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 Corynebacterium microorganism 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 Corynebacterium microorganism 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 into how to effectively increase L-tryptophan production capacity is still needed. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US 8945907 B2 [Patent Document 2] US 2023-0134555 A1 [Patent Document 3] US Patent US 7662943 B2 [Patent Document 4] US Patent US 10584338 B2 [Patent Document 5] 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

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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 Corynebacterium capable of producing L-tryptophan, 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 the step of culturing in a medium an L-tryptophan-producing Corynebacterium microorganism into which an exogenous udhA protein or a polynucleotide encoding the same has been introduced.

[0009] The present application provides a composition for producing L-tryptophan, comprising a Corynebacterium microorganism capable of producing L-tryptophan 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. [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 Corynebacterium capable of producing 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 NAD + + 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, Erwinia, and Alteromonas, and more specifically, a microorganism selected from Pseudomonas aeruginosa, Erwinia amylovora, and Alteromonas mediterranea.

[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, WP_004154881.1 derived from Erwinia amylovora, and AFV86869.1 derived from Alteromonas mediterranea.

[0017] In the present application, the udhA protein may have, comprise, consist of, or essentially consist of any one amino acid sequence selected from SEQ ID NOs: 1, 3, and 5.

[0018] In the present application, the udhA protein may comprise the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5, 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. For example, the udhA protein of the present application may comprise any one or more selected from the group consisting of the amino acid sequence of any one of SEQ ID NOs: 1, 3, and 5, or an amino acid sequence having 87.5% or more homology or identity thereto. Furthermore, it is obvious that proteins having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted or added are also included within the scope of the present application, as long as the amino acid sequence has such homology or identity and exhibits efficacy corresponding to a protein containing any one of the amino acid sequences selected from SEQ ID NOs: 1, 3 and 5.

[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 may 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] By way of example, the udhA gene may be, but is not limited to, a polynucleotide encoding WP_003091177.1 from Pseudomonas aeruginosa, WP_004154881.1 from Erwinia amylovora, or AFV86869.1 from Alteromonas mediterranea. By way of another example, the udhA gene may be, but is not limited to, NZ_CP034244.1 from Pseudomonas aeruginosa, NC_013961.1 from Erwinia amylovora, or CP003917.1 from Alteromonas mediterranea. It is clear that the udhA gene includes 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 any one of the amino acid sequences selected from SEQ ID NOs: 1, 3, and 5. As an example of the present application, the polynucleotide of the present application may have or comprise any one of the nucleotide sequences selected from SEQ ID NOs: 2, 4, and 6. Furthermore, the polynucleotide of the present application may consist of or essentially consist of any one of the nucleotide sequences selected from SEQ ID NOs: 2, 4, and 6. Specifically, the udhA gene may be encoded by a polynucleotide described by any one of the nucleotide sequences selected from SEQ ID NOs: 2, 4, and 6.

[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, 4, and 6, 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 any one of SEQ ID NOs: 2, 4, and 6, 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 Corynebacterium 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 an activity of a foreign udhA protein that is not originally present due to the introduction of a udhA protein that is not endogenously expressed, or the enhanced udhA protein activity may be an activity that is 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 within the microorganism by introducing an exogenous udhA protein having increased water-soluble pyridine nucleotide transhydrogenase activity compared to wild-type Corynebacterium microorganisms, 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 may 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 activity 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 Corynebacterium microorganism, thereby enhancing water-soluble pyridine nucleotide transhydrogenase activity within the microorganism. The unmodified microorganism without the introduction of an exogenous udhA protein, 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 CM05-9157 (US 2023-0134555 A1) or Corynebacterium glutamicum ATCC13869, or strains of the above strains to which one or more genetic modifications have been added 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 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.

[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 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 the sequence with one 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.

[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 start codon or 5'-UTR region of the gene transcript encoding the polypeptide (3) may be, for example, but is not limited to, substituting a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start 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. Expression of the introduced polynucleotide in a host cell results in the production of 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 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.

[0052] Such enhanced polypeptide activity may be, but is not limited to, an increase in the activity or expression level 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 the polypeptide.

[0053] For example, the activity of the water-soluble pyridine nucleotide transhydrogenase of the present application may be enhanced by introducing an exogenous polynucleotide that exhibits water-soluble pyridine nucleotide transhydrogenase activity. As another example, the activity of the water-soluble pyridine nucleotide transhydrogenase of the present application may be enhanced by replacing the promoter of the gene encoding the water-soluble pyridine nucleotide transhydrogenase or by modifying the promoter. As another example, the activity may be enhanced by a combination of the above modifications. However, the present invention is not limited to this example.

