additive, a pharmaceutical ingredient for infusions, and a health food ingredient. L-tryptophan can be produced by chemical synthesis, enzymatic reaction, fermentation, etc., but currently, direct fermentation using microorganisms is the most widely used method.

[0004] Microorganisms possess an aromatic biosynthetic pathway for L-tryptophan biosynthesis, in which phosphoenol pyruvate (PEP), an intermediate in glycolysis, and erythrose-4-phosphate (E4P), a product of the pentose phosphate pathway, are polymerized by DAHP synthase (3-Deoxy-D-arabinoheptulosonate 7-phosphate synthase, EC 2.5.1.54). Previous studies have demonstrated that tryptophan biosynthesis requires the highest level of energy among the 20 amino acids through quantitative intracellular analysis (Proc. Natl. Acad. Sci. USA, (2002) V99, pp. 3695-3700).

[0005] Therefore, in order to stably supply the precursor E4P, methods are being researched to increase biosynthesis by strengthening the expression of the tktA gene (NCBI gene ID: 12931960), which encodes transketolase (EC 2.2.1.1) (Current Opinion in Biotechnology, (2009) V20, pp651-658). Research is also underway to reduce the use of ATP, a high-energy substance, to maintain intracellular energy levels (FEMS Microbiol Lett, (2009) V297, pp217-224).

[0006] However, it is generally known that the activity of glucose synthesis (gluconeogenesis) is extremely low in nutrient-rich culture environments (J Bacteriol. 2013 Sep, 195(18), 4283-4296.; Nature Communications volume 8, Article number: 14316, 2017). Furthermore, during the L-tryptophan production process, the production of by-products such as acetic acid reduces the amount of phosphoenolpyruvate, a tryptophan precursor, resulting in reduced L-tryptophan production.

[0007] Therefore, research into effective increases in L-tryptophan productivity is still needed. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 7,662,943 [Patent Document 2] U.S. Patent No. 10,584,338 [Patent Document 3] U.S. Patent No. 10,273,491 [Patent Document 4] Korean Patent Registration No. 10-2278000 [Non-patent literature]

[0009] [Non-Patent Document 1] Proc. Natl. Acad. Sci. USA,(2002) V99,pp3695-3700 [Non-patent document 2] Current Opinion in Biotechnology,(2009) V20,pp651-658 [Non-patent document 3] FEMS Microbiol Lett,(2009)V297,pp217-224 [Non-patent document 4] J Bacteriol. 2013 Sep, 195(18), 4283-4296.;Nature Communications volume 8,Article number:14316,2017 [Non-Patent Document 5] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444 [Non-patent document 6] Rice et al., 2000, Trends Genet. 16:276-277

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[0010] The present inventors completed the present application by confirming that the L-tryptophan production ability of Corynebacterium microorganisms into which the pyruvate, phosphate dikinase protein (PPDK) derived from Komagataibacter xylinus or a polynucleotide encoding it has been introduced is increased. [Means for solving the problem]

[0011] One aspect of the present application provides a Corynebacterium microorganism capable of producing L-tryptophan, into which a pyruvate phosphate dikinase protein derived from Komagataibacter xylinus or a polynucleotide encoding the same has been introduced.

[0012] In one embodiment, the pyruvate phosphate dikinase from Komagataebacter xylinus may comprise the amino acid sequence of SEQ ID NO:1.

[0013] In another embodiment, the pyruvate phosphate dikinase from Komagataebacter xylinus may be encoded by the ppdK gene.

[0014] In any one of the above-mentioned embodiments, the Corynebacterium microorganism may be Corynebacterium glutamicum.

[0015] In any one of the above-mentioned embodiments, the Corynebacterium microorganism may have an increased ability to produce L-tryptophan compared to a non-modified microorganism.

[0016] Another aspect of the present application provides a method for producing L-tryptophan, comprising culturing in a medium a Corynebacterium microorganism capable of producing L-tryptophan, into which a pyruvate phosphate dikinase protein derived from Komagataibacter xylinus or a polynucleotide encoding the same has been introduced.

[0017] In one embodiment, the method may further comprise the step of recovering L-tryptophan from the cultured microorganism, the culture of the microorganism, the fermentation product of the microorganism, or the culture medium.

[0018] Another aspect of the present application provides a composition for producing L-tryptophan, comprising: a Corynebacterium microorganism capable of producing L-tryptophan, into which a pyruvate phosphate dikinase protein derived from Komagataibacter xylinus or a polynucleotide encoding the same has been introduced; a culture of the microorganism; a fermentation product of the microorganism; or a combination of two or more of these.

[0019] Another aspect of the present application provides a Corynebacterium microorganism capable of producing L-tryptophan, into which a pyruvate phosphate dikinase protein derived from Komagataibacter xylinus or a polynucleotide encoding the same has been introduced; and use of a composition comprising a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more of these, for the production of L-tryptophan. [Effects of the Invention]

[0020] A Corynebacterium microorganism capable of producing L-tryptophan, into which the pyruvate phosphate dikinase protein derived from Komagataebacter xylinus or a polynucleotide encoding it of the present application has been introduced, can produce L-tryptophan with a high yield and can be usefully utilized in the industrial production of L-tryptophan. DETAILED DESCRIPTION OF THE INVENTION

[0021] This will be explained in more detail as follows: Meanwhile, each description and embodiment disclosed in this application also applies to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the specific descriptions set forth below are not deemed to limit the category of this application. Furthermore, numerous papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the state of the art to which this application pertains and the contents of this application.

[0022] definition As used in this specification and the appended claims, the singular articles "a," "an," and "the" include plural referents unless otherwise stated. Also, singular terms include plurals and plural terms include the singular, unless otherwise stated. Also, in this specification and the appended claims, the use of "or" is intended to include "and / or" unless otherwise stated.

[0023] In this application, the term "about" may be used before a specific numerical value. As used in this application, the term "about" includes not only the exact number listed after the term, but also approximately that number or a range close to that number. Whether a number is close to or approximately the specific number mentioned can be determined by considering the context in which the number is provided. As an example, the term "about" may refer to a range of 10% to +10% of the numerical value. As another example, the term "about" may refer to a range of -5% to +5% of the given numerical value. However, the present invention is not limited thereto.

[0024] In this application, terms such as "first, second, third...", "i), ii), iii)...", or "(a), (b), (c), (d)..." are used to distinguish between components. When used in connection with steps of a method, use, or analysis, these terms do not imply sequential or orderly execution; for example, the steps may be performed without any time interval between them, simultaneously, sequentially, reversely, or randomly, with intervals of seconds, minutes, hours, days, or months between them.

[0025] In this application, the term "consisting of" means that the percentage of the specified features, steps, components, or other components listed after the term totals 100%. The features, steps, components, or other components listed after the term "consisting of" may be essential or mandatory. For example, any other features, steps, components, or other components, or non-essential features, steps, components, or other components, other than the features, steps, components, or other components that follow "consisting of," are excluded.

[0026] In this application, the term "consisting essentially of" means that one or more unspecified features, steps, components, or other components may be present when the features, steps, components, or other components of the subject matter claimed in this application are not substantially affected by the presence of the unspecified features, steps, components, or other components.

[0027] In this application, the term "comprising" means the presence of a feature, step, component, or other element described below that term, but does not exclude the presence of one or more additional features, steps, components, or other elements. In this application, a feature, step, component, or other element described below as "comprising" may be essential or mandatory, but some embodiments may further include other optional or non-essential features, steps, components, or other elements.

[0028] Proteins, polypeptides As used herein, the term "protein" or "polypeptide" refers to a polymer or oligomer of consecutive amino acid residues. In this application, "polypeptide," "protein," and "peptide" are used interchangeably.

[0029] In some cases, the term "active amino acid sequence" refers to the "polypeptide," "protein," or "peptide," and when the "polypeptide," "protein," "peptide," or "active amino acid sequence" has catalytic activity, it is referred to as an "enzyme."

