Microorganism into which exogenous soluble pyridine nucleotide transhydrogenase is introduced, and method for producing L-tryptophan using same

Abstract

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

Description

TECHNICAL FIELD

[0001] The present disclosure relates to an Escherichia microorganism into which an exogenous soluble pyridine nucleotide transhydrogenase is introduced, and a method for producing L-tryptophan using the same. BACKGROUND

[0002] In order to produce L-amino acids and other beneficial substances, various studies have been conducted to develop microorganisms that produce efficiently and techniques for fermentation processes. For example, when producing L-tryptophan, a target-specific method such as a method of increasing expression of a gene encoding an enzyme involved in L-tryptophan biosynthesis or a method of removing a gene unnecessary for biosynthesis is mainly used (US 8945907 B2).

[0003] However, as the demand for L-tryptophan increases, there is still a need for research to effectively increase L-tryptophan production capacity. SUMMARY

[0004] TECHNICAL PROBLEM

[0005] The present inventors have confirmed that when an exogenous udhA protein is introduced into a microorganism, the L-tryptophan production capacity of the microorganism is increased compared to that of an unmodified microorganism, thereby completing the present disclosure.

[0006] TECHNICAL SOLUTION

[0007] An object of the present disclosure is to provide an Escherichia microorganism into which an exogenous udhA protein or a polynucleotide encoding the same is introduced.

[0008] Another object of the present disclosure is to provide a method for producing L-tryptophan, comprising culturing an Escherichia microorganism into which an exogenous udhA protein or a polynucleotide encoding the same is introduced in a culture medium.

[0009] Another object of the present disclosure is to provide a composition for producing L-tryptophan, comprising an Escherichia microorganism into which an exogenous udhA protein or a polynucleotide encoding the same is introduced; a culture medium in which the microorganism is cultured; or a combination thereof.

[0010] ADVANTAGEOUS EFFECTS

[0011] By introducing an exogenous udhA protein, the microorganism of the present disclosure can have increased L-tryptophan production capacity compared to existing unmodified microorganisms. DETAILED DESCRIPTION

[0012] The present disclosure will be described in detail below. Also, each description and embodiment disclosed herein can be applied to other descriptions and embodiments in terms of common features. That is, all combinations of various elements disclosed herein fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific descriptions described below. In addition, many papers and patent documents are referred to and cited throughout the specification. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety, and will more clearly describe the level of the technical field to which the present disclosure belongs and the contents of the present disclosure.

[0013] One aspect of the present disclosure provides an Escherichia microorganism into which an exogenous udhA protein or a polynucleotide encoding the same is introduced.

[0014] As used herein, the term "soluble pyridine nucleotide transhydrogenase (udhA)" refers to a protein having an activity of catalyzing the reaction of NADPH + NADP+ → NADP + NADH. + + The "soluble pyridine nucleotide transhydrogenase" can be used interchangeably with "udhA protein", "udhA", and the like.

[0015] The amino acid sequence of the udhA protein can be obtained from known databases such as GenBank of NCBI, etc.

[0016] In one example, the udhA protein of the present disclosure can be derived from a microorganism. The protein can be derived from a microorganism selected from the genus Pseudomonas and the genus Erwinia. More specifically, it can be derived from a microorganism selected from Pseudomonas aeruginosa and Erwinia amylovora.

[0017] In another example, the amino acid sequence of the udhA protein of the present disclosure can be WP_003091177.1 derived from Pseudomonas aeruginosa and WP_004154881.1 derived from Erwinia amylovora, etc.

[0018] In the present disclosure, the udhA protein can have, include, or consist of the amino acid sequence of SEQ ID NO: 1 or 3, or can essentially consist of the above-described amino acid sequence.

[0019] In the present disclosure, the udhA protein can include the amino acid sequence of SEQ ID NO: 1 or 3, or an amino acid sequence having 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. Also, it is obvious that any protein having an amino acid sequence in which part of the sequence is deleted, modified, substituted, conservatively substituted, or added can also fall within the scope of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits the efficacy corresponding to the protein including the amino acid sequence of SEQ ID NO: 1 or 3.

[0020] For example, it can be a sequence addition, a naturally occurring mutation, a silent mutation or a conservative substitution within the N-terminus, the C-terminus, and / or the amino acid sequence that does not change the function of the protein of the present disclosure.

[0021] As used herein, the term "conservative substitution" refers to the substitution of one amino acid for another amino acid having similar structure and / or chemical properties. Such amino acid substitution can typically occur based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues. Generally, conservative substitutions have little or no effect on the activity of the protein or polypeptide.

[0022] As used herein, the term "homology" or "identity" refers to the degree of relatedness between two given amino acid sequences or nucleotide sequences and can be expressed in percentage. The terms homology and identity can generally be used interchangeably with each other.

[0023] The sequence homology or identity of a conservative polynucleotide or polypeptide can be determined by standard alignment algorithms, and the default gap penalties established by the program used can be used together. Essentially, homologous or identical sequences are generally expected to hybridize under moderately or highly stringent conditions to all or a portion of the sequence. It is obvious that the hybridization of polynucleotides containing universal codons or degenerate codons in the hybridizing polynucleotides is also included.

[0024] Whether any two polynucleotide or polypeptide sequences are substantially identical, similar, or identical can be determined by known computer algorithms, such as the "FASTA" program, using the default parameters in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) can be used, which is implemented in the Needleman program in 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 (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, homology, similarity, or identity can be determined by using the National Center for Biotechnology Information's BLAST or ClustalW.

[0025] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, the GAP computer program, as disclosed in Needleman et al. (1970), J Mol Biol. 48:443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482. In sum, the GAP program defines homology, similarity or identity as a number that is obtained by dividing the number of similar aligned symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program can include: (1) a binary comparison matrix (comprising a value of 1 for identical day and a value of 0 for non-identical day), and the weighted comparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745 (or the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix), as disclosed in Atlas Of Protein Sequence And Structure, edited by Schwartz and Dayhoff, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty score of 3.0 for each gap, and an extra penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10, and a gap extension penalty of 0.5); and (3) no penalty for end gaps.