[0054] 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 and / or chemicals, such as ultraviolet light and radiation. 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.

[0055] 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 may be integrated into the genome itself.

[0056] 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.

[0057] 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 the insertion into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of the 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.

[0058] The term "transformation" as used herein 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. The 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.

[0059] 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.

[0060] In another example of the present application, the microorganism of the present application may be a microorganism capable of producing L-tryptophan.

[0061] The microorganism of the present application may be a microorganism with improved L-tryptophan production ability.

[0062] 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 Corynebacterium microorganism. For example, the microorganism of the present application may be a recombinant microorganism.

[0063] 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.

[0064] 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.

[0065] For example, the recombinant strain with increased L-tryptophan production ability may be increased by about 1% or more, specifically, about 2% or more, about 3% or more, about 4% 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 11% or more, about 12% or more, about 13% or more, about 14% or more, about 15% or more, about 16% or more, about 17% or more, about 18% or more, about 19% or more, about 20% or more, about 21% or more, or about 22% 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 L-tryptophan production ability may be increased by about 1.01-fold or more, about 1.02-fold or more, about 1.03-fold or more, about 1.04-fold or more, about 1.05-fold or more, about 1.06-fold or more, about 1.07-fold or more, about 1.08-fold or more, about 1.09-fold or more, about 1.1-fold or more, about 1.12-fold or more, about 1.13-fold or more, about 1.14-fold or more, about 1.15-fold or more, about 1.16-fold or more, about 1.17-fold or more, about 1.18-fold or more, about 1.19-fold or more, about 1.2-fold or more, about 1.21-fold or more, or about 1.22-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.

[0066] 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 an appropriate 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 an appropriate method known in the art.

[0067] 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 due to genetic mutations caused by 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."

[0068] In yet another example of the present application, the microorganism of the present application is Corynebacterium glutamicum, Corynebacterium stationis, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium The bacterial strain may be Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium testudinoris, or Corynebacterium flavescens, and specifically may be Corynebacterium glutamicum, but is not limited thereto.

[0069] 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 further enhanced or the activity of a part of a protein in the L-tryptophan degradation pathway is further weakened, thereby enhancing the ability to produce L-tryptophan.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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 or translation of the encoding polynucleotide into a 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 the 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.

[0074] 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.).

[0075] 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 in order 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); or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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, a substitution with a nucleotide sequence encoding another initiation codon that has a lower polypeptide expression rate than the endogenous initiation codon.

[0080] 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].

[0081] 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.

[0082] 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.

[0083] Another aspect of the present application provides a method for producing L-tryptophan, comprising culturing in a medium an L-tryptophan-producing Corynebacterium microorganism into which an exogenous udhA protein or a polynucleotide encoding the same has been introduced.

[0084] 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.

[0085] The exogenous udhA protein may have increased water-soluble pyridine nucleotide transhydrogenase activity compared to a wild-type Corynebacterium microorganism, or the exogenous udhA protein may be one that is not endogenously expressed in a wild-type Corynebacterium microorganism.

[0086] 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 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.

[0087] 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 are not particularly limited as long as they are media used for culturing conventional microorganisms. The microorganism of the present application can be cultured in a conventional culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins under aerobic conditions by adjusting the temperature, pH, etc.

[0088] Specifically, 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)].

[0089] 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. Various other carbon sources may also be used in appropriate amounts without limitation. These carbon sources may be used alone or in combination, and are not limited thereto.

[0090] 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.

[0091] 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.

[0092] 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 an anaerobic or microaerobic state, no gas can be injected or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limited to these.

[0093] 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.

[0094] The L-tryptophan produced by the culture of the present application is either secreted into the medium or remains intracellularly.

[0095] 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.

[0096] 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 carried out) or the cultured microorganism. The recovery step may be further included after the culturing step.

[0097] The recovery may involve collecting the target L-tryptophan using an appropriate 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. The target L-tryptophan can be recovered from the medium or the microorganism using an appropriate method known in the art.

[0098] 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.

[0099] In the method of the present application, the foreign udhA protein, polynucleotide, vector, microorganism, etc. are as described in the other aspects above.