[0030] As used herein, the term "mature polypeptide or mature protein" refers to a polypeptide or protein in a form that lacks a signal sequence or propeptide sequence. A mature polypeptide or mature protein may be a functional form of a polypeptide or protein. A mature polypeptide or mature protein refers to a polypeptide in its final form after translation and / or posttranslational modification. Examples of posttranslational modification include, but are not limited to, N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and removal of a leader sequence.

[0031] In this application, amino acid sequences are written in the N-terminal to C-terminal direction unless otherwise indicated.

[0032] In the present application, in relation to amino acid sequences, a polypeptide or protein "comprising" an amino acid sequence set forth in a particular SEQ ID NO, a polypeptide or protein "consisting of" an amino acid sequence set forth in a particular SEQ ID NO, or a polypeptide or protein "having" an amino acid sequence set forth in a particular SEQ ID NO, is understood to include polypeptides or proteins in which some amino acid(s) have been deleted, modified, substituted, conservatively substituted, or added, so long as they have the same or equivalent activity as a polypeptide or protein consisting of the amino acid sequence of the SEQ ID NO. For example, the polypeptides or proteins may also include polypeptides or proteins with additions or deletions of sequences within or before or after (at the N-terminus or C-terminus) the polypeptide or protein sequence that do not change the function of the protein, naturally occurring mutations, silent mutations, or conservative substitutions thereof, so long as they have the same or equivalent activity.

[0033] For example, polypeptides or proteins conjugated to an N-terminal signal (or leader) sequence involved in co-translationally or post-translationally protein (polypeptide) translocation, or polypeptides or proteins conjugated to other sequences or linkers that allow the polypeptide or protein to be identified, purified, or synthesized, may also be included within the scope of the polypeptide or protein having the amino acid sequence set forth in the specific SEQ ID NO.

[0034] As used herein, the term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and 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. As another example, amino acids can be classified into electrically charged amino acids (arginine, lysine, histidine, glutamic acid, and aspartic acid) and uncharged amino acids (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). Phenylalanine, tryptophan, and tyrosine can be classified as aromatic amino acids. Valine, leucine, and isoleucine can be classified as branched-chain amino acids.As another example, the 20 amino acids can be classified by size, and the amino acid group with the smallest volume 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, conservative substitutions are not necessarily limited to these. Typically, conservative substitutions have little or no effect on the activity of a polypeptide or protein.

[0035] Genes, polynucleotides As used herein, the term "gene" refers to a polynucleotide that encodes a functional molecule and includes regions preceding and following the polynucleotide, or to a functional RNA. In some embodiments, a gene may have sequences (introns) inserted between each coding region (exon).

[0036] As used herein, the terms "polynucleotide," "nucleic acid," or "nucleic acid molecule" refer to a DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA) chain of a certain length or greater that is a polymer of nucleotides in which nucleotide monomers are linked together in a long chain by covalent bonds. In this application, "polynucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably.

[0037] identity, homology As used herein, the terms "identity" or "homology" refer to the degree of similarity between two given amino acid or nucleotide sequences, which can be expressed as a percentage. In this application, "homology" and "identity" are often used interchangeably.

[0038] Conserved polynucleotide or polypeptide sequence homology or identity may be determined by standard sequence algorithms, with default gap penalties established by the program used.

[0039] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms such as the "FASTA" program using default parameters, e.g., as in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]:2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) can be used, as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later), or the Smith-Waterman algorithm (Smith and Waterman, Adv. Appl. Math (1981) 2:482) can be used to compare sequence information using a GAP computer program (GCG program package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] [ET AL, J MOLECULAR GENESIS]). 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). For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0040] Furthermore, whether any two polynucleotide sequences have homology, similarity, or identity can be confirmed by Southern hybridization experiments under defined, appropriate hybridization conditions, which can be determined by methods well 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. Polynucleotide sequences that are substantially homologous or identical can generally hybridize under stringent conditions over at least about 50%, 60%, 70%, 80%, or 90% of the entire sequence or the entire length.

[0041] In the present application, the term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8). For example, the conditions may be such that polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 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, hybridize with each other, but polynucleotides with lower homology or identity do not hybridize with each other; or the conditions may be such that washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization, which are 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.

[0042] Although hybridization requires that the two nucleotides have complementary sequences, hybridized polynucleotides may contain some mismatches between bases, depending on the stringency of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to each other. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. Thus, polynucleotides of the present application may include isolated nucleic acid fragments that are complementary to the entire sequence, as well as those with substantially similar base sequences.

[0043] For example, a polynucleotide having homology or identity to the polynucleotide of the present application can be hybridized and detected at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0044] The appropriate stringency for hybridizing such polynucleotides depends on the length and degree of complementation of the polynucleotides, variables well known in the art (eg, J. Sambrook et al., supra).

[0045] Nucleic acid constructs, vectors, and transformation As used herein, the term "nucleic acid construct" refers to a single- or double-stranded nucleic acid molecule that contains one or more regulatory sequences and that has been artificially synthesized, engineered to contain a specific sequence in a manner that does not occur in nature, or isolated from nature.

[0046] The term "vector" as used herein refers to a DNA construct for transferring a polynucleotide of interest into a suitable host or host cell. For example, the vector may include, but is not limited to, a base sequence of a polynucleotide encoding a polypeptide of interest operably linked to a suitable expression control region (or expression control sequence) so as to enable the expression of the polypeptide of interest in a suitable host.

[0047] The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding an appropriate mRNA ribosome binding site, and a sequence regulating the termination of transcription and decoding. After being transformed into an appropriate host cell (microorganism), the vector can replicate or function independently of the host genome, or it can be integrated into the genome itself.

[0048] The vectors used in this application are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pDZ, pDC, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Examples of vectors that can be used include pDZ, pDC, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC.

[0049] For example, a polynucleotide of interest can be inserted into a chromosome of a cell via a vector for chromosomal integration. The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for determining whether or not the polynucleotide has been inserted into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to determine whether or not the polynucleotide of interest has been inserted. A marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide, may be used. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0050] The term "transformation" as used herein refers to the introduction of a vector containing a polynucleotide of interest into a host cell (microorganism) to change the genetic traits of the host cell (microorganism). The transformed polynucleotide may be inserted into the chromosome of the host cell (microorganism) or may be extrachromosomally located. The polynucleotide may comprise DNA or RNA. The polynucleotide may be introduced in a form appropriate for the purpose of introduction. For example, a polynucleotide for expressing a polypeptide of interest may be introduced into a host cell (microorganism) in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. The expression cassette typically contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of an autonomously replicating expression vector. The polynucleotide may also be introduced into a host cell (microorganism) in its own form and operably linked to sequences necessary for expression in the host cell (microorganism), but is not limited thereto.

[0051] As used herein, the term "operably linked" refers to a configuration in which a regulatory sequence is positioned appropriately so that it controls the expression of a coding sequence. Thus, "operably linked" includes a regulatory region of a functional domain having a known or desired activity, such as a promoter, terminator, signal sequence, or enhancer region, attached or linked to a target (gene or polypeptide) so as to regulate the expression, secretion, or function of the target through the known or desired activity. For example, it refers to the operably linking of a polynucleotide sequence to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the polypeptide.

[0052] In this application, the term "expression" includes any step involved in the production of a polypeptide, such as, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0053] As used herein, the term "expression vector" refers to a linear or circular nucleic acid molecule containing a polynucleotide sequence of interest and a regulatory sequence operably linked thereto for its expression. For example, the vector may contain a polynucleotide sequence encoding a polypeptide of interest operably linked to an appropriate expression regulatory region (or expression regulatory sequence) so as to enable the expression of the polypeptide of interest in a suitable host.