[0026] The udhA protein of the present disclosure can be encoded by the udhA gene.

[0027] In one example, the udhA gene can be a polynucleotide encoding WP_003091177.1 derived from Pseudomonas aeruginosa or WP_004154881.1 derived from Erwinia amylovora, but is not limited thereto. In another example, the udhA gene can be NZ_CP034244.1 derived from Pseudomonas aeruginosa or NC_013961.1 derived from Erwinia amylovora, but is not limited thereto, and it is obvious that the udhA gene can include udhA genes of various origins encoding a protein having soluble pyridine nucleotide transhydrogenase activity.

[0028] As used herein, the term "polynucleotide" is a nucleotide polymer consisting of nucleotide monomers linked by covalent bonds into a long chain, which is a DNA or RNA chain having at least a certain length. More specifically, it can refer to a polynucleotide fragment encoding a protein.

[0029] The polynucleotide encoding the udhA protein of the present disclosure can include a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or 3. In one example of the present disclosure, the polynucleotide of the present disclosure can have or include the nucleotide sequence of SEQ ID NO: 2 or 4. Also, the polynucleotide of the present disclosure can consist of or consist essentially of the nucleotide sequence of SEQ ID NO: 2 or 4. Specifically, the udhA gene can be encoded by a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 2 or 4.

[0030] The polynucleotide of the present disclosure can be variously modified in the coding region within the range that does not change the amino acid sequence of the soluble pyridine nucleotide transhydrogenase, due to codon degeneracy or in consideration of preferred codons in an organism in which the soluble pyridine nucleotide transhydrogenase is to be expressed. Specifically, the polynucleotide of the present disclosure can have or include a nucleotide sequence having 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 homology or identity to any one of SEQ ID NO: 2 or 4, or can consist of or consist essentially of a nucleotide sequence having 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 homology or identity to any one of SEQ ID NO: 2 or 4, but is not limited thereto.

[0031] Furthermore, the polynucleotide of the present disclosure can include a probe that can be prepared from a known gene sequence, for example, any polynucleotide sequence that can hybridize to the complement of all or a part of the polynucleotide sequence of the present disclosure under stringent conditions without limitation. "Stringent conditions" refer to conditions that enable specific hybridization between polynucleotides. Such conditions are disclosed in detail in the literature (see J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., 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, 9.50-9.51, 11.7-11.8). For example, stringent conditions can include conditions in which polynucleotides having high homology or identity, i.e., polynucleotides having 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 homology or identity, hybridize to each other, and polynucleotides having less homology or identity than the above do not hybridize to each other; or can include washing conditions of ordinary Southern hybridization, i.e., one, in particular 2 or 3, washing at a salt concentration and temperature corresponding to 60°C, 1xSSC, 0.1% SDS, specifically 60°C, 0.1xSSC, 0.1% SDS, more specifically 68°C, 0.1xSSC, 0.1% SSD.

[0032] Hybridization requires that the two nucleic acids have complementary sequences, although 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 each other. For example, for DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the present disclosure can also include isolated nucleic acid fragments that are complementary to the entire sequence and nucleic acid sequences substantially similar thereto.

[0033] Specifically, a hybridization condition including a hybridization step of T m The hybridization condition of T m The value can be 60°C, 63°C, or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art according to their purposes.

[0034] The appropriate stringency for hybridization polynucleotides depends on several variables and is well known in the art (e.g., J. Sambrook et al., supra).

[0035] As used herein, the term "microorganism (or strain)" includes all wild-type microorganisms or naturally or artificially genetically modified microorganisms, and it can be a microorganism in which a specific mechanism is weakened or enhanced due to insertion of an exogenous gene, or enhancement or weakening of endogenous gene activity, etc., and can be a genetically modified microorganism including production of a desired polypeptide, protein, or product.

[0036] Thus, in one example of the microorganism of the present disclosure, it can be a recombinant microorganism into which an exogenous udhA protein is introduced, which has increased soluble pyridine nucleotide transhydrogenase activity compared to a wild-type Escherichia microorganism, and / or a recombinant microorganism having a genetic modification that enhances the activity of the udhA protein compared to its endogenous activity.

[0037] The microorganism of the present disclosure can have increased activity compared to the endogenous activity of the udhA protein. In one example, the microorganism can be a microorganism having increased L-tryptophan production capacity by introducing exogenous udhA protein activity. By introducing exogenous udhA protein activity expressed non-endogenously, the microorganism can have enhanced udhA protein activity.

[0038] Enhanced udhA protein activity can mean that exogenous udhA protein activity that the microorganism does not originally have is exhibited because a udhA protein expressed non-endogenously is introduced.

[0039] For example, by introducing an exogenous udhA protein having increased soluble pyridine nucleotide transhydrogenase activity compared to a wild-type Escherichia microorganism, the microorganism of the present disclosure can have enhanced soluble pyridine nucleotide transhydrogenase activity within the microorganism, but is not limited thereto.

[0040] As used herein, the term "enhancement" of a polypeptide (e.g., a protein designated by the name of each enzyme) refers to an increase in the activity of the polypeptide compared to its endogenous activity. Enhancement can be used interchangeably with terms such as activation, upregulation, overexpression, increase, etc. In particular, activation, enhancement, upregulation, overexpression, and increase can include exhibiting an activity that was not originally present, or an activity that is enhanced compared to the endogenous activity or the activity before modification. "Endogenous activity" refers to the activity of a particular polypeptide that a parent strain before transformation or an unmodified microorganism originally has when the trait is changed by genetic modification caused by natural or artificial factors, and can be used interchangeably with "activity before modification." "Enhancement," "upregulation," "overexpression," or "increase" of the activity of a polypeptide compared to its endogenous activity refers to an increase in the activity and / or concentration (expression level) of the polypeptide compared to the particular polypeptide that the parent strain before transformation or the unmodified microorganism originally has.

