Microorganisms into which exogenous water-soluble pyridine nucleotide transhydrogenase has been introduced and method for producing L-tryptophan using the same

Abstract

The present application relates to a microorganism of the genus Escherichia into which an exogenous water-soluble pyridine nucleotide transhydrogenase has been introduced, and a method for producing L-tryptophan using the same.

Description

[Technical Field]

[0001] The present application relates to a microorganism of the genus Escherichia into which an exogenous water-soluble pyridine nucleotide transhydrogenase has been introduced, and a method for producing L-tryptophan using the same. [Background technology]

[0002] Various studies have been conducted to develop highly efficient microorganisms and fermentation process technologies for the production of L-amino acids and other useful substances. For example, in the production of L-tryptophan, target-specific approaches have been mainly used, such as increasing the expression of genes encoding enzymes involved in L-tryptophan biosynthesis or deleting genes unnecessary for biosynthesis (US 8945907 B2).

[0003] However, due to the increasing demand for L-tryptophan, research is still needed to effectively increase L-tryptophan production capacity. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US 8945907 B2 [Patent Document 2] US 10995378 B2 [Patent Document 3] US 2023-0134555 A1 [Patent Document 4] US Patent US 7662943 B2 [Patent Document 5] US Patent US 10584338 B2 [Patent Document 6] US Patent US 10273491 B2 [Non-patent literature]

[0005] [Non-Patent Document 1] Pearson et al (1988)[Proc. Natl. Acad. Sci. USA 85]:2444

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Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

[0006] The present inventors have completed the present application by confirming that when a foreign udhA protein is introduced into a microorganism, the L-tryptophan-producing ability is increased compared to that of an untransformed microorganism. [Means for solving the problem]

[0007] The present application provides a microorganism of the genus Escherichia into which a foreign udhA protein or a polynucleotide encoding the same has been introduced.

[0008] The present application provides a method for producing L-tryptophan, which comprises culturing in a medium a microorganism of the genus Escherichia into which a foreign udhA protein or a polynucleotide encoding the same has been introduced.

[0009] The present application provides a composition for producing L-tryptophan, comprising an Escherichia microorganism into which a foreign udhA protein or a polynucleotide encoding the same has been introduced; a medium in which the microorganism has been cultured; or a combination thereof. [Effects of the Invention]

[0010] By introducing a foreign udhA protein into the microorganism of the present application, the L-tryptophan-producing ability can be increased compared to existing non-transformed microorganisms. DETAILED DESCRIPTION OF THE INVENTION

[0011] 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, throughout this specification, numerous papers and patent documents are referenced and citations thereof are displayed. The disclosure contents 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.

[0012] One aspect of the present application provides a microorganism of the genus Escherichia having the ability to produce L-tryptophan, into which a foreign udhA protein or a polynucleotide encoding the same has been introduced.

[0013] In this application, the term "soluble pyridine nucleotide transhydrogenase (udhA)" refers to an enzyme that + + NADPH ⇔ NADH + NADP + The term "water-soluble pyridine nucleotide transhydrogenase" may be used interchangeably with "udhA protein," "udhA," etc.

[0014] The amino acid sequence of the udhA protein can be obtained from a publicly known database such as NCBI's Genbank.

[0015] For example, the udhA protein of the present application may be derived from a microorganism, specifically, a microorganism selected from the group consisting of Pseudomonas and Erwinia, and more specifically, a microorganism selected from Pseudomonas aeruginosa and Erwinia amylovora.

[0016] As another example, the amino acid sequence of the udhA protein of the present application may be WP_003091177.1 derived from Pseudomonas aeruginosa and WP_004154881.1 derived from Erwinia amylovora.

[0017] In the present application, the udhA protein may have, comprise, consist of, or essentially consist of the amino acid sequence of SEQ ID NO: 1 or 3.

[0018] In the present application, the udhA protein may comprise the amino acid sequence of SEQ ID NO: 1 or 3, or an amino acid sequence having at least 70% or more, 75% or more, 76% or more, 85% or more, 86% or more, 87% or more, 87.5% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 92.5% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 97.5% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, or 99.9% or more homology or identity thereto. It is clear that proteins having such homology or identity and in which a portion of the amino acid sequence has been deleted, modified, substituted, conservatively substituted, or added are also within the scope of the present application, so long as the amino acid sequence has the same efficacy as the protein comprising the amino acid sequence of SEQ ID NO: 1 or 3.

[0019] For example, the amino acid sequence may have additions or deletions, naturally occurring mutations, silent mutations, or conservative substitutions at the N-terminus, C-terminus, and / or internally that do not alter the function of the protein of the present application.

[0020] The term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0021] As used herein, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid or nucleotide sequences, which can be expressed as a percentage. The terms homology and identity can often be used interchangeably.