[0054] In this application, the term "regulatory sequence" refers to a polynucleotide sequence required for regulating the expression of a target polynucleotide sequence. Each regulatory sequence may be native to the coding sequence (having the same origin), or may be a foreign (derived from another gene) sequence, or a mutant sequence thereof, or other artificial sequence. Examples of the regulatory sequence include a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination. The minimum unit of the regulatory sequence may include a promoter, a transcription and translation termination sequence.

[0055] As used herein, the term "genetic modification" refers to a natural or artificial process in which genetic elements, such as DNA or RNA, are altered in a manner that differs from their original sequence through disassembly and reassembly.

[0056] As used herein, the term "recombinant gene" refers to a gene having a new genomic configuration that is generated as a result of genetic recombination, such as by chemical synthesis or genetic engineering techniques. The terms "recombinant gene," "recombinant DNA," and "recombinant polynucleotide" are used interchangeably in this application. For example, a recombinant gene may include an artificial combination of nucleic acid fragments, such as regulatory sequences, that are not found together in nature.

[0057] In the present application, the term "recombinant protein" means a protein produced as a result of genetic recombination.

[0058] microorganisms In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and prokaryotic or eukaryotic microorganisms that have been genetically modified naturally or artificially, including microorganisms in which specific mechanisms have been weakened or enhanced by inserting an exogenous gene or by increasing or inactivating the activity of an endogenous gene, and may also be a microorganism that contains genetic modifications for the production of a desired polypeptide, protein, or product. In this application, the terms "microorganism," "strain," "host," and "host cell" are used interchangeably.

[0059] As used herein, the term "recombinant microorganism" refers to a microorganism that has been genetically altered and exhibits a different genotype and / or phenotype compared to a naturally occurring microorganism (e.g., when the genetic alteration affects the nucleic acid sequence coding for the microorganism), and can include all of the progeny or potential progeny of said microorganism. The terms "recombinant microorganism," "genetically altered microorganism," "recombinant host cell," "recombinant cell," and "recombinant strain" are used interchangeably in this application. The recombinant microorganism can, for example, express genes not found in their native (non-recombinant) form; not express genes expressed in their native form; or express native genes in a manner different from how they are expressed in their native form.

[0060] For example, the microorganism of the present application may be, but is not limited to, a microorganism into which a pyruvate phosphate dikinase protein or a polynucleotide encoding the same has been introduced (e.g., a recombinant microorganism).

[0061] In this application, the term "microorganism having the ability to produce L-tryptophan" refers to a microorganism that can produce L-tryptophan within the organism, and may include both microorganisms that do not inherently have the ability to produce L-tryptophan but have been imparted with the ability to produce L-tryptophan, and microorganisms that inherently have the ability to produce L-tryptophan. The ability to produce L-tryptophan may be imparted or enhanced by species improvement.

[0062] As used herein, the term "unaltered microorganism (strain)" does not exclude microorganisms (strains) containing naturally occurring mutations, but refers to a wild-type or naturally occurring microorganism (strain) itself, or a microorganism (strain) before its characteristics are altered by genetic mutation due to natural or artificial factors. In this application, the term "unaltered microorganism (strain)" is used interchangeably with "pre-altered microorganism (strain)," "unaltered microorganism (strain)," "parent microorganism," "parent strain," "wild-type microorganism (strain)," "reference microorganism (strain)," or "reference microorganism (strain)." In this application, "unaltered microorganism" refers to a microorganism (strain) into which the pyruvate phosphate dikinase protein or a polynucleotide encoding it of this application has not been introduced or before its introduction, but is not limited thereto. In addition, in this application, "unaltered microorganism" may be, but is not limited to, a microorganism that does not contain a polypeptide consisting of SEQ ID NO: 1 or a polynucleotide consisting of SEQ ID NO: 2.

[0063] Increased protein (polypeptide) activity In this application, the term "increase" in protein (polypeptide) activity means that the activity of a protein (polypeptide) in a host cell (microorganism) is increased compared to its endogenous activity. The increase may be used interchangeably with terms such as activation, up-regulation, overexpression, and enhancement. The host cell (microorganism) may be a prokaryotic or eukaryotic microorganism.

[0064] The increase in protein (polypeptide) activity can include both the display of a protein (polypeptide) activity that the host cell (microorganism) did not have endogenously, and the display of a protein (polypeptide) activity that is improved compared to the endogenous activity or the activity before transformation.

[0065] For example, the "exhibition of a protein (polypeptide) activity that is not endogenously possessed" or the "exhibition of improved protein (polypeptide) activity" may be due to "introduction of a protein (polypeptide)," but is not limited to this.

[0066] As used herein, the term "introduction" of a protein (polypeptide) means that a gene not originally possessed by a microorganism is expressed in the microorganism to exhibit a specific protein activity, or that the polypeptide activity is strengthened, increased, or improved compared to the endogenous activity of the protein or the activity before modification. For example, this may be achieved by introducing a gene encoding the protein (polypeptide) into a host cell (microorganism). For example, a polynucleotide encoding a specific protein (polypeptide) may be introduced into a chromosome in the host cell (microorganism), or a vector containing a polynucleotide encoding a specific protein (polypeptide) may be introduced into a host cell (microorganism) to exhibit or improve its activity.

[0067] The term "intrinsic activity" refers to the activity of a specific protein (polypeptide) that a host cell (microorganism) before transformation or a non-transformed host cell (microorganism) originally possesses when its trait has been altered by genetic mutation due to natural or artificial factors. This term may be used interchangeably with "activity before transformation."

[0068] The activity of a protein (polypeptide) being increased compared to its endogenous activity means that the activity and / or concentration (expression level) of the protein (polypeptide) in the host cell (microorganism) is improved compared to the activity and / or concentration (expression level) of the protein (polypeptide) originally possessed by the host cell (microorganism) before transformation or the untransformed host cell (microorganism).

[0069] For example, the increase may be such that the activity of the corresponding protein (polypeptide) was absent, or the activity or concentration may be increased by, but is not limited to, generally 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, based on the activity or concentration in the host cell (microorganism) before transformation or in a non-transformed host cell (microorganism).

[0070] The increase in the activity of the protein (polypeptide) can be achieved by introducing an exogenous protein (polypeptide) or by increasing the activity of an endogenous protein (polypeptide). The increase in the activity of the protein (polypeptide) can be confirmed by the increase in the activity, expression level, or amount of product resulting from the activity of the protein (polypeptide).

[0071] The increase in the activity of the protein (polypeptide) can be achieved by various methods well known in the art, and is not limited as long as the activity of the target protein (polypeptide) can be increased compared to that of the host cell (microorganism) before transformation. Specifically, the increase can be achieved by using genetic engineering and / or protein engineering, which are routine methods in molecular biology and well known to those of ordinary skill in the art, but is not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2, pp. 1-16; Sambrook et al., Molecular Cloning 2012, etc.).

[0072] Specifically, the activity of the protein (polypeptide) of the present application is increased by: 1) An increase in the intracellular copy number of a polynucleotide encoding a protein (polypeptide); 2) Modification of the expression regulatory region of a gene on a chromosome encoding a protein (polypeptide) (for example, introduction of a mutation within the expression regulatory region, replacement with a sequence with stronger activity, or insertion of a sequence with stronger activity); 3) A modification of the nucleotide sequence encoding the initiation codon or 5'UTR region of a gene transcript encoding a protein (polypeptide); 4) modifying the amino acid sequence of a protein (polypeptide) so as to increase the activity of the protein (polypeptide); 5) Modifying a polynucleotide sequence encoding a protein (polypeptide) so as to increase the activity of the protein (polypeptide) (e.g., modifying the polynucleotide sequence of a gene encoding the protein (polypeptide) so as to encode a protein (polypeptide) that has been modified so as to increase the activity of the protein (polypeptide); 6) Introduction of a foreign protein (polypeptide) exhibiting the activity of a protein (polypeptide) or a foreign polynucleotide encoding the same; 7) Codon optimization of polynucleotides encoding proteins (polypeptides); 8) Analyzing the tertiary structure of proteins (polypeptides) and selectively altering or chemically modifying exposed sites; or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0073] for example, The increase in intracellular copy number of a polynucleotide encoding a protein (polypeptide) in the above 1) method may be achieved by introducing a vector containing a polynucleotide encoding the protein (polypeptide) operably linked to an appropriate regulatory sequence into a host cell (microorganism). Alternatively, one or more copies of a polynucleotide encoding the protein (polypeptide) operably linked to an appropriate regulatory sequence may be introduced into a chromosome of the host cell (microorganism). The introduction into a chromosome can be achieved by, but is not limited to, introducing into the host cell (microorganism) a vector capable of inserting the polynucleotide into the chromosome of the host cell (microorganism). The vector is as described above. The regulatory sequence may be natural (having the same origin) or foreign (derived from another gene) sequences relative to the encoding polynucleotide sequence, or a mutant sequence thereof, or another artificial sequence, and may induce expression of the polynucleotide in the host cell (microorganism).