[0041] Enhancement can be achieved by introducing an exogenous polypeptide or by enhancing the activity and / or concentration (expression level) of an endogenous polypeptide. Enhancement of the activity of a polypeptide can be confirmed by an increase in the activity level, expression level, or amount of a product secreted by the polypeptide.

[0042] For the purposes of the present disclosure, the microorganism of the present disclosure has enhanced soluble pyridine nucleotide transhydrogenase activity within the microorganism by introducing an exogenous udhA protein having increased soluble pyridine nucleotide transhydrogenase activity compared to a wild-type Escherichia microorganism and an unmodified microorganism into which an exogenous udhA protein has not been introduced, which is a target strain for increasing L-tryptophan production ability or soluble pyridine nucleotide transhydrogenase activity, can be an L-tryptophan production strain CA04-4303 strain (US 10995378B2), E. coli W3110 strain, or a strain in which one or more genetic modifications are added to the above-mentioned strains to enhance the L-tryptophan production pathway, but is not limited thereto.

[0043] Enhancement of the activity of a polypeptide can be achieved by various methods well known in the art, provided that it can enhance the activity of the target polypeptide compared to the microorganism before modification, and the method is not limited. Specifically, genetic engineering and / or protein engineering well known to those skilled in the art can be used, which are conventional methods of molecular biology, but the method is not limited thereto (e.g., Sitnicka et al., Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16; Sambrook et al., Molecular Cloning 2012; etc.).

[0044] Specifically, enhancement of the activity of the polypeptide of the present disclosure can be achieved by:

[0045] 1) increasing the number of copies of a polynucleotide encoding a polypeptide in a cell;

[0046] 2) replacing an expression regulatory region of a gene encoding a polypeptide on a chromosome with a sequence having stronger activity;

[0047] 3) modifying a nucleotide sequence of a coding initiation codon or a 5'-UTR of a transcript of a gene encoding a polypeptide;

[0048] 4) modifying an amino acid sequence of a polypeptide so that the activity of the polypeptide is enhanced;

[0049] 5) modifying a polynucleotide sequence encoding a polypeptide so that the activity of the polypeptide is enhanced (for example, modifying a polynucleotide sequence of a polypeptide gene to encode a polypeptide that has been modified to enhance the activity of the polypeptide);

[0050] 6) introducing an exogenous polypeptide exhibiting the activity of a polypeptide or an exogenous polynucleotide encoding the polypeptide;

[0051] 7) codon optimization of a polynucleotide encoding a polypeptide;

[0052] 8) analyzing the tertiary structure of a polypeptide and selecting and modifying or chemically modifying exposed sites therefrom;

[0053] 9) regulating the cellular localization of a protein (polypeptide); or

[0054] 10) a combination of two or more selected from the above 1) to 9), but not particularly limited thereto.

[0055] More specifically,

[0056] 1) A method of increasing the number of copies of a polynucleotide encoding a polypeptide in a cell can be achieved by introducing a vector into a host cell, the vector being operably linked to a polynucleotide encoding a polypeptide and being capable of replication and functioning independently of the host cell. Alternatively, the method can be achieved by introducing one or two copies of a polynucleotide encoding a polypeptide into the chromosome of a host cell. Introduction into the chromosome can be performed by introducing a vector capable of inserting a polynucleotide into the chromosome of a host cell into a host cell, but is not limited thereto. The vector is as described above.

[0057] 2) The method of replacing the expression regulatory region (or expression regulatory sequence) of the gene encoding the polypeptide on the chromosome with a sequence having strong activity can be further enhanced by introducing a modification in the sequence by, for example, deletion, insertion, non-conservative substitution, or conservative substitution, or a combination thereof, to enhance the activity of the expression regulatory region, or by replacing the sequence with a sequence having stronger activity. The expression regulatory region can include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, a sequence regulating transcription and translation termination, etc. In one example, the method can include replacing the original promoter with a strong promoter, but is not limited thereto.

[0058] Examples of known strong promoters can include the PlysCm1 promoter (US 2023-0134555 A1), the CJ1 to CJ7 promoters (US 7662943 B2), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (US 10584338 B2), the O2 promoter (US 10273491 B2), the tkt promoter, the yccA promoter, etc., but the strong promoter is not limited thereto.

[0059] 3) The method of modifying the nucleotide sequence encoding the start codon of the transcript of the gene encoding the polypeptide or the 5'-UTR can be achieved by, for example, replacing the nucleotide sequence with a nucleotide sequence encoding another start codon having a higher polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0060] 4) and 5) The method of modifying the amino acid sequence or the polynucleotide sequence can be achieved by inducing a modification in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, or by replacing the sequence with a modified amino acid sequence or polynucleotide sequence having stronger activity, or an amino acid sequence or polynucleotide sequence modified to enhance activity, but is not limited thereto. Specifically, the replacement can be performed by inserting the polynucleotide into the chromosome by homologous recombination, but is not limited thereto. The vector used herein can further include a selection marker to confirm insertion into the chromosome.

[0061] 6) The method of introducing an exogenous polynucleotide exhibiting the activity of the polypeptide can be achieved by introducing an exogenous polynucleotide encoding a polypeptide exhibiting the same / similar activity as the polypeptide into a host cell. The exogenous polynucleotide can be used without limitation regardless of its origin or sequence, as long as it exhibits the same / similar activity as the polypeptide. The introduction can be performed by a transformation method known in the art, appropriately selected by one of ordinary skill in the art, and the expression of the introduced polynucleotide in the host cell can produce the polypeptide, thereby increasing its activity.

