Novel MdtH mutant and method for producing O-phosphoserine, cysteine ​​and their derivatives using the same

Abstract

The present application relates to novel MdtH mutants and methods for producing O-phosphoserine, cysteine, and cysteine ​​derivatives using the same.

Description

[Technical Field]

[0001] The present application relates to MdtH mutants and methods for producing O-phosphoserine, cysteine, and cysteine ​​derivatives using the same. [Background technology]

[0002] L-cysteine ​​is an important amino acid in sulfur metabolism in all living organisms, and is used not only in the synthesis of proteins such as hair keratin, glutathione, biotin, methionine, and other sulfur-containing metabolites, but also as a precursor for coenzyme A biosynthesis.

[0003] Known methods for producing L-cysteine ​​using microorganisms include: 1) biological conversion of D,L-ATC (D,L-2-aminothiazoline-4-carboxylic acid) using microorganisms; 2) direct fermentation of L-cysteine ​​using Escherichia coli (European Patent EP 0885962B; Wada M and Takagi H, Appl. Microbiol. Biochem., 73:48-54, 2006); and 3) fermentation of O-phosphoserine (hereinafter referred to as "OPS") using microorganisms, followed by conversion to L-cysteine ​​by reaction with sulfide under the catalysis of O-phosphoserine sulfhydrylase (hereinafter referred to as "OPSS") (European Patent EP 2444481).

[0004] However, due to the increasing demand for L-cysteine, research into effective methods for producing L-amino acids is still needed. In particular, in order to produce cysteine ​​at a high yield using method 3), it was necessary to overproduce the precursor OPS. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] European Registration Permit EP0885962B [License 2] European Open License EP 2444481 [License 3] US Open Patent (US 2012-0190081) [License 4] US Registry Permit US 7662943 B2 [Patent Document 5] US Registry Permit US 10584338 B2 [License 6] US Registry Permit US 10273491 B2 [License 7] US Registry Permit US 9127324 [Non-licensed literature]

[0006] [Non-licensed Document 1] Wada M andTakagi H, Appl. Microbiol. Biochem.,73:48-54,2006 [Non-licensed Document 2] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]:2444 [Non-licensed Document 3] Rice et al.,2000,Trends Genet. 16:276-277 [Non-licensed Document 4] Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453 [Non-licensed Document 5] Devereux,J.,et al,Nucleic Acids Research 12:387 (1984) [Non-licensed Document 6] Atschul,[S.] [F.,] [ET AL,J MOLEC BIOL 215]:403 (1990) [Non-licensed Document 7] Guide to Huge Computers,Martin J. Bishop,[ED.,] Academic Press,San Diego, 1994 [Non-licensed document 8] [CARILLO ETA / . ](1988) SIAM J Applied Math 48:1073

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

[0007] The present inventors made extensive efforts to overproduce OPS, a precursor of cysteine, in order to produce cysteine ​​at high yields. As a result, they confirmed that a novel MdtH mutant improves OPS production, leading to the completion of this application. [Means for solving the problem]

[0008] One object of the present application is to provide an MdtH mutant in which the amino acid corresponding to position 125 of the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid.

[0009] Another object of the present application is to provide polynucleotides encoding the variants of the present application.

[0010] Another object of the present application is to provide a vector containing the polynucleotide of the present application.

[0011] Another object of the present application is to provide a recombinant Escherichia microorganism comprising an MdtH mutant in which the amino acid corresponding to position 125 of the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid, or a polynucleotide encoding said mutant.

[0012] Another object of the present application is to provide a method for producing O-phosphoserine, which includes a step of culturing in a medium an MdtH mutant in which the amino acid corresponding to position 125 of the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, or a microorganism containing a polynucleotide encoding the mutant.

[0013] Another object of the present application is to provide a method for producing cysteine ​​or a derivative thereof, the method comprising: a) culturing in a medium a microorganism containing an MdtH mutant in which the amino acid corresponding to position 125 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, or a polynucleotide encoding the mutant, to produce O-phosphoserine or a medium containing the same; and b) reacting O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing the same and the O-phosphoserine produced in step a) or the medium containing the same with sulfide. [Effects of the Invention]

[0014] When a microorganism capable of producing OPS is cultured using the novel mutant polypeptide having OPS export activity of the present application, OPS can be produced in a higher yield than when existing unmodified or mutant proteins are used. DETAILED DESCRIPTION OF THE INVENTION

[0015] This will be explained in more detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application belong to the scope of this application. Furthermore, the specific descriptions described below are not considered to limit the category of this application.

[0016] One embodiment of the present application is an MdtH mutant in which the amino acid corresponding to position 125 of the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid.

[0017] In one embodiment, the other amino acid may be isoleucine.