[0074] The 2) replacement of a gene expression regulatory region (or expression regulatory sequence) on a chromosome encoding a protein (polypeptide) with a sequence with stronger activity may involve, for example, introducing a mutation into the sequence by deletion, insertion, substitution, or a combination thereof to further increase the activity of the expression regulatory region, or replacing it with a sequence with stronger activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. For example, the original promoter may be replaced with a strong promoter, but this is not limited thereto.

[0075] Examples of known strong promoters include, but are not limited to, the cj1 to cj7 promoters (U.S. Patent No. 7,662,943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (U.S. Patent No. 10,584,338 B2), the O2 promoter (U.S. Patent No. 10,273,491 B2), the tkt promoter, and the yccA promoter.

[0076] The 3) modification of the nucleotide sequence of the region encoding the start codon or 5'UTR of the gene encoding the protein (polypeptide) may be, for example, a modification to encode a different start codon that results in a higher protein (polypeptide) expression rate than the endogenous start codon, or a modification to encode a ribosome binding site (RBS) sequence that results in a higher protein (polypeptide) expression rate than the endogenous RBS sequence, but is not limited thereto.

[0077] The modification of the amino acid sequence or polynucleotide sequence of the protein (polypeptide) in 4) and 5) above may be, but is not limited to, introducing a mutation into the amino acid sequence of the protein (polypeptide) or the polynucleotide sequence encoding the protein (polypeptide) by deletion, insertion, substitution, or a combination thereof so as to increase the activity of the protein (polypeptide), or replacing the amino acid sequence or polynucleotide sequence with an amino acid sequence or polynucleotide sequence modified to increase activity. The replacement can be performed, for example, by inserting a polynucleotide into a chromosome by homologous recombination, but is not limited to these.

[0078] The introduction of an exogenous polynucleotide that exhibits the activity of a protein (polypeptide) as described above in 6) above may involve the introduction into a host cell (microorganism) of an exogenous polynucleotide that encodes a protein (polypeptide) that exhibits the same or similar activity as the protein (polypeptide). There are no limitations on the origin or sequence of the exogenous polynucleotide, as long as it exhibits the same or similar activity as the protein (polypeptide). The method used for the introduction can be any known transformation method appropriately selected by those skilled in the art. Expression of the introduced polynucleotide in the host cell results in the production of the protein (polypeptide), and its activity can be increased.

[0079] The codon optimization of the polynucleotide encoding the protein (polypeptide) may be that of an endogenous polynucleotide that has been codon-optimized to increase transcription and translation within a host cell (microorganism), or that of an exogenous polynucleotide that has been codon-optimized to optimize transcription and translation within a host cell (microorganism).

[0080] 8) Analyzing the tertiary structure of a protein (polypeptide) and selecting and modifying or chemically modifying exposed sites may involve, for example, comparing the sequence information of the protein (polypeptide) to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and modifying exposed sites to be modified or chemically modified.

[0081] Such an increase in protein (polypeptide) activity may be, but is not limited to, an increase in the activity or concentration of the corresponding protein (polypeptide) relative to the activity or concentration of the protein (polypeptide) expressed in a wild-type or untransformed host cell (microorganism), or an increase in the amount of a product resulting from the activity of the protein (polypeptide).

[0082] Modification of a part or all of a polynucleotide in the microorganism of the present application can be induced by, but is not limited to, (a) a method using homologous recombination using a vector for chromosomal insertion in the microorganism or genome editing using engineered nuclease (e.g., CRISPR-Cas9) and / or (b) treatment with light, such as ultraviolet light and radiation, and / or chemicals.

[0083] culture In the present application, the term "culturing" means growing a microorganism under appropriately controlled environmental conditions. The culturing process can be carried out using an appropriate medium and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected microorganism. Specifically, the culturing may be, but is not limited to, a batch, continuous, and / or fed-batch culture.

[0084] As used herein, the term "culture medium" refers to a mixture of nutrients, primarily those required for culturing microorganisms, and provides nutrients, including water, and growth factors essential for survival and growth. Specifically, the culture medium and other culture conditions used to culture the microorganisms of the present application can be any medium commonly used for culturing microorganisms, without any particular limitations. For example, the microorganisms of the present application can be cultured in a conventional medium containing appropriate carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins under aerobic conditions, with temperature, pH, and other parameters adjusted. For example, culture media for Corynebacterium microorganisms can be found in the literature, "Manual of Methods for General Bacteriology" by the American Society for Bacteriology (Washington, DC, USA, 1981).

[0085] In the present application, the carbon source may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid may also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used. A variety of other carbon sources may also be used in appropriate amounts without limitation. These carbon sources may be used alone or in combination of two or more, and are not limited thereto.

[0086] Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its degradation products, and defatted soybean cake or its degradation products. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0087] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, and other compounds, including amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, the present invention is not limited to these.

[0088] During the cultivation of the microorganism of the present application, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the medium in an appropriate manner to adjust the pH of the medium. During cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. To maintain an aerobic state in the medium, oxygen or an oxygen-containing gas can be injected into the medium, or to maintain an anaerobic or microaerobic state, no gas can be injected or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limiting.

[0089] In the culture of the present application, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and the culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0090] In this application, the term "culture" refers to a culture solution, concentrated culture solution, dried culture solution, culture filtrate, concentrated culture filtrate, or dried culture filtrate obtained by culturing a specific microorganism in a culture medium, and the culture solution contains the specific microorganism, while the culture filtrate is substantially free of the specific microorganism (here, "substantially" means that the specific microorganism separated by filtration or the like is excluded, but does not mean that the microorganism is completely excluded in the filtrate). The form of the culture is not limited, and may be, for example, a liquid, emulsion, or solid.

[0091] In this application, the term "fermentation" refers to the process in which microorganisms use their own enzymes to decompose organic matter, but is not a putrefaction reaction. Fermentation and putrefaction proceed through similar processes, but if useful substances are produced as a result of decomposition, it is called fermentation, and if foul odors or harmful substances are produced, it is called putrefaction.

[0092] In the present application, the method for obtaining a fermented product from the microorganism is not particularly limited, and the product can be obtained by a method commonly used in the technical field or a similar field.

[0093] In the present application, the term "fermentate" includes not only the fermented substance itself but also any kind of substance containing a fermentate generated from said microorganism, such as a substance containing a fermented microorganism, a culture generated from the fermented microorganism, a fermentation of a culture, a concentrated fermentate, a dried fermentate, a fermentation filtrate, a concentrated fermentation filtrate, or a dried fermentation filtrate, an extract of a fermentate, or a dilution of a fermentate.

[0094] Specific Description of the Application A specific example of the present application will be described in more detail below.

[0095] One aspect of the present application provides a Corynebacterium microorganism capable of producing L-tryptophan, into which a pyruvate phosphate dikinase protein derived from Komagataibacter xylinus or a polynucleotide encoding the same has been introduced.