[0062] 7) The method of codon optimization of a polynucleotide encoding a polypeptide can be achieved by codon optimization of an endogenous polynucleotide to increase transcription or translation in a host cell, or by optimization of codons so that optimized transcription and translation of an exogenous polynucleotide can be achieved in a host cell.

[0063] 8) The method of analyzing the tertiary structure of a polypeptide and thereby selecting and modifying or chemically modifying exposed sites can be achieved by, for example, comparing sequence information of the polypeptide to be analyzed with a database storing known protein sequence information to determine template protein candidates according to the degree of sequence similarity, and thereby confirming the structure based on the information, so as to select and transform or modify the exposed sites to be modified or chemically modified.

[0064] 9) The method of regulating the cellular localization of a protein (polypeptide) can be achieved by targeting the protein (polypeptide) to a specific intracellular organelle or a specific intracellular space, for example, by adding or removing a leader sequence that plays a role in the targeting of the protein (polypeptide) to target the protein (polypeptide) to the periplasm or cytoplasm, but is not limited thereto.

[0065] Such enhancement of the activity of the polypeptide can mean 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 strain or a microorganism before modification, or an increase in the amount of a product produced by a microorganism in which the activity of the polypeptide is increased, but is not limited thereto.

[0066] In one example, the enhancement of the activity of the soluble pyridine nucleotide transhydrogenase of the present disclosure can be enhanced by introducing an exogenous polynucleotide exhibiting the activity of the soluble pyridine nucleotide transhydrogenase, but is not limited thereto.

[0067] The partial or total modification of the polynucleotide in the microorganism of the present disclosure can be achieved by (a) homologous recombination using a vector for chromosomal insertion in the microorganism or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) can be induced by light, such as ultraviolet rays and irradiation, etc., and / or chemical treatment, but is not limited thereto. The method of modifying part or all of the gene can include a method using a DNA recombination technique. For example, by injecting a nucleotide sequence or a vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination, part or all of the gene can be deleted. The injected nucleotide sequence or vector can include a dominant selection marker, but is not limited thereto.

[0068] The vector of the present disclosure can include a DNA construct containing a nucleotide sequence of a polynucleotide encoding a target polypeptide, which is operably linked to a suitable expression regulatory region (expression regulatory sequence) so that the target polypeptide can be expressed in a suitable host cell. The expression regulatory region can include a promoter capable of initiating transcription, any operator sequence regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating transcription and translation termination. Once transformed into a suitable host cell, the vector can replicate independently of the host genome or function, or can be integrated into its genome.

[0069] The vector used in the present disclosure is not particularly limited, and any vector known in the art can be used. Examples of the commonly used vector can include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A, etc. can be used as bacteriophage vectors or cosmid vectors; those based on pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET, etc. can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0070] In one example, the polynucleotide encoding target polypeptide can be inserted into the chromosome by the vector for chromosome insertion in the cell. The polynucleotide being inserted into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. The carrier can also include a selective marker to confirm that it is inserted into the chromosome. The selective marker is used to select the cell transformed by the carrier, that is, for confirming whether the target nucleic acid molecule has been inserted, and the marker providing a selectable phenotype (such as drug resistance, auxotrophy, cytotoxic agent resistance or surface polypeptide expression) can be used. Only the cell expressing the selective marker can survive or show different phenotypes under the environment of processing with a selection agent, so the cell transformed can be selected.

[0071] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. As long as the transformed polynucleotide can be expressed in the host cell, it does not matter whether the transformed polynucleotide is integrated into and located in the host cell's chromosome or located extrachromosomally, and both situations are included. In addition, the polynucleotide may include DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form, as long as it can be introduced into the host cell and expressed therein. 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 the elements required for its autonomous expression. The expression cassette may generally contain a promoter, a transcription terminator, a ribosome binding site, or a translation terminator operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. In addition, the polynucleotide may be introduced into the host cell as is and operably linked to sequences required for expression in the host cell, but is not limited thereto.

[0072] Furthermore, as used herein, the term "operably linked" means that a polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the polypeptide of interest of the present disclosure.

[0073] In another example of the present disclosure, the microorganism of the present disclosure may be a microorganism having the ability to produce L-tryptophan.

[0074] The microorganism of the present disclosure may be a microorganism having increased L-tryptophan-producing ability.

[0075] The microorganism of the present disclosure can be a microorganism having enhanced soluble pyridine nucleotide transhydrogenase activity. Specifically, the microorganism of the present disclosure can be a microorganism in which an exogenous udhA protein or a udhA gene encoding the same is enhanced; or a microorganism genetically modified to further enhance the exogenous udhA protein or the udhA gene encoding the same, but is not limited thereto. The microorganism in which the exogenous udhA protein or the udhA gene encoding the same is enhanced can have increased soluble pyridine nucleotide transhydrogenase activity compared to a wild-type or unmodified Escherichia microorganism. For example, the microorganism of the present disclosure can be a recombinant microorganism.

[0076] The microorganism of the present disclosure can be a microorganism in which L-tryptophan production ability is increased or conferred by introducing an exogenous udhA protein or a polynucleotide encoding the same into a microorganism naturally having L-tryptophan production ability or a parent strain not having L-tryptophan production ability, but is not limited thereto.

[0077] For the purpose of the present disclosure, the recombinant microorganism of the present disclosure can be a microorganism having increased L-tryptophan production ability compared to a naturally wild-type microorganism or a microorganism producing L-tryptophan (which comprises a protein having endogenous soluble pyridine nucleotide transhydrogenase activity or a polynucleotide encoding the same) by introducing an exogenous udhA protein or a polynucleotide encoding the same into the naturally wild-type microorganism or the microorganism producing L-tryptophan (which comprises a protein having endogenous soluble pyridine nucleotide transhydrogenase activity or a polynucleotide encoding the same), but is not limited thereto. In one example, the naturally wild-type microorganism or the microorganism producing L-tryptophan (which comprises a protein having endogenous soluble pyridine nucleotide transhydrogenase activity or a polynucleotide encoding the same) can be a target strain for comparing L-tryptophan production ability or soluble pyridine nucleotide transhydrogenase activity increase, but is not limited thereto.