[0022] Homology or identity of conserved polynucleotide or polypeptide sequences can be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences are generally capable of hybridizing to all or a portion of the sequence under moderately or highly stringent conditions. Hybridization obviously includes hybridization to polynucleotides containing common codons in polynucleotides or codons that take codon degeneracy into account.

[0023] 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, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), as implemented in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later), as implemented in the GCG program package (Devereux, J., et al., Nucleic Acids Research 12:387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] [ET AL, J MOLEC BIOL 215]:403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.] Academic Press, San Diego, 1994, and [CARILLO ET AL.] (1988) SIAM J Applied Math 48:1073. For example, BLAST or ClustalW from the National Database Center for Biotechnology Information can be used to determine homology, similarity or identity.

[0024] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that of Needleman et al. (1970), J Mol Biol. 48:443, as known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines a match as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a unitary matrix (containing a value of 1 for identity and 0 for non-identity) and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0025] The udhA protein of the present application may be encoded by the udhA gene.

[0026] For example, the udhA gene may be, but is not limited to, WP_003091177.1 from Pseudomonas aeruginosa or WP_004154881.1 from Erwinia amylovora. For example, the udhA gene may be, but is not limited to, NZ_CP034244.1 from Pseudomonas aeruginosa or NC_013961.1 from Erwinia amylovora. It is clear that the udhA gene may be, but is not limited to, udhA genes from various sources that encode proteins having water-soluble pyridine nucleotide transhydrogenase activity.

[0027] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and refers to a DNA or RNA chain of a certain length or more, and more specifically refers to a polynucleotide fragment that encodes the protein.

[0028] A polynucleotide encoding the udhA protein of the present application may comprise a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or 3. As an example of the present application, the polynucleotide of the present application may have or comprise the nucleotide sequence of SEQ ID NO: 2 or 4. Furthermore, the polynucleotide of the present application may consist of or essentially consist of the nucleotide sequence of SEQ ID NO: 2 or 4. Specifically, the udhA gene may be encoded by a polynucleotide described by the nucleotide sequence of SEQ ID NO: 2 or 4.

[0029] The polynucleotide of the present application may be modified in various ways in the coding region without changing the amino acid sequence of the water-soluble pyridine nucleotide transhydrogenase, taking into consideration codon degeneracy or codons preferred in the organism in which the water-soluble pyridine nucleotide transhydrogenase of the present application is to be expressed. Specifically, the polynucleotide of the present application may have or comprise a nucleotide sequence that is 70% or more, 75% or more, 76% 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 any one of SEQ ID NOs: 2 or 4, or may consist of or essentially consist of a nucleotide sequence that is 70% or more, 75% or more, 76% 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 sequence of SEQ ID NOs: 2 or 4, but is not limited thereto.

[0030] Furthermore, the polynucleotides of the present application may include, without limitation, any sequences that can hybridize under stringent conditions to probes prepared from known gene sequences, for example, sequences complementary to all or part of the polynucleotide sequences of 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 J. Sambrook et al., "Molecular Cloning, A Laboratory Manual," 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., "Current Protocols in Molecular Biology," John Wiley & Sons, Inc., New York, pp. 9.50-9.51, 11.7-11.8). For example, conditions include conditions under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 70% or more, 75% or more, 76% 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 conditions under which washing is performed once, specifically 2 to 3 times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization, such as 60°C, 1X SSC, and 0.1% SDS, specifically 60°C, 0.1X SSC, and 0.1% SDS, more specifically 68°C, 0.1X SSC, and 0.1% SDS.

[0031] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to one another. For example, for DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can include isolated nucleic acid fragments that are complementary to entire sequences, as well as substantially similar nucleic acid sequences.

[0032] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the above-mentioned hybridization conditions, including a hybridization step 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.

[0033] 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).

[0034] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially, and may be microorganisms in which a specific mechanism has been enhanced or weakened by inserting an exogenous gene or by enhancing or weakening the activity of an endogenous gene, and may also be microorganisms that contain genetic modifications for the production of a desired polypeptide, protein, or product.

[0035] Therefore, examples of the microorganism of the present application include a recombinant microorganism into which an exogenous udhA protein having increased water-soluble pyridine nucleotide transhydrogenase activity compared to a wild-type Escherichia microorganism has been introduced and / or a recombinant microorganism having genetic modifications that enhance the activity of the udhA protein compared to the endogenous activity.

[0036] The microorganism of the present application may have increased udhA protein activity compared to endogenous activity. For example, the microorganism may be one whose L-tryptophan-producing ability is improved by introducing the activity of an exogenous udhA protein. The microorganism may have enhanced udhA protein activity by introducing the activity of an exogenous udhA protein that is not endogenously expressed.

[0037] The enhanced udhA protein activity means that the udhA protein exhibits the activity of a foreign udhA protein that is not originally present because a udhA protein that is not endogenously expressed has been introduced, or the enhanced udhA protein activity may be increased compared to the endogenous udhA protein.