[0100] Another aspect of the present application provides a composition for producing L-tryptophan, comprising a Corynebacterium microorganism into which an exogenous udhA protein or a polynucleotide encoding the same has been introduced; a culture medium in which the same has been cultured; or a combination thereof.

[0101] 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.

[0102] Another aspect of the present application provides a method for producing an L-tryptophan-producing Corynebacterium microorganism, the method comprising the step of introducing an exogenous udhA protein or a polynucleotide encoding the same.

[0103] The foreign udhA protein, the polynucleotide encoding it, and the microorganism are as described above.

[0104] The method can include transforming a microorganism to have increased water-soluble pyridine nucleotide transhydrogenase activity compared to its endogenous activity by introducing an exogenous udhA protein into the Corynebacterium microorganism.

[0105] Another aspect of the present application provides use of a Corynebacterium microorganism, into which a foreign udhA protein or a polynucleotide encoding the same has been introduced, for producing L-tryptophan.

[0106] The foreign udhA protein, the Corynebacterium microorganism, etc. are as described above in the other embodiment. [Example]

[0107] 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.

[0108] 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.

[0109] [Table 1]

[0110] Example 2: Construction of plasmids for gene insertion To insert the gene into the Corynebacterium chromosome, a plasmid was constructed using pDCM2 (US 2023-0134555 A1) to delete the transposon site of wild-type Corynebacterium glutamicum and to insert the udhA gene.

[0111] Using wild-type Corynebacterium glutamicum ATCC13869 chromosomal DNA as a template, the upstream region where homologous recombination occurs on the chromosome was amplified using the primer pair of sequence numbers 13 and 14, and the downstream region was amplified using the primer pair of sequence numbers 15 and 16, and the respective gene fragments were obtained.

[0112] The polymerase is Solg TM PCR amplification 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.

[0113] The primer sequences used here are as shown in Table 2 below.

[0114] [Table 2]

[0115] The upstream and downstream fragments of the region where homologous recombination occurs on the chromosome obtained in this process and the chromosomal transformation vector pDCM2 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. Cloning was performed by mixing the Gibson assembly reagent with each gene fragment in the calculated molar amounts and storing at 50°C for 1 hour. The recombinant plasmid constructed in this manner was designated pDCM2-△Tn.

[0116] Example 3: Construction of Corynebacterium microorganisms carrying an exogenous udhA gene 3-1: Construction of microorganisms incorporating the udhA gene from Pseudomonas aeruginosa To insert the udhA gene from P. aeruginosa selected in Example 1 into the genomic DNA of Corynebacterium glutamicum, PCR was performed using the udhA gene of the P. aeruginosa strain synthesized using the gene synthesis service of Bionix Co., Ltd. as a template and the primer pair of SEQ ID NO: 17 and SEQ ID NO: 18 to amplify the udhA gene from P. aeruginosa (NZ_CP034244.1, SEQ ID NO: 2). 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.

[0117] In addition, to obtain the PlysCm1 promoter (US 2023-0134555 A1) for enhancing the activity of the udhA protein, PCR was performed using pDCM2-Tn::PlysCm1_pheA (US 2023-0134555 A1) as a template and the primer pair of SEQ ID NO: 19 and SEQ ID NO: 20. 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 30 seconds, repeated 27 times, followed by polymerization at 72°C for 5 minutes.

[0118] The amplified PlysCm1 promoter region, the P. aeruginosa udhA gene fragment, and pDCM2-△Tn (Example 2), digested with ScaI restriction enzyme, were cloned using the Gibson assembly method to obtain a recombinant plasmid. 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. The recombinant plasmid containing the P. aeruginosa udhA gene constructed as described above was designated pDCM2-△Tn::PlysCm1-udhA(P. aeruginosa).

[0119] The constructed pDCM2-△Tn::PlysCm1-udhA(P.ae) plasmid was transformed into the L-tryptophan-producing strain CM05-9157 (US 2023-0134555 A1) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545). A secondary crossover process was then carried out to obtain a strain with one copy of PlysCm1-udhA(A.ht) inserted between the transposon genes on the chromosome. The genetic manipulation was confirmed by PCR using the primer pair of SEQ ID NOs: 21 and 22, which amplify the upstream and downstream regions of the homologous recombination region where the gene was inserted, respectively, and by genome sequencing. The strain obtained above into which PlysCm1-udhA(P.ae) was introduced was designated CM05-9157△Tn::PlysCm1-udhA(P.ae).