[0078] In one example, the recombinant strain having increased production capacity can have an L-tryptophan production capacity that is 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 (not particularly limited as long as the upper limit is, 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 capacity of the parent strain before modification or the unmodified microorganism, but not limited thereto, as long as the increase has a + value compared to the production capacity of the parent strain before modification or the unmodified microorganism. In another example, the microorganism having increased production capacity can have an L-tryptophan production capacity 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 (not particularly limited as long as the upper limit is, 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 L-tryptophan production capacity of the parent strain before modification or the unmodified microorganism, but not limited thereto.

[0079] The production capacity can be evaluated by measuring the yield of the desired product obtained by culturing in a culture medium. The yield of the desired product can be measured using a suitable method known in the art to evaluate. For example, HPLC (high performance liquid chromatography), GC (gas chromatography), GC / MS (gas chromatography-mass spectrometry), LC / MS (liquid chromatography-mass spectrometry), GPC (gel permeation chromatography), or a combination thereof can be used, and the yield of the desired product can be measured using a suitable method known in the art.

[0080] As used herein, the term "unmodified microorganism" does not exclude a strain containing mutations that can occur naturally in the microorganism, and can refer to a wild-type strain or a natural-type strain as it is, or a strain before its properties are changed due to genetic modification by natural or artificial factors. For example, an unmodified microorganism can refer to a strain in which the soluble pyridine nucleotide transhydrogenase described herein or a polynucleotide encoding the same is not enhanced, or a strain before enhancement. The "unmodified microorganism" can be used interchangeably with "strain before modification", "microorganism before modification", "unmutated strain", "unmodified strain", "unmutated microorganism", or "reference microorganism".

[0081] In another example of the present disclosure, the microorganism of the present disclosure is not particularly limited in its type as long as it can produce L-tryptophan, but it can be a microorganism belonging to the genus Escherichia. In one example, it can be Escherichia coli.

[0082] In another example of the present disclosure, the recombinant microorganism of the present disclosure can be a microorganism having enhanced L-tryptophan production capacity by further enhancing the activity of some proteins in the L-tryptophan biosynthesis pathway or by further attenuating the activity of some proteins in the L-tryptophan degradation pathway.

[0083] In one embodiment, the recombinant microorganism of the present disclosure can be a microorganism in which the activity of genes in the competing pathway, a regulator in the L-tryptophan operon directed pathway, genes for importing L-tryptophan, or genes for importing and decomposing L-tryptophan is attenuated or inactivated, thereby enhancing the L-tryptophan biosynthesis pathway; and / or can be a microorganism in which the L-tryptophan operon activity is overexpressed.

[0084] In another embodiment, the recombinant microorganism of the present disclosure can be a microorganism in which the activity of trpR (a gene for regulating a group of enzymes for tryptophan synthesis, which inhibits the expression of L-tryptophan biosynthesis genes (trpEDCBA)) or Mtr (i.e., a membrane protein) that imports extracellular L-tryptophan into the cell can be attenuated or removed compared to its endogenous activity, but is not limited thereto.

[0085] As used herein, the term "attenuation" of polypeptide activity is a comprehensive concept including reduced activity or no activity compared to its endogenous activity. Attenuation can be used interchangeably with terms such as inactivation, lack, down-regulation, reduction, decrease, weakening, etc.

[0086] Attenuation can also include cases where the polypeptide activity itself is reduced or eliminated compared to the polypeptide activity that the microorganism originally has due to mutation of the polynucleotide encoding the polypeptide, cases where the overall level of polypeptide activity and / or concentration (expression level) in the cell is reduced compared to the natural strain due to inhibition of gene expression of the polynucleotide encoding the polypeptide or inhibition of translation into the polypeptide, etc., cases where the polynucleotide is not expressed at all, and / or cases where no polypeptide activity is observed even when the polynucleotide is expressed. As used herein, the term "endogenous activity" refers to the activity of a specific polypeptide that the parent strain before transformation, wild type, or unmodified microorganism originally has when the trait is changed by genetic modification caused by natural or artificial factors, and can be used interchangeably with "activity before modification." The expression that the polypeptide activity is "attenuated, inactivated, deficient, reduced, down-regulated, decreased, or weakened" compared to its endogenous activity means that the polypeptide activity is reduced compared to the activity of the specific polypeptide that the parent strain before transformation or unmodified microorganism originally has.

[0087] Attenuation of the polypeptide activity can be performed by any method known in the art, but the method is not limited thereto, and can be achieved by applying various 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.).

[0088] In particular, attenuation of the polypeptide activity of the present disclosure can be achieved by:

[0089] 1) deletion of part or all of the gene encoding the polypeptide;

[0090] 2) modification of the expression regulatory region (expression regulatory sequence) so that the expression of the gene encoding the polypeptide is reduced;

[0091] 3) modification of the amino acid sequence constituting the polypeptide so that the polypeptide activity is eliminated or attenuated (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence);

[0092] 4) modification of the gene sequence encoding the polypeptide so that the polypeptide activity is eliminated or attenuated (e.g., deletion / substitution / addition of one or more nucleotides in the nucleotide sequence of the polypeptide gene to encode a modified polypeptide to eliminate or attenuate the activity of the polypeptide);

[0093] 5) modification of the nucleotide sequence of the coding start codon, Shine-Dalgarno (SD) sequence, or 5'-UTR of the transcript of the gene encoding the polypeptide;

[0094] 6) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to a transcript of a gene encoding a polypeptide;

[0095] 7) adding a sequence complementary to a Shine-Dalgarno (SD) sequence of a gene encoding a polypeptide to the front of the SD sequence to form a secondary structure, thereby inhibiting ribosome attachment;

[0096] 8) reverse transcription engineering (RTE) that adds a promoter to be reverse-transcribed to the 3' end of an open reading frame (ORF) of a gene sequence encoding a polypeptide;

[0097] 9) regulating the cellular localization of a protein (polypeptide); or

[0098] 10) a combination selected from two or more of the above-described methods 1) to 9), but is not particularly limited thereto.