[0038] For example, the microorganism of the present application may have enhanced water-soluble pyridine nucleotide transhydrogenase activity in the microorganism by introducing an exogenous udhA protein having increased water-soluble pyridine nucleotide transhydrogenase activity compared to a wild-type Escherichia microorganism, but is not limited thereto.

[0039] As used herein, the term "enhancement" of a polypeptide (including, for example, the proteins specified in the names of each enzyme) means that the activity of the polypeptide is increased compared to its endogenous activity. The term "enhancement" may be used interchangeably with terms such as "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and increase can all encompass the display of an activity not inherently possessed, or the display of an activity that is improved compared to the endogenous activity or the activity before modification. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or an unaltered microorganism before the trait has been altered due to genetic mutation caused by natural or artificial factors. This term may be used interchangeably with "activity before modification." The term "enhancement," "up-regulation," "overexpression," or "increase" of a polypeptide compared to its endogenous activity means that the activity and / or concentration (expression level) of a specific polypeptide is improved compared to the activity and / or concentration (expression level) of a specific polypeptide inherently possessed by a parent strain or an unaltered microorganism before the trait has been altered.

[0040] The enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. The enhancement of the activity of the polypeptide can be confirmed by an increase in the activity, expression level, or amount of a product secreted from the polypeptide.

[0041] For the purposes of this application, the microorganism of this application has improved L-tryptophan production ability due to the introduction of an exogenous udhA protein having increased water-soluble pyridine nucleotide transhydrogenase activity compared to a wild-type Escherichia microorganism, thereby enhancing water-soluble pyridine nucleotide transhydrogenase activity within the microorganism. The unmodified microorganism into which the exogenous udhA protein has not been introduced, which is the subject strain for comparing the increase in L-tryptophan production ability or water-soluble pyridine nucleotide transhydrogenase activity, may be, but is not limited to, L-tryptophan-producing strains such as CA04-4303 (US 10995378 B2) and E. coli W3110, or strains into which one or more genetic modifications have been added to the above strains to enhance the L-tryptophan production pathway.

[0042] The activity of the polypeptide can be enhanced by various methods well known in the art, and is not limited thereto, as long as the activity of the polypeptide of interest can be enhanced compared to that of the microorganism before transformation. Specifically, the enhancement may be achieved by using genetic engineering and / or protein engineering, which are routine methods in molecular biology and well known to those skilled 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.).

[0043] Specifically, the activity of the polypeptide of the present application is enhanced by: 1) an increase in the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the expression regulatory region of a gene on a chromosome that encodes a polypeptide with a sequence with strong activity; 3) a modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the polypeptide activity is enhanced; 5) modifying a polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide's activity (e.g., modifying the polynucleotide sequence of the polypeptide gene so as to encode a polypeptide modified so as to enhance the polypeptide's activity); 6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same; 7) codon optimization of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide and selectively deforming or chemically modifying exposed sites; 9) Regulation of the intracellular localization of proteins (polypeptides); or 10) It may be a combination of two or more selected from the above 1) to 9), but is not particularly limited thereto.

[0044] More specifically, The 1) increase in the intracellular copy number of a polynucleotide encoding a polypeptide may be achieved by introducing into a host cell a vector operably linked to the polynucleotide encoding the polypeptide, which vector is capable of replicating and functioning independently of the host. Alternatively, the increase may be achieved by introducing one or more copies of the polynucleotide encoding the polypeptide into a chromosome in the host cell. The introduction into a chromosome can be achieved by, but is not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell. The vector is as described above.

[0045] The 2) replacement of the expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide with a sequence with stronger activity may be, for example, by introducing a mutation in the sequence through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or by 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 limiting.

[0046] Examples of known strong promoters include, but are not limited to, the PlysCm1 promoter (US 2023-0134555 A1), CJ1 to CJ7 promoters (US Patent No. 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US Patent No. 10584338 B2), O2 promoter (US Patent No. 10273491 B2), tkt promoter, and yccA promoter.

[0047] The modification of the nucleotide sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, but is not limited to, substituting a nucleotide sequence encoding another initiation codon that has a higher polypeptide expression rate than the endogenous initiation codon.

[0048] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) above may be, but is not limited to, a sequence mutation such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, or an amino acid sequence or polynucleotide sequence modified to have stronger activity or to have increased activity. Specifically, the replacement can be achieved by, but is not limited to, inserting the polynucleotide into a chromosome by homologous recombination. The vector used in this case may further contain a selection marker to confirm whether or not the polynucleotide has been inserted into the chromosome.

[0049] The introduction of an exogenous polynucleotide that exhibits the activity of a polypeptide (6) may be the introduction of an exogenous polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide into a host cell. The exogenous polynucleotide is not limited in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be any known transformation method appropriately selected by those skilled in the art. The introduced polynucleotide is expressed in a host cell to produce a polypeptide, and its activity can be increased.