[0018] The variant of the present application may be a variant in which the amino acid valine corresponding to the 125th position in the amino acid sequence set forth in SEQ ID NO: 1, which is the parent sequence, is substituted with isoleucine, and which has at least 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, or less than 100% homology or identity to the amino acid sequence set forth in SEQ ID NO: 1. For example, the variant of the present application may have, include, or consist essentially of an amino acid sequence having at least 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.7% or more, or less than 100% homology or identity to the amino acid sequence of SEQ ID NO: 1. It is clear that variants having partial deletions, modifications, substitutions, conservative substitutions, or additions in the amino acid sequence are also included within the scope of the present application, as long as they have such homology or identity and exhibit the efficacy corresponding to the variant of the present application.

[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 variant of the present application.

[0020] The term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made 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 term "variant" refers to a protein that differs from the parent sequence of a specific protein by conservative substitution and / or modification of one or more amino acids, thereby maintaining the functions or properties of the specific protein. A variant differs from an identified sequence by the substitution, deletion, or addition of a few amino acids. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the specific protein and evaluating the properties of the modified protein. That is, the performance of the variant may be increased, unchanged, or decreased compared to the native protein. Some variants may also include variants in which one or more portions, such as the N-terminal leader sequence or transmembrane domain, have been removed. Other variants may include variants in which portions have been removed from the N- and / or C-termini of the mature protein. The term "mutant" may refer to a mutant type, a variant, a mutated protein, a mutant polypeptide, a mutation, etc. (in English, modification, modified protein, modified polypeptide, mutant, mutein, divergent, variant, etc.), as long as it is a term used to mean a mutation, but is not limited thereto. For purposes of this application, the mutant may be a mutated protein with increased activity compared to a native wild-type or non-mutated protein, but is not limited thereto.

[0022] The variants may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be conjugated to a signal (or leader) sequence involved in co- or post-translational protein translocation. The variants may also be conjugated to other sequences or linkers to allow for identification, purification, or synthesis.

[0023] In the present application, the term "parent sequence" refers to a reference sequence into which modifications are introduced to form a mutant polypeptide. That is, the parent sequence may be used as a starting sequence into which mutations such as substitutions, insertions, and / or deletions are introduced. The parent sequence may be a naturally occurring or wild-type sequence, or may be a variant of the naturally occurring or wild-type sequence in which one or more substitutions, insertions, or deletions have occurred, or may be an artificially synthesized sequence.

[0024] In this application, 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 are often used interchangeably. Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, with default gap penalties established by the program used. Substantially homologous or identical sequences can generally hybridize to all or part 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.

[0025] 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 ETA / .] (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.

[0026] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that described in Needleman et al. (1970), J Mol Biol. 48:443, as known in, for example, 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 binary comparison 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.

[0027] As used herein, the term "corresponding to" refers to the amino acid residue at the recited position in the polypeptide, or an amino acid residue that is similar, identical, or homologous to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position may be determining the specific amino acid of a sequence that references a particular sequence. As used herein, a "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0028] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on this, each amino acid residue in the amino acid sequence can be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as that described in this application can identify the position of an amino acid relative to a query sequence (also referred to as a "reference sequence"), or the position where a variation such as a substitution, insertion, or deletion occurs.

[0029] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) or the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) can be used, but is not limited to these. Sequence alignment programs, pairwise sequence comparison algorithms, etc. known in the art can be used as appropriate.

[0030] In the present application, the term "MdtH" refers to a type of transporter in the major facilitator superfamily (MFS), a superfamily of membrane transport proteins that facilitates the movement of small solutes across the cell membrane in response to a chemical demand gradient. It is known as a protein that exhibits OPS export activity in Escherichia coli cells that are free from growth inhibition in the presence of excessive OPS. In the present application, MdtH refers to a membrane protein that has the activity of exporting O-phosphoserine (OPS) to the outside of cells. The sequence of MdtH can be obtained from GenBank, a publicly known database at NCBI. For example, MdtH may be, but is not limited to, a polypeptide with OPS export activity encoded by the mdtH gene.

[0031] The variants of the present application can have an activity that increases OPS efflux capacity compared to the wild-type polypeptide.

[0032] In this application, the term "O-phosphoserine (hereinafter "OPS")" refers to a phosphoric acid ester of serine and is a component of various proteins. OPS is a precursor of L-cysteine ​​and can be converted to cysteine ​​by reacting with sulfide under the catalytic action of OPS sulfhydrylase (OPSS) (U.S. Patent Publication US 2012-0190081), but is not limited thereto.

[0033] The variant of the present application may further include a substitution of one or more amino acids among the amino acids corresponding to positions 60, 180, and 398 in the amino acid sequence of SEQ ID NO: 1 with other amino acids. As an example, the variant of the present application may include a substitution of the amino acid corresponding to position 125 in the amino acid sequence of SEQ ID NO: 1 with other amino acids, and further including a substitution of one or more, two or more, or three amino acids among the amino acids corresponding to positions 60, 180, and 398 with other amino acids, but is not limited thereto.

[0034] Specifically, the variant of the present application may be a mutant polypeptide that exhibits OPS-efflux activity, in which i) the amino acid residue valine corresponding to the 125th position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 has been substituted with an amino acid residue other than valine, and in addition, ii) the amino acid residue glutamine corresponding to the 60th position, iii) the amino acid residue phenylalanine corresponding to the 180th position, and / or iv) the amino acid residue leucine corresponding to the 398th position has been substituted with another amino acid residue.