[0099] For example,

[0100] 1) The method of deleting part or all of a gene encoding a polypeptide can be achieved by deleting all of the polynucleotides encoding an endogenous target polypeptide within a chromosome, or by replacing the polynucleotides with polynucleotides in which nucleotides are partially deleted or with marker genes.

[0101] 2) The method of modifying an expression regulatory region (expression regulatory sequence) can be achieved by inducing modification of the expression regulatory region (expression regulatory sequence) by deletion, insertion, non-conservative substitution, or conservative substitution, or a combination thereof; or by replacing the sequence with a sequence having weaker activity. The expression regulatory region can include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating transcription and translation termination, but is not limited thereto.

[0102] 3) and 4) The method of modifying an amino acid sequence or a polynucleotide sequence can be achieved by inducing modification of the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof of the amino acid sequence of a polypeptide or the polynucleotide sequence encoding the polypeptide to weaken the activity of the polypeptide, or by replacing the sequence with an amino acid sequence or a polynucleotide sequence modified to have weaker activity or an amino acid sequence or a polynucleotide sequence modified to have no activity, but is not limited thereto. For example, the expression of a gene can be inhibited or weakened by introducing a mutation in the polynucleotide sequence to form a stop codon, but is not limited thereto.

[0103] 5) The method of modifying the nucleotide sequence of the coding start codon or the 5'-UTR of the transcript of the gene encoding the polypeptide can be achieved by, for example, replacing the nucleotide sequence with a nucleotide sequence encoding another start codon having a lower polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0104] 6) The method of introducing an antisense oligonucleotide (e.g., antisense RNA) complementary to the transcript of the gene encoding the polypeptide can be found in the literature (Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews-Trends in Genetics, Vol. 1(1) 1986).

[0105] 7) The method of adding a sequence complementary to the Shine-Dalgarno (SD) sequence of the gene encoding the polypeptide to the front of the SD sequence to form a secondary structure, thereby inhibiting ribosome attachment, can be achieved by inhibiting mRNA translation or reducing its speed.

[0106] In addition, 8) reverse transcription engineering (RTE), which adds a promoter to be reverse-transcribed to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide, can be achieved by forming an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide to weaken activity.

[0107] 9) The method of regulating the cellular localization of a protein (polypeptide) can be achieved by targeting the protein (polypeptide) to a specific intracellular organelle or a specific intracellular space, for example, by adding or removing a leader sequence that plays a role in the targeting of the protein (polypeptide), targeting the protein (polypeptide) to the periplasm or cytoplasm, but is not limited thereto.

[0108] Such weakening of the activity of the polypeptide can mean that the activity or concentration of the corresponding polypeptide is reduced relative to the activity or concentration of the polypeptide expressed in the wild-type strain or the microorganism before modification, or that the amount of a product produced by the microorganism in which the activity of the polypeptide is increased is reduced, but is not limited thereto.

[0109] Another aspect of the present disclosure provides a method of producing L-tryptophan, comprising: culturing an Escherichia microorganism for producing L-tryptophan in a culture medium, the microorganism into which an exogenous udhA protein or a polynucleotide encoding the same has been introduced.

[0110] The method of producing L-tryptophan of the present disclosure can include culturing a microorganism in which an exogenous udhA protein or a udhA gene encoding the same is enhanced; or a microorganism genetically modified to further enhance an exogenous udhA protein or a udhA gene encoding the same, as described above.

[0111] The exogenous udhA protein can have increased soluble pyridine nucleotide transhydrogenase activity compared to a wild-type Escherichia microorganism. In addition, the exogenous udhA protein can not be endogenously expressed in a wild-type Escherichia microorganism.

[0112] As used herein, the term "culturing" refers to growing the microorganism of the present disclosure under suitably controlled environmental conditions. The culturing process of the present disclosure can be performed under suitable culture media and culture conditions known in the art. Such a culturing process can be easily adjusted for use by those skilled in the art depending on the microorganism to be selected. Specifically, the culturing can be batch culture, continuous culture, and / or fed-batch culture, but is not limited thereto.

[0113] As used herein, the term "culture medium" refers to a mixture of substances containing nutrients necessary for culturing the microorganism of the present disclosure as a main component, which provides nutrients and growth factors, as well as water necessary for survival and growth. Specifically, the culture medium and other culture conditions for culturing the microorganism of the present disclosure can be any culture medium used for conventional culturing of microorganisms, without any particular limitation. However, the microorganism of the present disclosure can be cultured in a conventional culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin under aerobic conditions, while adjusting the temperature, pH, etc.

[0114] In the present disclosure, the carbon source can include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, the carbon source can include natural organic nutrients such as starch hydrolysate, molasses, jaggery paste, rice bran, cassava, sugar cane molasses, and corn syrup, etc. Specifically, carbohydrates such as glucose and sterile pretreated molasses (i.e., molasses converted into reducing sugar) can be used, and in addition, an appropriate amount of various other carbon sources can be used without limitation. These carbon sources can be used alone or in combination of two or more, but are not limited thereto.

[0115] The nitrogen source can include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; amino acids such as glutamic acid, methionine, glutamine, etc.; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources can be used alone or in combination of two or more, but are not limited thereto.