[0096] As used herein, the term "pyruvate, phosphate dikinase (PPDK)" refers to an enzyme in the transferase family that catalyzes the reaction [ATP + pyruvate + phosphate ⇔ phosphoenolpyruvate (PEP) + diphosphate]. The term "pyruvate, phosphate dikinase" as used herein may be used interchangeably with "PPDK." Specifically, the term "pyruvate, phosphate dikinase" as used herein may be a protein having pyruvate, phosphate dikinase activity encoded by the ppdk gene, but is not particularly limited as long as it has activity equivalent to that of pyruvate, phosphate dikinase. The pyruvate, phosphate dikinase encoded by the ppdk gene is known in the art, and the amino acid and polynucleotide sequences of the pyruvate, phosphate dikinase can be obtained from publicly available databases, including, but not limited to, GenBank at NCBI.

[0097] For example, the pyruvate phosphate dikinase protein derived from Komagataeibacter xylinus may include the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60% or more homology or identity thereto, but is not limited thereto as long as it has pyruvate phosphate dikinase protein activity. Specifically, even if a protein includes a sequence in which a portion of the amino acid sequence of SEQ ID NO: 1 is deleted, modified, substituted, or added, it can still be included in the pyruvate phosphate dikinase protein as long as it exhibits the activity corresponding to the pyruvate phosphate dikinase protein. Furthermore, the pyruvate phosphate dikinase protein may include a protein that has, comprises, consists of, or essentially consists of an amino acid sequence that has at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity to the amino acid sequence of SEQ ID NO: 1, and that exhibits activity equivalent to that of the pyruvate phosphate dikinase protein.

[0098] Furthermore, the sequence of a polynucleotide encoding the pyruvate phosphate dikinase protein derived from Komagataibacter xylinus having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 60% or more homology or identity thereto can be obtained, for example, based on codon information publicly known in the art. For example, the pyruvate phosphate dikinase protein may be encoded by a polynucleotide having, comprising, consisting of, or essentially consisting of 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 to the sequence of SEQ ID NO: 2, but is not limited thereto. The nucleotide sequence of SEQ ID NO: 2 can also be obtained from publicly known databases, such as, but not limited to, NCBI's GenBank.

[0099] In this application, the polynucleotide (gene) comprising the base sequence of SEQ ID NO: 2 may be used interchangeably with a polynucleotide (gene) having the base sequence of SEQ ID NO: 2, a polynucleotide (gene) consisting of the base sequence of SEQ ID NO: 2, or ppdk.

[0100] The polynucleotides of the present application may have various modifications in the coding region thereof, taking into consideration codon degeneracy or preferred codons in the organism in which the pyruvate phosphate dikinase protein of the present application is to be expressed, without changing the amino acid sequence of the pyruvate phosphate dikinase protein of the present application. Therefore, it is clear that polynucleotides of the present application also include polynucleotides that, due to codon degeneracy, can be translated into a polypeptide consisting of the amino acid sequence of the pyruvate phosphate dikinase protein of the present application or a polypeptide having homology or identity thereto. For example, the polynucleotide of the present application may be SEQ ID NO: 2 or a degenerated sequence thereof.

[0101] As another example, the polynucleotide of the present application may have or comprise a nucleotide sequence that is 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 homologous or identical to SEQ ID NO:2, or may consist of or essentially consist of a nucleotide sequence that is 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 homologous or identical to SEQ ID NO:2, but is not limited thereto.

[0102] Furthermore, the polynucleotide of the present application may include, without limitation, a probe prepared from a known gene sequence, for example, a sequence that hybridizes under stringent conditions with a complementary sequence to all or part of the polynucleotide sequence of the present application and encodes the pyruvate phosphate dikinase protein of the present application.

[0103] For purposes of this application, the microorganism of this application includes all microorganisms capable of producing the desired L-tryptophan by introducing a pyruvate phosphate dikinase protein or a polynucleotide encoding the same. For example, the microorganism of this application may be a genetically modified or recombinant microorganism characterized by increased L-tryptophan production ability due to the introduction of a pyruvate phosphate dikinase protein or a polynucleotide encoding the same, but is not limited thereto. Specifically, the recombinant strain with increased L-tryptophan production ability may be, but is not limited to, a naturally occurring wild-type microorganism or a microorganism that has endogenous pyruvate phosphate dikinase activity or that lacks endogenous pyruvate phosphate dikinase activity and has increased L-tryptophan production ability compared to a non-modified microorganism.

[0104] For example, a microorganism capable of producing L-tryptophan is a prokaryotic or eukaryotic microbial strain capable of producing L-tryptophan within the organism, and may include both microorganisms that inherently have the ability to produce L-tryptophan and microorganisms in which the ability to produce L-tryptophan has been imparted to a parent strain lacking the ability to produce L-tryptophan through the activity of the pyruvate phosphate dikinase protein introduced into the present application. The ability to produce L-tryptophan may also be imparted or enhanced by species improvement.

[0105] The microorganisms of the present application may include any microorganism into which a pyruvate phosphate dikinase protein or a polynucleotide encoding the same has been introduced by a variety of known methods.

[0106] For example, the recombinant microorganism capable of producing L-tryptophan of the present application can include any microorganism that is transformed through a vector and into which an exogenous gene encoding the pyruvate phosphate dikinase of the present application, specifically an exogenous gene encoding the pyruvate phosphate dikinase derived from Komagataibacter xylinus, is introduced and that can produce L-tryptophan.

[0107] For example, the L-tryptophan-producing microorganism may be a microorganism into which a polynucleotide sequence encoding a protein comprising the amino acid sequence of SEQ ID NO:1, or a protein comprising an amino acid sequence having 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 to the amino acid sequence of SEQ ID NO:1 has been introduced.

[0108] For example, the L-tryptophan-producing microorganism may be a microorganism into which a polynucleotide capable of encoding a protein comprising an amino acid sequence having at least 80% homology to the amino acid sequence of SEQ ID NO: 1 has been introduced; or a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence that is 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 homologous or identical to the nucleotide sequence of SEQ ID NO: 2 has been introduced.

[0109] For example, the microorganism having increased L-tryptophan production ability of the present application may be, but is not limited to, a microorganism having increased L-tryptophan production ability compared to a non-modified microorganism. For example, the non-modified microorganism, which is the subject strain for comparing the increased L-tryptophan production ability, may be, but is not limited to, the CM05-9157 strain.

[0110] For example, the microorganism with increased L-tryptophan production ability may be increased 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 16% or more, about 17% or more, or about 18% or more (the upper limit is not particularly limited, and may be, 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), compared to the L-tryptophan production ability of the parent microorganism (parent strain) or unmodified microorganism before mutation, but is not limited thereto as long as there is an increase in the + value compared to the production ability of the parent microorganism (parent strain) or unmodified microorganism. In another example, the recombinant strain with increased L-tryptophan production ability may have an L-tryptophan production ability that is increased by about 1.1 times or more, about 1.15 times or more, about 1.16 times or more, about 1.17 times or more, or about 1.18 times or more (there is no particular upper limit, and the increase may be, for example, 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 1.3 times or less, or about 1.2 times or less) compared to the parent microorganism (parent strain) before mutation or an untransformed microorganism, but is not limited thereto.

[0111] For example, the microorganism capable of producing L-tryptophan of the present application may be either a prokaryotic cell or a eukaryotic cell, and specifically may be a prokaryotic cell. Examples of prokaryotic cells include, but are not limited to, microorganisms belonging to the genera Escherichia, Erwinia, Serratia, Providencia, Corynebacteria, Pseudomonas, Leptospira, Salmonella, Brevibacteria, Hypomonas, Chromobacterium, and Norcardia, or fungi or yeast. Specifically, the prokaryotic cells may be microorganisms and yeasts belonging to the genera Escherichia, Corynebacterium, and Leptospira. More specifically, it may be a microorganism of the genus Corynebacterium.