[0120] 3-2: Construction of microorganisms carrying the Erwinia amylovora udhA gene To insert the udhA gene from E. amylovora selected in Example 1 into the genomic DNA of Corynebacterium glutamicum, PCR was performed using the primer pair of SEQ ID NO: 23 and SEQ ID NO: 24 as a template using the udhA gene of the E. amylovora strain synthesized using the gene synthesis service of Bionix Co., Ltd., in the same manner as in Example 3-1 to amplify the udhA gene from E. amylovora (NC_013961.1, SEQ ID NO: 4).

[0121] In addition, to obtain the PlysCm1 promoter for enhancing the activity of the udhA protein, PCR was performed using pDCM2-Tn::PlysCm1_pheA as a template and the primer pair of SEQ ID NO: 19 and SEQ ID NO: 25, as described in Example 3-1.

[0122] The amplified PlysCm1 promoter region, the E. amylovora udhA gene fragment, and pDCM2-△Tn (Example 2), digested with ScaI restriction enzyme, were cloned using the Gibson assembly method to obtain a recombinant plasmid. 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. The recombinant plasmid containing the E. amylovora udhA gene constructed above was designated pDCM2-△Tn::PlysCm1-udhA(E.am).

[0123] The constructed pDCM2-△Tn::PlysCm1-udhA(E.am) plasmid was transformed into the tryptophan-producing strain CM05-9157 by electroporation, followed by a second crossover process to obtain a strain with one copy of PlysCm1-udhA(E.am) inserted between the transposon genes on the chromosome. The relevant genetic manipulation was confirmed through PCR using the primer pair of SEQ ID NOs: 21 and 22, which amplify the upstream and downstream regions of the homologous recombination region where the gene was inserted, respectively, and genome sequencing. The resulting strain with PlysCm1-udhA(E.am) inserted was designated CM05-9157△Tn::PlysCm1-udhA(E.am).

[0124] 3-3: Construction of microorganisms incorporating the udhA gene from Alteromonas mediterranea To insert the udhA gene derived from A. mediterranea selected in Example 1 into the genomic DNA of Corynebacterium glutamicum, PCR was performed using the udhA gene of the A. mediterranea strain synthesized using the gene synthesis service of Bionix Co., Ltd. as a template and the primer pair of SEQ ID NO: 26 and SEQ ID NO: 27 in the same manner as in Example 3-1 to amplify the udhA gene derived from A. mediterranea (CP003917.1, SEQ ID NO: 6).

[0125] In addition, to obtain the PlysCm1 promoter for enhancing the activity of the udhA protein, PCR was performed using pDCM2-Tn::PlysCm1_pheA as a template and the primer pair of SEQ ID NO: 19 and SEQ ID NO: 28, as described in Example 3-1.

[0126] The amplified PlysCm1 promoter region, the A. mediterranea udhA gene fragment, and pDCM2-△Tn (Example 2), digested with ScaI restriction enzyme, were cloned using the Gibson assembly method to obtain a recombinant plasmid. 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. The recombinant plasmid containing the A. mediterranea udhA gene constructed as described above was designated pDCM2-△Tn::PlysCm1-udhA(A.me).

[0127] The constructed pDCM2-△Tn::PlysCm1-udhA(A.me) plasmid was transformed into the tryptophan-producing strain CM05-9157 by electroporation, followed by a second crossover process to obtain a strain with one copy of PlysCm1-udhA(A.me) inserted between the transposon genes on the chromosome. The genetic manipulation was confirmed through PCR using the primer pair of SEQ ID NOs: 21 and 22, which amplify the upstream and downstream regions of the homologous recombination region where the gene was inserted, respectively, and genome sequencing. The resulting strain with PlysCm1-udhA(A.me) inserted was designated CM05-9157△Tn::PlysCm1-udhA(A.me).

[0128] 3-4: Construction of microorganisms containing the udhA gene from Spongiibacter sp. IMCC21906 To insert the udhA gene derived from Spongiibacter sp. IMCC21906 selected in Example 1 into the genomic DNA of Corynebacterium glutamicum, PCR was performed using the udhA gene of Spongiibacter sp. IMCC21906 strain synthesized using the gene synthesis service of Bionics Co., Ltd. as a template and the primer pair of SEQ ID NO: 29 and SEQ ID NO: 30 in the manner described in Example 3-1 to amplify the udhA gene derived from Spongiibacter sp. IMCC21906 (NZ_CP011477.1, SEQ ID NO: 8).