[0116] The phosphorus source can include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a corresponding sodium-containing salt, etc. Examples of inorganic compounds can include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors can be included. These constituent ingredients or precursors can be added to the culture medium in a batch or continuous manner, but these phosphorus sources are not limited thereto.

[0117] In addition, in the process of culturing the microorganism of the present disclosure in an appropriate manner, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid, etc. In addition, an antifoaming agent such as a fatty acid polyethylene glycol ester can be used to prevent the formation of bubbles during the culturing process. In addition, oxygen or an oxygen-containing gas can be injected into the culture medium to maintain aerobic conditions of the culture medium; or nitrogen, hydrogen, carbon dioxide, or no gas can be injected to maintain anaerobic or microaerophilic conditions, but the gas is not limited thereto.

[0118] The temperature during the culturing of the present disclosure can be in the range of 20℃ to 45℃, specifically 25℃ to 40℃, and the culturing can be performed for about 10 to 160 hours, but the culturing is not limited thereto.

[0119] The L-tryptophan produced by the culturing of the present disclosure can be released into the culture medium or retained in the cells.

[0120] The method of producing L-tryptophan of the present disclosure can further include a step of preparing the microorganism of the present disclosure, a step of preparing a culture medium for culturing the microorganism, or a combination thereof (regardless of the order, in any order), for example, before the culturing step.

[0121] The method of producing L-tryptophan of the present disclosure can further include a step of recovering L-tryptophan from the culture medium (on which the culture is grown) or the cultured microorganism. The recovery step can also be included after the culturing step.

[0122] In the recovery step, the method of culturing the microorganism of the present disclosure can be used, for example, according to a batch culture method, a continuous culture method, or a fed-batch culture method, and the desired L-tryptophan can be collected using a suitable method known in the art. For example, methods such as centrifugation, filtration, treatment with a protein crystallization precipitant (salting-out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography (e.g., molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC, and the like, or a combination thereof can be used, and a suitable method known in the art can be used to recover the desired L-tryptophan from the culture medium or the microorganism.

[0123] Further, the method for producing L-tryptophan of the present disclosure can further include a purification step, which can be performed using a suitable method known in the art. In one example, when the method for producing L-tryptophan of the present disclosure includes both a recovery step and a purification step, the recovery step and the purification step can be performed continuously or intermittently without regard to the order, or can be performed simultaneously, or can be integrated into one step, but the method is not limited thereto.

[0124] In the method of the present disclosure, the exogenous udhA protein, polynucleotide, vector, and microorganism, etc. are as described in the other aspects above.

[0125] Another aspect of the present disclosure provides a composition for producing L-tryptophan, comprising: an Escherichia microorganism into which an exogenous udhA protein or a polynucleotide encoding the same is introduced; a culture medium for culturing the microorganism; or a combination thereof.

[0126] The composition of the present disclosure can further include any suitable excipient generally used in a composition for producing L-tryptophan, and such an excipient includes, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, or an isotonic agent, etc., but is not limited thereto.

[0127] Another aspect of the present disclosure provides a method of preparing an Escherichia microorganism for producing L-tryptophan, comprising: introducing an exogenous udhA protein or a polynucleotide encoding the same.

[0128] The exogenous udhA protein, polynucleotide encoding the same, and microorganism are as described above.

[0129] The preparation method can include a step of modifying the microorganism such that the microorganism has increased soluble pyridine nucleotide transhydrogenase activity compared to its endogenous activity. The step includes introducing an exogenous udhA protein into an Escherichia microorganism.

[0130] Another aspect of the present disclosure provides the use of an Escherichia microorganism into which an exogenous udhA protein or a polynucleotide encoding the same is introduced for producing L-tryptophan.

[0131] The exogenous udhA protein, the Escherichia microorganism, etc. are as described in other aspects above.

[0132] Mode of carrying out the present application

[0133] The present disclosure will be described in detail by way of examples. However, the examples are provided only for the purpose of illustrating preferred embodiments, and thus the scope of the present disclosure is not intended to be limited to or by the examples or the embodiments. Meanwhile, those skilled in the art of the present disclosure or in a similar technical field can fully understand and easily implement technical features not described herein.

[0134] Example 1: Screening and selection of a gene encoding soluble pyridine nucleotide transhydrogenase (udhA)

[0135] As a result of PSI-BLAST screening based on the NCBI and KEGG databases, using the amino acid sequence of the udhA protein derived from Escherichia coli as a query sequence, candidate proteins expected to have activity of catalyzing the reaction of converting 2-keto-L-gulonate to L-ascorbic acid, genes encoding the proteins, and microorganisms having the genes were determined. Among them, six candidate proteins were selected in consideration of the biosafety level suitable for the production of amino acids and the availability of the produced amino acids, and the results are shown in Table 1 below. [Table 1]

[0136] [Table 1]

[0137]

[0138]

[0139] Example 2: Preparation of a plasmid for gene insertion

[0140] In order to insert the genes into the chromosome of Escherichia, a plasmid was prepared using pSKH (US 8945907 B2).

[0141] In order to extract the genomic DNA of wild-type Escherichia coli W3110, a genomic-tip system from Qiagen, Inc. was used. Based on the obtained genomic DNA as a template, each gene fragment was obtained by amplifying the upstream region and the downstream region where homologous recombination occurs on the chromosome of the udhA gene derived from Escherichia coli. Solg TM Pfu-X DNA polymerase was used as a polymerase, and PCR was performed under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of denaturation at 95°C for 20 seconds, annealing at 60°C for 40 seconds, and polymerization at 72°C for 1 minute, and then polymerization at 72°C for 5 minutes.