[0112] As a microorganism according to any one of the above-mentioned embodiments, the microorganism of the present application may be a microorganism of the genus Corynebacterium.

[0113] As an example of the present application, the microorganism of the present application is Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium The microorganism may be, but is not limited to, a Corynebacterium species, such as Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens. Specifically, the microorganism of the present application may be a Corynebacterium microorganism, more specifically, Corynebacterium glutamicum.

[0114] Specifically, the microorganism of the present application may be a microorganism of the genus Corynebacterium, more specifically, Corynebacterium glutamicum, but is not limited thereto.

[0115] Meanwhile, the Corynebacterium microorganisms of the present application having the ability to produce L-tryptophan may include all of the following: natural wild-type microorganisms themselves; Corynebacterium microorganisms that have improved L-tryptophan production ability by increasing or decreasing the activity of genes related to the L-tryptophan production mechanism; and Corynebacterium microorganisms that have improved L-tryptophan production ability by introducing or increasing the activity of exogenous genes.

[0116] Another aspect of the present application provides a method for producing L-tryptophan, comprising culturing in a medium a Corynebacterium microorganism capable of producing L-tryptophan, into which the pyruvate phosphate dikinase protein derived from Komagataebacter xylinus of the present application or a polynucleotide encoding the same has been introduced.

[0117] In the method of the present application, the microorganism can be cultured under any culture conditions and by any culture method known in the art, and such a culture process can be easily adjusted by a person skilled in the art depending on the selected microorganism.

[0118] The L-tryptophan produced by the culture of the present invention is either secreted into the medium or remains intracellularly.

[0119] In one embodiment, the method for producing L-tryptophan of the present application may further include a step of preparing the microorganism of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order), for example, before the culturing step.

[0120] The method for producing L-tryptophan of the present application may further include a step of recovering the target substance, specifically L-tryptophan, from the cultured microorganism, the culture product of the microorganism, the fermentation product of the microorganism, or the culture medium. The recovery step may be further included after the culturing step.

[0121] The recovery may involve collecting the target L-tryptophan using an appropriate method known in the art through the microbial culture method of the present application, such as a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting-out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination of these methods can be used. The target substance, specifically, L-tryptophan, can be recovered from the medium or the microorganism using an appropriate method known in the art.

[0122] The method for producing L-tryptophan of the present application may further include a purification step. The purification can be performed using an appropriate method known in the art. In one example, when the method for producing L-tryptophan of the present application includes both a recovery step and a purification step, the recovery step and the purification step can be performed continuously or discontinuously, in any order, simultaneously, or integrated into one step, but are not limited thereto.

[0123] In the method of the present application, pyruvate, phosphate dikinase, transfection, L-tryptophan, etc. are as described above in other embodiments.

[0124] Another aspect of the present application provides a Corynebacterium microorganism capable of producing L-tryptophan, into which the pyruvate phosphate dikinase protein derived from Komagataebacter xylinus of the present application or a polynucleotide encoding the same has been introduced; or a composition for producing L-tryptophan, comprising a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more of these.

[0125] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing L-tryptophan, and such excipients may be, for example, but are not limited to, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, or isotonic agents.

[0126] In one embodiment, each component present in the compositions of the present application may be included in a microbiologically effective amount, or in an amount that may be suitably present in a production composition.

[0127] In the composition of the present application, pyruvate, phosphate dikinase, transfection, L-tryptophan, etc. are as described above in other aspects.

[0128] Another aspect of the present application provides the use of a Corynebacterium microorganism capable of producing L-tryptophan, into which the pyruvate phosphate dikinase protein derived from Komagataebacter xylinus of the present application or a polynucleotide encoding the same has been introduced, for the production of L-tryptophan.

[0129] For purposes of this application, pyruvate, phosphate dikinase, transfection, and L-tryptophan are as described above in other embodiments. Example

[0130] The present application will be described in more detail below with reference to experimental examples. However, the following examples are merely preferred embodiments for illustrating the present application, and are not intended to limit the scope of the present application. Meanwhile, technical matters not described in this specification can be fully understood and easily implemented by those of ordinary skill in the technical field of the present application or a similar technical field.

[0131] Example 1. Search and selection of phosphoenolpyruvate synthase (ppsA) or pyruvate, phosphate dikinase (ppdK) genes To select phosphoenolpyruvate synthase or pyruvate phosphate dikinase with high gluconeogenic activity, we searched for acetic acid bacteria and methanogen-derived organisms that use carbon sources consisting of 1 to 3 carbons based on literature survey results. Among them, considering the biosafety level applicable to the production strain and the possibility of ensuring safety, we selected four microorganisms that are expected to have phosphoenolpyruvate synthase or pyruvate phosphate dikinase, as shown in Table 1 below.

[0132] [Table 1]

[0133] Example 2. Construction of L-tryptophan-producing microorganisms incorporating exogenous pyruvate phosphate dikinase Example 2-1. Construction of a plasmid for gene insertion Plasmids were constructed to introduce the pyruvate phosphate dikinase or phosphoenolpyruvate synthase genes into the transposon gene site in the chromosome of Corynebacterium glutamicum by homologous recombination.

[0134] Specifically, PCR was performed using the chromosomal DNA of Corynebacterium glutamicum ATCC13869 as a template and the primer pair of SEQ ID NO: 11 and SEQ ID NO: 12, and the primer pair of SEQ ID NO: 13 and SEQ ID NO: 14 to obtain the respective fragments. TM The PCR was performed using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 4 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 30 seconds, and polymerization at 72°C for 50 seconds, repeated 27 times, followed by polymerization at 72°C for 5 minutes. The primer sequences used are shown in Table 2 below.

[0135] [Table 2]

[0136] The two fragments amplified by PCR and the transformation vector pDCM2 (Korean Patent No. 10-2278000) digested with SmaI restriction enzyme were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid, designated pDCM2-△Tn.

[0137] Example 2-2. Construction of Corynebacterium spp. microorganisms incorporating pyruvate phosphate dikinase derived from Komagataeibacter xylinus To introduce the ppdK gene (NZ_ CP024644.1, SEQ ID NO: 2) derived from Komagataeibacter xylinus (K. xylinus) encoding pyruvate phosphate dikinase (SEQ ID NO: 1) into Corynebacterium glutamicum, the ppdK gene was first amplified by PCR using the primer pair SEQ ID NO: 15 and SEQ ID NO: 16, with the K. xylinus ppdK gene (SEQ ID NO: 2) synthesized using the gene synthesis service of Bionix Co., Ltd. as a template.

[0138] The polymerase is Solg TM PCR amplification was performed using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 2 minutes, denaturation at 95°C for 20 seconds, annealing at 60°C for 40 seconds, and polymerization at 72°C for 1 minute, repeated 27 times, followed by polymerization at 72°C for 5 minutes.

[0139] [Table 3]

[0140] To obtain the Pcj7 promoter, PCR was performed using p117-cj7-gfp (US 7,662,943 B2) as a template and primers of SEQ ID NO: 17 and SEQ ID NO: 18. The polymerase was Solg TM Pfu-X DNA polymerase (SolGent Co.) was used, and the PCR amplification conditions were as follows: 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 30 seconds, repeated 27 times, followed by polymerization at 72°C for 5 minutes.

[0141] [Table 4]

[0142] The K. xylinus ppdK gene amplified from the above, the Pcj7 promoter region, and pDCM2-△Tn constructed in Example 2-1, digested with ScaI restriction enzyme, were then cloned using Gibson assembly (D.G. Gibson et al., NATURE METHODS, VOL. 6, NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid named pDCM2-△Tn::Pcj7-ppdK(K. xylinus). Cloning was performed by mixing the Gibson assembly reagent and each fragment in the calculated molar amounts and storing the mixture at 50°C for 1 hour.