[0129] In addition, to obtain the PlysCm1 promoter for enhancing the activity of the udhA protein, PCR was performed using pDCM2-Tn::PlysCm1_pheA as a template and the primer pair of SEQ ID NO: 19 and SEQ ID NO: 31, as described in Example 3-1.

[0130] The amplified PlysCm1 promoter region, the udhA gene fragment from Spongiibacter sp. IMCC21906, and pDCM2-△Tn (Example 2), digested with ScaI restriction enzyme, were cloned using the Gibson assembly method to obtain a recombinant plasmid. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated molar amounts and storing the mixture at 50°C for 1 hour. The recombinant plasmid containing the udhA gene from Spongiibacter sp. IMCC21906 was designated pDCM2-△Tn::PlysCm1-udhA(S.po).

[0131] The constructed pDCM2-△Tn::PlysCm1-udhA(S.po) plasmid was transformed into the tryptophan-producing strain CM05-9157 by electroporation, followed by a second crossover process to obtain a strain with one copy of PlysCm1-udhA(S.po) inserted between the transposon genes on the chromosome. The genetic manipulation was confirmed through PCR using the primer pair of SEQ ID NOs: 21 and 22, which amplify the upstream and downstream regions of the homologous recombination region where the gene was inserted, respectively, and genome sequencing. The resulting strain with PlysCm1-udhA(S.po) inserted was designated CM05-9157△Tn::PlysCm1-udhA(S.po).

[0132] 3-5: Construction of microorganisms incorporating the udhA gene from Amycolatopsis albispora To insert the udhA gene from A. albispora selected in Example 1 into the genomic DNA of Corynebacterium glutamicum, PCR was performed using the udhA gene of an A. albispora strain synthesized using the gene synthesis service of Bionix Co., Ltd. as a template and the primer pair of SEQ ID NO: 32 and SEQ ID NO: 33 in the same manner as in Example 3-1 to amplify the udhA gene from A. albispora (CP015163.1, SEQ ID NO: 10).

[0133] In addition, to obtain the PlysCm1 promoter for enhancing the activity of the udhA protein, PCR was performed using pDCM2-Tn::PlysCm1_pheA as a template and the primer pair of SEQ ID NO: 19 and SEQ ID NO: 34, as described in Example 3-1.

[0134] The amplified PlysCm1 promoter region, the A. albispora udhA gene fragment, and pDCM2-△Tn (Example 2), digested with ScaI restriction enzyme, were cloned using the Gibson assembly method to obtain a recombinant plasmid. 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. The recombinant plasmid containing the A. albispora udhA gene constructed above was designated pDCM2-△Tn::PlysCm1-udhA(A. al).

[0135] The constructed pDCM2-△Tn::PlysCm1-udhA(A. al) plasmid was transformed into the tryptophan-producing strain CM05-9157 by electroporation, followed by a second crossover process to obtain a strain with one copy of PlysCm1-udhA(A. al) inserted between the transposon genes on the chromosome. The genetic manipulation was confirmed through PCR using the primer pair of SEQ ID NOs: 21 and 22, which amplify the upstream and downstream regions of the homologous recombination region where the gene was inserted, respectively, and genome sequencing. The resulting strain with PlysCm1-udhA(A. al) inserted was designated CM05-9157△Tn::PlysCm1-udhA(A. al).

[0136] 3-6: Construction of microorganisms carrying the udhA gene from Azotobacter vinelandii To insert the udhA gene from A. vinelandii selected in Example 1 into the genomic DNA of Corynebacterium glutamicum, PCR was performed using the udhA gene of the A. vinelandii strain synthesized using the gene synthesis service of Bionix Co., Ltd. as a template and the primer pair of SEQ ID NOs: 35 and 36 in the manner described in Example 3-1 to amplify the udhA gene from A. vinelandii (NZ_FPKM01000023.1, SEQ ID NO: 12).

[0137] In addition, to obtain the PlysCm1 promoter for enhancing the activity of the udhA protein, PCR was performed using pDCM2-Tn::PlysCm1_pheA as a template and the primer pair of SEQ ID NO: 19 and SEQ ID NO: 37, as described in Example 3-1.