[0050] The codon optimization of the polynucleotide encoding the polypeptide (7) may be that of an endogenous polynucleotide that has been codon-optimized to increase transcription or translation in a host cell, or that of an exogenous polynucleotide that has been codon-optimized to optimize transcription or translation in a host cell.

[0051] 8) Analyzing the tertiary structure of a polypeptide and selecting and modifying or chemically modifying exposed sites may involve, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins based on 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.

[0052] The 9) intracellular localization of a protein (polypeptide) may be targeted to a specific organelle or specific intracellular space within a cell, for example, to the periplasm or cytoplasm through the addition or removal of a leader sequence that functions in targeting the protein (polypeptide), but is not limited thereto.

[0053] Such enhanced polypeptide activity may be, but is not limited to, an increase in the activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in a wild-type or untransformed microbial strain, or an increase in the amount of a product produced from a microorganism in which the activity of the polypeptide is enhanced.

[0054] As an example, the enhancement of the activity of the water-soluble pyridine nucleotide transhydrogenase of the present application may be achieved by introducing an exogenous polynucleotide that exhibits water-soluble pyridine nucleotide transhydrogenase activity, but is not limited thereto.

[0055] Modification of a portion or all of a polynucleotide in the microorganism of the present application can be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal insertion in the microorganism or genome editing using engineered nucleases (e.g., CRISPR-Cas9) and / or (b) treatment with light, such as ultraviolet light or radiation, and / or chemicals. Methods for modifying a portion or all of the gene include DNA recombination techniques. For example, a nucleotide sequence or vector containing a nucleotide sequence homologous to a target gene can be injected into the microorganism to cause homologous recombination, thereby deleting a portion or all of the gene. The injected nucleotide sequence or vector may contain, but is not limited to, a dominant selectable marker.

[0056] The vector of the present application may comprise a DNA construct comprising 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 expression of the polypeptide of interest in a suitable host. The expression control region may comprise a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating the termination of transcription and translation. After being transformed into a suitable host cell, the vector can replicate or function independently of the host genome, or it can be integrated into the genome itself.

[0057] 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, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0058] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome of a cell using 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 confirming the presence or absence of insertion into the chromosome may also be included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of insertion of the target nucleic acid molecule. 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.

[0059] As used herein, the term "transformation" refers to introducing a vector containing a polynucleotide encoding a polypeptide of interest into a host cell or microorganism, thereby enabling the expression of the polypeptide encoded by the polynucleotide in the host cell. A transformed polynucleotide may include any polynucleotide, whether it is located intrachromosomally or extrachromosomally, as long as it is expressible in the host cell. The polynucleotide may also include DNA and / or RNA encoding the polypeptide of interest. The polynucleotide may be introduced in any form that can be introduced and expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. The expression cassette typically includes 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 the host cell in its own form, operably linked to sequences necessary for expression in the host cell, but is not limited thereto.

[0060] In addition, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the target polypeptide of the present application.

[0061] In another example of the present application, the microorganism of the present application may be a microorganism capable of producing L-tryptophan.

[0062] The microorganism of the present application may be a microorganism with improved L-tryptophan production ability.

[0063] The microorganism of the present application may have enhanced water-soluble pyridine nucleotide transhydrogenase activity. Specifically, the microorganism of the present application may be, but is not limited to, a microorganism in which an exogenous udhA protein or the udhA gene encoding it has been enhanced; or a microorganism that has been genetically modified to further enhance the exogenous udhA protein or the udhA gene encoding it. The microorganism in which the exogenous udhA protein or the udhA gene encoding it has been enhanced may have increased water-soluble pyridine nucleotide transhydrogenase activity compared to a wild-type or unmodified Escherichia microorganism. For example, the microorganism of the present application may be a recombinant microorganism.

[0064] The microorganism of the present application may be, but is not limited to, a microorganism that naturally has the ability to produce L-tryptophan or a microorganism in which the ability to produce L-tryptophan has been increased or imparted by introducing an exogenous udhA protein or a polynucleotide encoding the same into a parent strain that does not have the ability to produce L-tryptophan.

[0065] For purposes of this application, the recombinant microorganism of this application may be, but is not limited to, a microorganism in which the exogenous udhA protein or a polynucleotide encoding it has been introduced into a natural wild-type microorganism or a microorganism that produces L-tryptophan and contains a protein having endogenous water-soluble pyridine nucleotide transhydrogenase activity or a polynucleotide encoding it, thereby increasing L-tryptophan production ability compared to the natural wild-type microorganism or a microorganism that produces L-tryptophan and contains a protein having endogenous water-soluble pyridine nucleotide transhydrogenase activity or a polynucleotide encoding it. For example, the natural wild-type microorganism or a microorganism that produces L-tryptophan and contains a protein having endogenous water-soluble pyridine nucleotide transhydrogenase activity or a polynucleotide encoding it may be, but is not limited to, a subject strain for comparing the presence or absence of an increase in L-tryptophan production ability or water-soluble pyridine nucleotide transhydrogenase activity, as described above.