[0143] The constructed pDCM2-△Tn::Pcj7-ppdK(K.xy) vector was transformed into the tryptophan-producing strain CM05-9157 (Korean Patent No. 10-2278000) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), followed by a second crossover process to obtain a strain with one copy of the Pcj7-ppdK(K.xy) gene inserted between the transposon genes on the chromosome. This strain was identified by PCR and sequencing using primers of SEQ ID NO: 19 and SEQ ID NO: 20, which can amplify the upstream and downstream regions of the gene insertion site, respectively.

[0144] [Table 5]

[0145] The strain obtained in the above manner was designated CM05-9157::Pcj7-ppdK(K.xy).

[0146] Example 2-3. Construction of Corynebacterium microorganisms incorporating pyruvate phosphate dikinase derived from Acetobacter pasteurianus subsp. To introduce the gene (NZ_CP021922.1, SEQ ID NO: 4) encoding pyruvate phosphate dikinase (SEQ ID NO: 3) derived from Acetobacter pasteurianus subsp. into Corynebacterium glutamicum, the pddK gene (SEQ ID NO: 4) of Acetobacter pasteurianus subsp. was synthesized using the gene synthesis service of Bionix Co., Ltd., and PCR was performed using the primer pair SEQ ID NO: 21 and SEQ ID NO: 22 as a template to amplify the ppdK gene. The polymerase used was Solg TM PCR amplification was performed using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 2 minutes, denaturation at 95°C for 20 seconds, annealing at 60°C for 40 seconds, and polymerization at 72°C for 1 minute, repeated 27 times, followed by polymerization at 72°C for 5 minutes.

[0147] [Table 6]

[0148] To obtain the Pcj7 promoter, PCR was performed using p117-cj7-gfp (US 7,662,943 B2) as a template and primers of SEQ ID NO: 17 and SEQ ID NO: 23. The polymerase was Solg TM Pfu-X DNA polymerase (SolGent Co.) was used, and the PCR amplification conditions were as follows: 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 30 seconds, repeated 27 times, followed by polymerization at 72°C for 5 minutes.

[0149] [Table 7]

[0150] The ppdK gene of Acetobacter pasteurianus subsp. amplified from the above, the Pcj7 promoter region, and pDCM2-△Tn constructed in Example 2-1, digested with ScaI restriction enzyme, were then cloned using Gibson assembly (D.G. Gibson et al., NATURE METHODS, VOL. 6, NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid named pDCM2-△Tn::Pcj7-ppdK(A.pa). Cloning was performed by mixing the Gibson assembly reagent and each fragment in calculated molar amounts and storing at 50°C for 1 hour.

[0151] The constructed pDCM2-△Tn::Pcj7-ppdK(A.pa) vector was then transformed into the tryptophan-producing strain CM05-9157 (Korean Patent No. 10-2278000) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), followed by a second crossover process to obtain a strain with one copy of the Pcj7-ppdK(A.pa) gene inserted between the transposon genes on the chromosome. This strain was identified by PCR and sequencing using primers of SEQ ID NOs: 19 and 20, which can amplify the upstream and downstream regions of the inserted gene, respectively.

[0152] The strain obtained in the above manner was designated CM05-9157::Pcj7-ppdK(A.pa).

[0153] Example 2-4. Construction of Corynebacterium spp. microorganisms incorporating pyruvate phosphate dikinase derived from Methanosarcina acetivorans str. C2A To introduce the gene (NZ_AE010299.1, SEQ ID NO: 6) encoding pyruvate phosphate dikinase (SEQ ID NO: 5) derived from Methanosarcina acetivorans str. C2A into Corynebacterium glutamicum, the ppdK gene was first amplified by PCR using the primer pair SEQ ID NO: 24 and SEQ ID NO: 25, with the ppdK gene (SEQ ID NO: 6) of Methanosarcina acetivorans str. C2A synthesized using the gene synthesis service of Bionics Co., Ltd. as a template.

[0154] The polymerase is Solg TM PCR amplification was performed using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 2 minutes, denaturation at 95°C for 20 seconds, annealing at 60°C for 40 seconds, and polymerization at 72°C for 1 minute, repeated 27 times, followed by polymerization at 72°C for 5 minutes.

[0155] [Table 8]

[0156] To obtain the Pcj7 promoter, PCR was performed using p117-cj7-gfp (US 7,662,943 B2) as a template and primers of SEQ ID NO: 17 and SEQ ID NO: 26. The polymerase was Solg TM Pfu-X DNA polymerase (SolGent Co.) was used, and the PCR amplification conditions were as follows: 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 30 seconds, repeated 27 times, followed by polymerization at 72°C for 5 minutes.

[0157] [Table 9]

[0158] Next, the ppdK gene of Methanosarcina acetivorans str. C2A amplified from the above, the Pcj7 promoter region, and pDCM2-△Tn constructed in Example 2-1 above, digested with ScaI restriction enzyme, were cloned using Gibson assembly (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-△Tn::Pcj7-ppdK(M.ac). Cloning was performed by mixing the Gibson assembly reagent and each fragment in the calculated molar amounts and storing at 50°C for 1 hour.

[0159] The constructed pDCM2-△Tn::Pcj7-ppdK(M.ac) vector was then transformed into the tryptophan-producing strain CM05-9157 (Korean Patent No. 10-2278000) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), followed by a second crossover process to obtain a strain with one copy of the Pcj7-ppdK(M.ac) gene inserted between the transposon genes on the chromosome. This strain was identified by PCR and sequencing using primers of SEQ ID NO: 19 and SEQ ID NO: 20, which can amplify the upstream and downstream regions of the gene insertion site, respectively.

[0160] The strain obtained in the above manner was designated CM05-9157::Pcj7-ppdK(M.ac).

[0161] Example 2-5. Construction of Corynebacterium microorganisms incorporating pyruvate phosphate dikinase derived from Escherichia coli To introduce the ppsA gene (NZ_CP084899.1, SEQ ID NO: 8) encoding phosphoenolpyruvate synthase (SEQ ID NO: 7) from Escherichia coli, which is an ortholog of the pyruvate phosphate dikinase gene (ppdk) into Corynebacterium glutamicum, the ppsA gene was first amplified by PCR using the primer pair SEQ ID NO: 27 and SEQ ID NO: 28 and Escherichia coli W3110 chromosomal DNA as a template. TM PCR amplification was performed using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 2 minutes, denaturation at 95°C for 20 seconds, annealing at 60°C for 40 seconds, and polymerization at 72°C for 1 minute, repeated 27 times, followed by polymerization at 72°C for 5 minutes.

[0162] [Table 10]

[0163] To obtain the Pcj7 promoter, PCR was performed using p117-cj7-gfp (US 7,662,943 B2) as a template and primers of SEQ ID NO: 17 and SEQ ID NO: 29. The polymerase was Solg TM Pfu-X DNA polymerase (SolGent Co.) was used, and the PCR amplification conditions were as follows: 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 30 seconds, repeated 27 times, followed by polymerization at 72°C for 5 minutes.

[0164] [Table 11]

[0165] The Escherichia coli ppsA gene amplified from the above, the Pcj7 promoter region, and pDCM2-△Tn constructed in Example 2-1, digested with ScaI restriction enzyme, were then cloned using Gibson assembly (DG Gibson et al., NATURE METHODS, VOL. 6, NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid designated pDCM2-△Tn::Pcj7-ppsA(E. coli). Cloning was performed by mixing the Gibson assembly reagent and each fragment in the calculated molar amounts and storing the mixture at 50°C for 1 hour.

[0166] The constructed pDCM2-△Tn::Pcj7-ppsA(E.co) vector was then transformed into the tryptophan-producing strain CM05-9157 (Korean Patent No. 10-2278000) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), followed by a second crossover process to obtain a strain with one copy of the Pcj7-ppsA(E.co) gene inserted between the transposon genes on the chromosome. This strain was identified by PCR and sequencing using primers of SEQ ID NO: 19 and SEQ ID NO: 20, which can amplify the upstream and downstream regions of the gene insertion site, respectively.