[0142] In addition, in order to amplify the exogenous udhA gene fragment shown in Table 1 above to replace the existing E. coli-derived udhA gene, as templates for P. aeruginosa, E. amylovora, A. mediterranei, Spongiobacter sp. IMCC21906, N. pseudonovoguineense, and A. vinelandii strains' udhA genes synthesized using a gene synthesis service of Bionics Co., Ltd. were used, PCR was performed using the primer pairs shown in Table 2 below to amplify the P. aeruginosa-derived udhA gene (NZ_CP034244.1, SEQ ID NO: 2), the E. amylovora-derived udhA gene (NC_013961.1, SEQ ID NO: 4), the A. mediterranei-derived udhA gene (CP003917.1, SEQ ID NO: 6), the Spongiobacter sp. IMCC21906-derived udhA gene (NZ_CP011477.1, SEQ ID NO: 8), the N. pseudonovoguineense-derived udhA gene (CP015163.1, SEQ ID NO: 10), and the A. vinelandii-derived udhA gene (NZ_FPKM01000023.1, SEQ ID NO: 12). Solg TM Pfu-X DNA polymerase was used as a polymerase, and PCR was performed under the following PCR amplification conditions: denaturation at 95°C for 2 minutes, followed by 27 cycles of denaturation at 95°C for 20 seconds, annealing at 60°C for 40 seconds, and polymerization at 72°C for 1 minute, and then polymerization at 72°C for 5 minutes.

[0143] The primer sequences used herein are shown in Table 2 below.

[0144] [Table 2]

[0145]

[0146]

[0147]

[0148] The upstream gene fragment and the downstream gene fragment of the amplified E. coli-derived udhA gene thus obtained, the exogenous udhA gene fragment, and the vector pSKH for chromosome transformation cleaved by EcoRV restriction enzyme were cloned using the Gibson assembly method (DG Gibson et al., NATURE METHODS, VOL. 6 NO. 5, May 2009, NE Builder HiFi DNA Assembly Master Mix) to obtain a recombinant plasmid. The cloning was performed by mixing the Gibson assembly reagent and each gene fragment in a calculated molar number, and then incubating at 50°C for 1 hour. The resulting recombinant plasmid was 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, according to the kind of the replacement udhA gene.

[0149] Example 3: Preparation of an Escherichia microorganism into which a foreign udhA gene is introduced

[0150] 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 prepared in Example 2 were transformed into the L-tryptophan-producing strain CA04-4303 (US10995378 B2) by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), and then secondary crossing was performed to obtain strains in which the E. coli-derived udhA gene on the chromosome was replaced with the exogenous udhA gene. The corresponding genetic manipulation was confirmed by genome sequencing and a PCR method using a primer pair of SEQ ID NOs: 39 and 40, which can amplify the outer regions of the upstream region and the downstream region of the homologous recombination in which the corresponding gene was inserted, respectively. The resulting strains in which the E. coli-derived udhA gene was replaced with the exogenous 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.

[0151] The primer sequences used herein are shown in Table 3 below.

[0152] [Table 3]

[0153] SEQ ID NO: Sequence name Sequence (5' -> 3') 39 udhA-confirmation F CAGTAAACGCACGCGGCA 40 udhA-confirmation R GTGATGTTGCTGGAGCGG

[0154] Example 4: Confirmation of L-tryptophan-producing ability of an Escherichia microorganism into which a foreign udhA gene is introduced

[0155] To confirm the L-tryptophan production ability of the 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) strains into which the foreign udhA gene was introduced prepared in Example 3 and the parent strain CA04-4303 as a control, the strains were cultured in the following manner.

[0156] Specifically, one platinum loop of each strain cultured overnight in LB solid medium in a 37°C incubator was inoculated into 25 mL of the titer medium shown in Table 4 below, and then cultured in an incubator at a rate of 200 rpm at 37°C for 48 hours. After completion of the culture, the production of L-tryptophan was measured by HPLC, and the results are shown in Table 5 below.

[0157] [Table 4]

[0158]

[0159]

[0160] [Table 5]

[0161]

[0162] As shown in Table 5 above, among the six production strains into which the foreign udhA gene was introduced, the CA04-4303::udhA (P.ae) strain into which the udhA gene derived from P. aeruginosa was introduced finally produced 1.87 g / L of L-tryptophan during the shake flask culture, showing an increase of about 37% in the fermentation yield compared to the control CA04-4303 strain. In addition, the CA04-4303::udhA (E.am) strain into which the udhA gene derived from E. amylovora was introduced finally produced 1.62 g / L of L-tryptophan during the shake flask culture, showing an increase of about 18% in the fermentation yield compared to the control CA04-4303.

[0163] Based on the above 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 of E. coli.

[0164] According to the foregoing, it will be understood by those skilled in the art that the present disclosure can be implemented in other specific forms without modifying the technical concept or essential characteristics of the present disclosure. In this regard, the exemplary embodiments disclosed herein are merely for illustrative purposes and should not be construed as limiting the scope of the present disclosure. Rather, the present disclosure is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalent substitutions, and other embodiments that can be included within the spirit and scope of the present disclosure as defined by the appended claims.

Claims

1. An Escherichia microorganism, into which an exogenous udhA protein or a polynucleotide encoding the protein is introduced. 2 . The microorganism according to 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 identity thereto. The microorganism according to claim 1 , wherein the protein is derived from any one or more microorganisms selected from the group consisting of Pseudomonas aeruginosa and Erwinia amylovora. The microorganism according to claim 1 , wherein the Escherichia microorganism has an increased L-tryptophan-producing ability compared to the Escherichia microorganism before modification. The microorganism according to claim 1 , wherein the Escherichia microorganism is Escherichia coli.

6. A method for producing L-tryptophan, comprising: The Escherichia microorganism into which the exogenous udhA protein or the polynucleotide encoding the protein has been introduced is cultured in a culture medium. The method according to claim 6 , wherein the protein is derived from any 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 an exogenous udhA protein or a polynucleotide encoding the protein is introduced; a culture medium for culturing the microorganism; or a combination thereof.

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

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