[0066] For example, the recombinant strain with increased L-tryptophan production ability may be increased by about 1% or more, specifically, about 2.5% or more, about 5% or more, about 7.5% or more, about 10% or more, about 12.5% ​​or more, about 15% or more, about 17.5% or more, about 18% or more, about 20% or more, about 22.5% or more, about 25% or more, about 27.5% or more, about 30% or more, about 32.5% or more, about 35% or more, or about 37% or more (there is no particular upper limit, and it may be, for example, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 15% or less), compared to the L-tryptophan production ability of the 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 parent strain or unmodified microorganism. In another example, the microorganism with increased production ability may have an L-tryptophan production ability that is increased by about 1.01-fold or more, about 1.025-fold or more, about 1.05-fold or more, about 1.075-fold or more, about 1.1-fold or more, about 1.125-fold or more, about 1.15-fold or more, about 1.175-fold or more, about 1.18-fold or more, about 1.2-fold or more, about 1.225-fold or more, about 1.25-fold or more, about 1.275-fold or more, about 1.3-fold or more, about 1.325-fold or more, about 1.35-fold or more, or about 1.37-fold or more (there is no particular upper limit, and the increase may be, for example, about 10-fold or less, about 5-fold or less, about 3-fold or less, or about 2-fold or less) compared to the parent strain or unmodified microorganism before mutation, but is not limited thereto.

[0067] Such productivity can be evaluated by measuring the amount of the target product produced after culturing in a medium. The evaluation can be performed by measuring the amount of the target product produced using a suitable method known in the art. For example, high performance liquid chromatography (HPLC), gas chromatography (GC), gas chromatography-mass spectrometry (GC / MS), liquid chromatography-mass spectrometry (LC / MS), gel permeation chromatography (GPC), or a combination of these methods can be used. The amount of the target product produced can be measured using a suitable method known in the art.

[0068] In the present application, the term "unmodified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type or naturally occurring strain itself, or a strain before its traits are changed by genetic mutation due to natural or artificial factors. For example, the unmodified microorganism refers to a strain in which the water-soluble pyridine nucleotide transhydrogenase described herein or the polynucleotide encoding it has not been enhanced or has not been enhanced. The term "unmodified microorganism" may be used interchangeably with "strain before modification," "microorganism before modification," "non-mutated strain," "non-modified strain," "non-mutated microorganism," or "reference microorganism."

[0069] In yet another example of the present application, the microorganism of the present application is not particularly limited as long as it is capable of producing L-tryptophan, and may be a microorganism belonging to the genus Escherichia, for example, Escherichia coli.

[0070] As another example of the present application, the recombinant microorganism of the present application may be a microorganism in which the activity of a part of a protein in the L-tryptophan biosynthetic pathway is additionally enhanced or the activity of a part of a protein in the L-tryptophan degradation pathway is additionally attenuated, thereby enhancing the ability to produce L-tryptophan.

[0071] In one embodiment, the recombinant microorganism of the present application may be a microorganism in which the activity of a competing pathway gene, a directional pathway regulator of the L-tryptophan operon, an L-tryptophan influx gene, or an L-tryptophan influx and degradation gene is weakened or inactivated, and / or the activity of the L-tryptophan operon is overexpressed, in order to enhance the L-tryptophan biosynthetic pathway.

[0072] Another example of the present invention is a microorganism in which the activity of a tryptophan synthase regulatory gene (trpR) that suppresses the expression of an L-tryptophan biosynthesis gene (e.g., trpEDCBA) or the activity of the Mtr membrane protein that transports extracellular L-tryptophan into the cell is weakened or eliminated compared to the endogenous activity, but is not limited to these.

[0073] In the present application, the term "attenuation" of the activity of a polypeptide is a concept that encompasses a decrease in activity or no activity compared to the endogenous activity. The term "attenuation" may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0074] The term "attenuation" can also refer to cases where the activity of a polypeptide itself is reduced or eliminated compared to the activity of the polypeptide originally possessed by a microorganism due to, for example, a mutation in the polynucleotide encoding the polypeptide; where the overall polypeptide activity and / or concentration (expression level) in cells is lower than that of a wild-type strain due to, for example, inhibition of gene expression of the encoding polynucleotide or inhibition of translation into the polypeptide; where the polynucleotide is not expressed at all; and / or where the polypeptide activity is absent despite the expression of the polynucleotide. The term "endogenous activity" refers to the activity of a specific polypeptide originally possessed by a parent strain, wild-type, or unaltered microorganism before the trait has been altered due to genetic mutation caused by natural or artificial factors. This term may be used interchangeably with "activity before transformation." A polypeptide activity that is "attenuated, inactivated, deficient, reduced, down-regulated, decreased, or attenuated" compared to its endogenous activity means that the activity of the specific polypeptide is reduced compared to that originally possessed by a parent strain or unaltered microorganism before the trait has been altered.