[0167] The strain obtained in the above manner was designated CM05-9157::Pcj7-ppsA(E.co).

[0168] Example 2-6. Construction of Corynebacterium microorganisms transfected with pyruvate phosphate dikinase derived from Corynebacterium glutamicum ATCC13869 To introduce the ppsA gene (NZ_CP016335.1, SEQ ID NO: 10) from Corynebacterium glutamicum ATCC13869, which encodes phosphoenolpyruvate synthase (SEQ ID NO: 9), an ortholog of the pyruvate phosphate dikinase gene (ppdk), into Corynebacterium glutamicum, the ppsA gene was amplified by PCR using the primer pair SEQ ID NO: 30 and SEQ ID NO: 31 and Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template. TM PCR amplification was performed using Pfu-X DNA polymerase under the following conditions: denaturation at 95°C for 2 minutes, denaturation at 95°C for 20 seconds, annealing at 60°C for 40 seconds, and polymerization at 72°C for 1 minute, repeated 27 times, followed by polymerization at 72°C for 5 minutes.

[0169] [Table 12]

[0170] To obtain the Pcj7 promoter, PCR was performed using p117-cj7-gfp (US 7,662,943 B2) as a template and primers of SEQ ID NO: 17 and SEQ ID NO: 32. The polymerase was Solg TM Pfu-X DNA polymerase (SolGent Co.) was used, and the PCR amplification conditions were as follows: 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 30 seconds, repeated 27 times, followed by polymerization at 72°C for 5 minutes.

[0171] [Table 13]

[0172] The amplified ppsA gene of Corynebacterium glutamicum ATCC13869, the Pcj7 promoter region, and pDCM2-△Tn (constructed in Example 2-1) digested with ScaI restriction enzyme were then cloned using Gibson assembly (D.G. Gibson et al., NATURE METHODS, VOL. 6, NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid designated pDCM2-△Tn::Pcj7-ppsA(C.gl). Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated molar amounts and storing at 50°C for 1 hour.

[0173] The constructed pDCM2-△Tn::Pcj7-ppsA(C.gl) vector was then transformed into the tryptophan-producing strain CM05-9157 (Korean Patent No. 10-2278000) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), followed by a second crossover process to obtain a strain with one copy of the Pcj7-ppsA(C.gl) gene inserted between the transposon genes on the chromosome. This strain was identified by PCR and sequencing using primers of SEQ ID NO: 19 and SEQ ID NO: 20, which can amplify the upstream and downstream regions of the gene insertion site, respectively.

[0174] The strain obtained in the above manner was designated CM05-9157::Pcj7-ppsA(C.gl).

[0175] Example 3. Evaluation of L-tryptophan productivity of L-tryptophan-producing microorganisms into which an exogenous pyruvate phosphate dikinase gene or its ortholog has been introduced To confirm the L-tryptophan-producing ability of the 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) strains prepared in Examples 2-2, 2-3, 2-4, and 2-5, respectively, the parent strain CM05-9157 without an exogenous gene introduced therein, and the CM05-9157::Pcj7-ppsA(C.gl) strain prepared in Example 1-6, they were cultured as follows.

[0176] Each strain was inoculated into a 250 ml corner-baffled flask containing 25 ml of seed medium and cultured with shaking at 200 rpm at 30°C for 20 hours. Then, 1 ml of the seed culture was inoculated into a 250 ml corner-baffled flask containing 25 ml of production medium and cultured with shaking at 200 rpm at 30°C for 24 hours. After culture was completed, the amount of L-tryptophan produced was measured by HPLC. The compositions of the seed medium and production medium were as follows, and the L-tryptophan concentrations in the culture media for each strain tested are shown in Table 14 below.

[0177] <Seed medium (pH 7.0)> Dextrose 20g, peptone 10g, yeast extract 5g, urea 1.5g, KH2PO4 4g, K2HPO4 8g, MgSO4 7H2O 0.5g, biotin 100μg, thiamine HCl 1000μg, calcium pantothenate 2000μg, nicotinamide 2000μg (based on 1 liter of distilled water).

[0178] <Production medium (pH 7.0)> Glucose 30g, (NH4)2SO4 15g, MgSO47H2O 1.2g, KH2PO4 1g, yeast extract 5g, biotin 900μg, thiamine hydrochloride 4500μg, calcium pantothenate 4500μg, CaCO3 30g (based on 1 liter of distilled water).

[0179] [Table 14]

[0180] As a result, as shown in Table 14, the tryptophan production of the CM05-9157::Pcj7-ppsA(C.gl) strain into which the phosphoenolpyruvate synthase gene derived from Corynebacterium glutamicum was introduced was 1.89 g / L, which was confirmed to be almost the same as the tryptophan production of the parent strain, CM05-9157.

[0181] Furthermore, among the pyruvate phosphate dikinase genes or their orthologous phosphoenolpyruvate synthase genes derived from various microorganisms, only the CM05-9157::Pcj7-ppdK (K.xy) strain, which had been introduced with the pyruvate phosphate dikinase gene derived from Komagataibacter xylinus, produced a final L-tryptophan level of 2.35 g / L in flask culture, demonstrating an approximately 18% increase in L-tryptophan production compared to the parent strain CM05-9157 and / or CM05-9157::Pcj7-ppsA (C.gl) strain.

[0182] In contrast, three strains (CM05-9157::Pcj7-ppdK(A.pa), CM05-9157::Pcj7-ppdK(M.ac), CM05-9157::Pcj7-ppsA(E.co)) into which exogenous phosphoenolpyruvate synthase or pyruvate phosphate dikinase other than those derived from Komagataibacter xylinus had been introduced showed a slight decrease in tryptophan production or a relatively small increase compared to the control group, CM05-9157.

[0183] These results indicate that the L-tryptophan production ability is specifically increased only when the pyruvate phosphate dikinase gene from the specific Komagataebacter xylinus is introduced into Corynebacterium spp.

[0184] From the above description, those skilled in the art to which the present application pertains will understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present application should be interpreted as including within the meaning and scope of the claims below, and any modifications or variations derived from the equivalent concepts thereof, rather than the above detailed description.

Claims

1. A Corynebacterium microorganism capable of producing L-tryptophan, into which a pyruvate phosphate dikinase protein derived from Komagataebacter xylinus or a polynucleotide encoding the same has been introduced.

2. The Corynebacterium microorganism according to claim 1, wherein the pyruvate phosphate dikinase derived from Komagataebacter xylinus comprises an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having 90% or more sequence identity thereto.

3. The Corynebacterium microorganism according to claim 1, wherein the pyruvate phosphate dikinase derived from Komagataebacter xylinus is encoded by the ppdK gene.

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

5. The Corynebacterium microorganism according to any one of claims 1 to 4, wherein the Corynebacterium microorganism has an increased ability to produce L-tryptophan compared to a non-transformed microorganism.

6. A method for producing L-tryptophan, comprising the step of culturing in a medium an L-tryptophan-producing Corynebacterium microorganism into which a pyruvate phosphate dikinase protein derived from Komagataibacter xylinus or a polynucleotide encoding the same has been introduced.

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

8. A Corynebacterium microorganism capable of producing L-tryptophan, into which a pyruvate phosphate dikinase protein derived from Komagataebacter xylinus or a polynucleotide encoding the same has been introduced; or a composition for producing L-tryptophan, comprising a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more of these.

9. A Corynebacterium microorganism capable of producing L-tryptophan, into which a pyruvate phosphate dikinase protein derived from Komagataebacter xylinus or a polynucleotide encoding the same has been introduced; and use of a composition comprising a culture of the microorganism, a fermentation product of the microorganism, or a combination of two or more of these, for the production of L-tryptophan.