[0138] The amplified PlysCm1 promoter region, the A. vinelandii udhA gene fragment, and pDCM2-△Tn (Example 2), digested with ScaI restriction enzyme, were cloned using the Gibson assembly method to obtain a recombinant plasmid. Cloning was performed by mixing the Gibson assembly reagent and each gene fragment in the calculated molar amounts and storing the mixture at 50°C for 1 hour. The recombinant plasmid containing the A. vinelandii udhA gene constructed above was designated pDCM2-△Tn::PlysCm1-udhA(A.vi).

[0139] The constructed pDCM2-△Tn::PlysCm1-udhA(A.vi) plasmid was transformed into the tryptophan-producing strain CM05-9157 by electroporation, followed by a second crossover process to obtain a strain with one copy of PlysCm1-udhA(A.vi) inserted between the transposon genes on the chromosome. The genetic manipulation was confirmed through PCR using the primer pair of SEQ ID NOs: 21 and 22, which amplify the upstream and downstream regions of the homologous recombination region where the gene was inserted, respectively, and genome sequencing. The resulting strain with PlysCm1-udhA(A.vi) inserted was designated CM05-9157△Tn::PlysCm1-udhA(A.vi).

[0140] The primer sequences used in Examples 3-1 to 3-6 are as shown in Table 3 below.

[0141] [Table 3]

[0142] Example 4: Confirmation of L-tryptophan production ability of Corynebacterium spp. microorganisms into which a foreign udhA gene has been introduced To confirm the L-tryptophan producing ability of the strains into which the foreign udhA gene was introduced prepared in Examples 3-1 to 3-6 and the parent strain CM05-9157 as a control, they were cultured as follows.

[0143] 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 to obtain a seed culture. 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 the culture was completed, the amount of L-tryptophan produced was measured by HPLC, and the results are shown in Table 4 below.

[0144] <Seed medium (pH 7.0)> Glucose 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, pH 7.0 (based on 1 liter of distilled water)

[0145] <Production medium (pH 7.0)> Glucose 30g, (NH4)2SO4 15g, MgSO4 7H2O 1.2g, KH2PO4 1g, yeast extract 5g, biotin 900μg, thiamine hydrochloride 4500μg, calcium pantothenate 4500μg, CaCO3 30g, pH 7.0 (based on 1 liter of distilled water).

[0146] [Table 4]

[0147] As shown in Table 4, of the six production strains into which the exogenous udhA gene was introduced, the CM05-9157△Tn::Plyscm1_udhA(P.ae) strain, which contained the P. aeruginosa udhA gene, produced a final L-tryptophan level of 2.33 g / L in flask culture, a fermentation yield that was approximately 22% higher than that of the control strain CM05-9157. The CM05-9157△Tn::Plyscm1_udhA(E.am) strain, which contained the E. amylovora udhA gene, produced a final L-tryptophan level of 2.17 g / L in flask culture, a fermentation yield that was approximately 14% higher than that of the control strain CM05-9157. Furthermore, the CM05-9157△Tn::Plyscm1_udhA(A.me) strain, into which the udhA gene from A. mediterranea had been introduced, produced a final L-tryptophan yield of 2.05 g / L in flask culture, a fermentation yield that was approximately 9% higher than that of the control strain, CM05-9157.

[0148] From these results, it was confirmed that among the six foreign udhA genes, the udhA genes from P. aeruginosa, E. amylovora, and A. mediterranea specifically increased the L-tryptophan production ability in Corynebacterium glutamicum strains.

[0149] 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 Corynebacterium having an ability to produce L-tryptophan, into which a foreign udhA protein or a polynucleotide encoding the same has been introduced.

2. The microorganism according to claim 1, wherein the protein comprises any one of the amino acid sequences selected from SEQ ID NOs: 1, 3 and 5, or an amino acid sequence having 87.5% 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, Erwinia amylovora, and Alteromonas mediterranea.

4. The microorganism according to claim 1, wherein the Corynebacterium genus microorganism has an increased ability to produce L-tryptophan compared to a wild-type Corynebacterium genus microorganism.

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

6. A method for producing L-tryptophan, comprising the step of culturing in a medium an L-tryptophan-producing Corynebacterium microorganism 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, Erwinia amylovora, and Alteromonas mediterranea.

8. A composition for producing L-tryptophan, comprising: a Corynebacterium microorganism capable of producing L-tryptophan, 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, Erwinia amylovora, and Alteromonas mediterranea.

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