[0075] The activity of such polypeptides can be attenuated by any method known in the art, but is not limited to these, and can be achieved by applying a variety of methods well known in the art (e.g., Nakashima N et al., Bacterial cellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793, Sambrook et al. Molecular Cloning 2012, etc.).

[0076] Specifically, the activity of the polypeptide of the present application can be attenuated by: 1) Deletion of all or part of a gene encoding a polypeptide; 2) modification of the expression control region (or expression control sequence) so that expression of the gene encoding the polypeptide is reduced; 3) modification of the amino acid sequence constituting the polypeptide (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence) so as to eliminate or attenuate the activity of the polypeptide; 4) modification of the gene sequence encoding the polypeptide so that the activity of the polypeptide is eliminated or attenuated (e.g., deletion / substitution / addition of one or more nucleic acid bases in the nucleic acid base sequence of the polypeptide gene so as to encode a polypeptide that has been altered so that the activity of the polypeptide is eliminated or attenuated); 5) A modification of the nucleotide sequence encoding the initiation codon, Shine-Dalgarno sequence, or 5'-UTR region of a gene transcript encoding a polypeptide; 6) introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure to which ribosomes cannot attach; 8) Addition of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE); 9) Regulation of the intracellular localization of proteins (polypeptides); or 10) It may be a combination of two or more selected from the above 1) to 9), but is not particularly limited thereto.

[0077] for example, The deletion of a part or all of the gene encoding the polypeptide (1) may be removal of the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, replacement with a polynucleotide lacking some nucleotides, or replacement with a marker gene.

[0078] The modification of the expression regulatory region (or expression regulatory sequence) in 2) above may be the generation of a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacement with a sequence having a weaker activity. The expression regulatory region includes, 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.

[0079] The modifications of the amino acid sequence or polynucleotide sequence in 3) and 4) above may be, but are not limited to, mutations in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, such as deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to attenuate the activity of the polypeptide, or replacement with an amino acid sequence or polynucleotide sequence that has been improved to have weaker activity or no activity. For example, but not limited to, introducing a mutation into a polynucleotide sequence to form a stop codon can inhibit or attenuate gene expression.

[0080] The 5) modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, a substitution with a nucleotide sequence encoding another start codon that has a lower polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0081] 6) The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the transcript of the gene encoding the polypeptide can be carried out by referring to, for example, the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0082] 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure that prevents ribosome attachment may disable or slow down mRNA translation.

[0083] Furthermore, 8) the addition of a promoter transcribed in the opposite direction to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE) may create an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide, thereby weakening the activity.

[0084] The 9) intracellular localization of a protein (polypeptide) may be targeted to a specific organelle or specific intracellular space within a cell, for example, to the periplasm or cytoplasm through the addition or removal of a leader sequence that functions in targeting the protein (polypeptide), but is not limited thereto.

[0085] Such attenuation of polypeptide activity may be, but is not limited to, a decrease in the activity or concentration of the corresponding polypeptide relative to the activity or concentration of the polypeptide expressed in a wild-type or untransformed microbial strain, or an increase in the amount of a product produced from a microorganism in which the activity of the polypeptide is attenuated.

[0086] Another aspect of the present application provides a method for producing L-tryptophan, comprising the step of culturing in a medium an L-tryptophan-producing Escherichia microorganism into which an exogenous udhA protein or a polynucleotide encoding the same has been introduced.

[0087] The method for producing L-tryptophan of the present application may include culturing in a medium a microorganism in which an exogenous udhA protein or the udhA gene encoding it has been enhanced; or a microorganism that has been genetically modified so that the exogenous udhA protein or the udhA gene encoding it has been further enhanced, and the microorganism is as described above.

[0088] The exogenous udhA protein may have increased water-soluble pyridine nucleotide transhydrogenase activity compared to a wild-type Escherichia microorganism, or the exogenous udhA protein may not be endogenously expressed in a wild-type Escherichia microorganism.

[0089] In the present application, the term "culturing" means growing the microorganism of the present application under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using appropriate media 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.

[0090] In the present application, the term "culture medium" refers to a substance containing a mixture of nutrients, primarily those required for culturing the microorganism of the present application, and provides nutrients such as water essential for survival and growth, as well as growth factors, etc. Specifically, the culture medium and other culture conditions used for culturing the microorganism of the present application can be any medium used for culturing ordinary microorganisms without any particular limitations. The microorganism of the present application can be cultured in an ordinary culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins, etc., under aerobic conditions while controlling the temperature, pH, etc.

[0091] In the present application, examples of the carbon source 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 can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugars) can be used. Various other carbon sources can also be used in appropriate amounts without limitation. These carbon sources can be used alone or in combination, and are not limited thereto.

[0092] 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 to these.

[0093] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or the 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 culture medium in a batch or continuous manner. However, they are not limited to these.

[0094] 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, and to maintain anaerobic and microaerobic states, no gas can be injected or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limited to these.

[0095] 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.

[0096] The L-tryptophan produced by the culture of the present application is either secreted into the medium or remains intracellularly.

[0097] 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 microorganism, or a combination thereof (in any order), for example, before the culturing step.

[0098] The method for producing L-tryptophan of the present application may further include a step of recovering L-tryptophan from the culture medium (the medium in which the culture was performed) or the cultured microorganism. The recovery step may be further included after the culturing step.

[0099] The recovery may involve collecting the target L-tryptophan using a suitable method known in the art based on 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), 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 to recover the target L-tryptophan from the medium or the microorganism using a suitable method known in the art.

[0100] The method for producing L-tryptophan of the present application may further include a purification step. The purification can be performed using a suitable 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.

[0101] In the method of the present application, the foreign udhA protein, polynucleotide, vector, microorganism, etc. are as described in the other aspects above.

[0102] Another aspect of the present application provides a composition for producing L-tryptophan, comprising: an Escherichia microorganism into which an exogenous udhA protein or a polynucleotide encoding the same has been introduced; a culture medium in which the microorganism has been cultured; or a combination thereof.

[0103] 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.

[0104] Another aspect of the present application provides a method for producing an L-tryptophan-producing microorganism of the genus Escherichia, comprising the step of introducing an exogenous udhA protein or a polynucleotide encoding the same.

[0105] The foreign udhA protein, the polynucleotide encoding it, and the microorganism are as described above.

[0106] The method can include transforming a microorganism to have increased water-soluble pyridine nucleotide transhydrogenase activity relative to its endogenous activity by introducing an exogenous udhA protein into the Escherichia microorganism.

[0107] Another aspect of the present application provides use of a microorganism of the genus Escherichia, into which a foreign udhA protein or a polynucleotide encoding the same has been introduced, for producing L-tryptophan.

[0108] The foreign udhA protein, the Escherichia microorganism, etc. are as described above in the other embodiments. [Example]

[0109] 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 performed by those of ordinary skill in the technical field of the present application or a similar technical field.

[0110] Example 1: Search and selection of a gene encoding soluble pyridine nucleotide transhydrogenase (udhA) The amino acid sequence of the udhA protein derived from Escherichia coli was used as a query sequence, and PSI-BLAST was performed based on the NCBI and Kegg databases to identify NAD + + NADPH ⇔ NADH + NADP + We identified candidate proteins that are expected to have the activity of catalyzing the above reaction, the genes encoding them, and the microorganisms that possess them. Among them, six candidate proteins were selected in consideration of the biosafety level applicable to amino acid production and the possibility of securing the produced amino acids, as shown in Table 1 below.

[0111] [Table 1]

[0112] Example 2: Construction of plasmids for gene insertion For the insertion of the gene into the E. coli chromosome, a plasmid was constructed using pSKH (US Pat. No. US 8945907 B2).

[0113] The genomic DNA of wild-type E. coli W3110 was extracted using the Qiagen Genomic-tip system. The resulting genomic DNA was used as a template to amplify the upstream and downstream regions of the E. coli udhA gene chromosome where homologous recombination occurs, using the PCR HL premix kit (BIONEER). 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.

[0114] In addition, to amplify the foreign udhA gene fragments shown in Table 1 for replacing the existing udhA gene derived from E. coli, PCR was performed using the udhA genes of P. aeruginosa, E. amylovora, A. mediterranea, Spongiibacter sp. IMCC21906, A. albispora, and A. vinelandii strains, which were synthesized using the gene synthesis service of Vinyx Co., Ltd., as templates, with the primer pairs shown in Table 2 below to obtain the udhA gene derived from P. aeruginosa (NZ_CP034244.1, SEQ ID NO: 2), the udhA gene derived from E. amylovora (NC_013961.1, SEQ ID NO: 4), the udhA gene derived from A. mediterranea (CP003917.1, SEQ ID NO: 6), the udhA gene derived from Spongiibacter sp. IMCC21906 (NZ_CP011477.1, SEQ ID NO: 8), and the udhA gene derived from A. The udhA gene from A. albisporus (CP015163.1, SEQ ID NO: 10) and the udhA gene from A. vinelandii (NZ_FPKM01000023.1, SEQ ID NO: 12) were amplified. 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.

[0115] The primer sequences used here are as shown in Table 2 below.

[0116] [Table 2-1] [Table 2-2]

[0117] The upstream and downstream fragments of the E. coli udhA gene, the foreign udhA gene fragment, and the EcoRV-digested chromosomal transformation vector pSKH were cloned using the Gibson assembly method (D.G. Gibson et al., NATURE METHODS, VOL. 6, NO. 5, MAY 2009, NEBuilder HiFi DNA Assembly Master Mix) to obtain recombinant plasmids. Cloning was performed by mixing the Gibson assembly reagent with each gene fragment in calculated molar amounts and storing at 50°C for 1 hour. The recombinant plasmids constructed above were named pSKH-udhA(P. ae), pSKH-udhA(E. am), pSKH-udhA(A. me), pSKH-udhA(S. po), pSKH-udhA(A. al), and pSKH-udhA(A. vi), respectively, based on the species of the udhA gene exchanged.

[0118] Example 3: Construction of Escherichia microorganisms into which a foreign udhA gene has been introduced The pSKH-udhA(P. ae), pSKH-udhA(E. am), pSKH-udhA(A. me), pSKH-udhA(S. po), pSKH-udhA(A. al), and pSKH-udhA(A. vi) plasmids constructed in Example 2 were transformed into the L-tryptophan-producing strain CA04-4303 (US 10995378 B2) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), followed by a second crossover process to obtain a strain in which the E. coli udhA gene was replaced on the chromosome with the foreign udhA gene. The genetic manipulation was confirmed by PCR using the primer pair of SEQ ID NOs: 39 and 40, which can amplify the upstream and downstream regions of the homologous recombination region where the gene was inserted, respectively, and by genome sequencing. The strains obtained above, in which the E. coli udhA gene had been replaced with the foreign udhA gene, were named CA04-4303::udhA(P. ae), CA04-4303::udhA(E. am), CA04-4303::udhA(A. me), CA04-4303::udhA(S. po), CA04-4303::udhA(A. al), and CA04-4303::udhA(A. vi), respectively.

[0119] The primer sequences used here are as shown in Table 3 below.

[0120] [Table 3]

[0121] Example 4: Confirmation of L-tryptophan production ability of Escherichia microorganisms into which a foreign udhA gene has been introduced The strains CA04-4303::udhA(P. ae), CA04-4303::udhA(E. am), CA04-4303::udhA(A. me), CA04-4303::udhA(S. po), CA04-4303::udhA(A. al), and CA04-4303::udhA(A. vi) into which the foreign udhA gene prepared in Example 3 had been introduced, and the parent strain CA04-4303 as a control, were cultured as follows to confirm their L-tryptophan producing ability.

[0122] Specifically, each strain was cultured overnight on LB solid medium in an incubator at 37°C, and one platinum loop was inoculated into 25 mL of the titer medium shown in Table 4 below, and then cultured in an incubator at 37°C and 200 rpm for 48 hours. After the culture was completed, the amount of L-tryptophan produced was measured by HPLC, and the results are shown in Table 5 below.

[0123] [Table 4]

[0124] [Table 5]

[0125] As shown in Table 5, among the six production strains into which an exogenous udhA gene was introduced, the CA04-4303::udhA (P. ae) strain, into which the P. aeruginosa-derived udhA gene was introduced, produced a final L-tryptophan level of 1.87 g / L in flask culture, a fermentation yield that was approximately 37% higher than that of the control CA04-4303 strain. The CA04-4303::udhA (E. am) strain, into which the E. amylovora-derived udhA gene was introduced, produced a final L-tryptophan level of 1.62 g / L in flask culture, a fermentation yield that was approximately 18% higher than that of the control CA04-4303 strain.

[0126] From these results, it was confirmed that among the six foreign udhA genes, the udhA genes derived from P. aeruginosa and E. amylovora specifically increased the L-tryptophan production ability in E. coli.

[0127] 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 microorganism of the genus Escherichia into which a foreign udhA protein or a polynucleotide encoding the same has been introduced.

2. The microorganism of claim 1, wherein the protein comprises the amino acid sequence of SEQ ID NO: 1 or 3 or an amino acid sequence having 90% or more sequence identity thereto.

3. 2. The microorganism according to claim 1, wherein the protein is derived from one or more microorganisms selected from the group consisting of Pseudomonas aeruginosa and Erwinia amylovora.

4. 2. The microorganism according to claim 1, wherein the microorganism of the genus Escherichia has increased L-tryptophan-producing ability compared to the microorganism of the genus Escherichia before transformation.

5. The microorganism according to claim 1 , wherein the Escherichia microorganism is Escherichia coli.

6. A method for producing L-tryptophan, which comprises the step of culturing in a medium a microorganism of the genus Escherichia into which a foreign udhA protein or a polynucleotide encoding the same has been introduced.

7. 7. The method of claim 6, wherein the protein is derived from one or more microorganisms selected from the group consisting of Pseudomonas aeruginosa and Erwinia amylovora.

8. A composition for producing L-tryptophan, comprising: a microorganism of the genus Escherichia into which a foreign udhA protein or a polynucleotide encoding the same has been introduced; a medium in which the microorganism has been cultured; or a combination thereof.

9. 9. The composition of claim 8, wherein the protein is derived from one or more microorganisms selected from the group consisting of Pseudomonas aeruginosa and Erwinia amylovora.

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