[0035] From the N-terminus of the amino acid sequence of SEQ ID NO: 1, i) the amino acids other than valine, which is the amino acid residue corresponding to the 125th position, include glycine, alanine, leucine, isoleucine, serine, proline, phenylalanine, tryptophan, methionine, arginine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, histidine, aspartic acid, and glutamic acid; ii) the amino acids other than glutamine, which is the amino acid residue corresponding to the 60th position, include glycine, alanine, leucine, isoleucine, serine, proline, phenylalanine, tryptophan, methionine, arginine, threonine, cysteine, tyrosine, asparagine, valine, lysine, histidine, aspartic acid, and glutamic acid; iii) the amino acids other than glutamine, which is the amino acid residue corresponding to the 18th position, include glycine, alanine, leucine, isoleucine, serine, proline, phenylalanine, tryptophan, methionine, arginine, threonine, cysteine, tyrosine, asparagine, valine, lysine, histidine, aspartic acid, and glutamic acid; The amino acids other than phenylalanine, which is the amino acid residue corresponding to position 0, may include glycine, alanine, leucine, isoleucine, serine, proline, valine, tryptophan, methionine, arginine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, histidine, aspartic acid, and glutamic acid, and / or iv) the amino acids other than leucine, which is the amino acid residue corresponding to position 398, may include, but are not limited to, glycine, alanine, isoleucine, serine, proline, phenylalanine, valine, tryptophan, methionine, arginine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, histidine, aspartic acid, and glutamic acid.

[0036] More specifically, the variant of the present application may be such that, from the N-terminus of the amino acid sequence of SEQ ID NO: 1, i) the amino acid residue valine corresponding to the 125th position is substituted with isoleucine, and in addition, ii) the amino acid residue corresponding to the 60th position is glutamine or arginine, iii) the amino acid residue corresponding to the 180th position is phenylalanine or leucine, and iv) the amino acid residue corresponding to the 398th position is leucine or proline.

[0037] More specifically, the variant of the present application may be one in which, from the N-terminus of the amino acid sequence of SEQ ID NO: 1, i) the amino acid residue valine corresponding to the 125th position is substituted with isoleucine, and in contrast thereto, ii) the amino acid glutamine corresponding to the 60th position is substituted with arginine, iii) the amino acid phenylalanine corresponding to the 180th position is substituted with leucine, and / or iv) the amino acid leucine corresponding to the 398th position is substituted with proline.

[0038] In one embodiment, the variant of the present application may be a variant consisting of an amino acid sequence having 99% or more sequence identity with any one or more amino acid sequences selected from SEQ ID NO: 2 or SEQ ID NO: 3.

[0039] Furthermore, the variants of the present application may have, contain, consist of, or essentially consist of one or more amino acid sequences selected from SEQ ID NO: 2 or SEQ ID NO: 3, but are not limited thereto.

[0040] Another aspect of the present application is a polynucleotide encoding the variant of the present application. The mutant is as described above.

[0041] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide units are linked in a long chain by covalent bonds, such as a DNA or RNA chain of a certain length or more, and more specifically, a polynucleotide fragment encoding the variant.

[0042] A polynucleotide encoding a variant of the present application may comprise a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3. As an example of the present application, a polynucleotide of the present application may have or comprise the nucleic acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5. Alternatively, a polynucleotide of the present application may consist of or consist essentially of the nucleic acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

[0043] The polynucleotide of the present application may be modified in various ways in the coding region without changing the amino acid sequence of the variant of the present application, taking into consideration codon degeneracy or codons preferred in an organism in which the variant of the present application is to be expressed. Specifically, the polynucleotide of the present application may have or contain a nucleotide sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, and less than 100% homologous or identical to the nucleobase sequence of SEQ ID NO: 4 or SEQ ID NO: 5, or may consist of or essentially consist of a nucleotide sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, and less than 100% homologous or identical to the nucleobase sequence of SEQ ID NO: 4 or SEQ ID NO: 5, but is not limited thereto. In this case, in the sequence having homology or identity, the codon encoding the amino acid corresponding to the 125th position of SEQ ID NO: 1 may be one of the codons encoding isoleucine.

[0044] Furthermore, the polynucleotides of the present application include, without limitation, sequences that can hybridize under stringent conditions with probes that can be 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 homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, but polynucleotides with lower homology or identity do not hybridize with each other; or conditions under which washing is performed once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions for standard 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.

[0045] 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 are capable of hybridizing to one another. For example, with respect to DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can also include isolated nucleic acid fragments that are complementary to entire sequences, as well as substantially similar nucleic acid sequences.

[0046] 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.

[0047] The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables well known in the art (eg, J. Sambrook et al., supra).

[0048] Another aspect of the present application is a vector comprising the polynucleotide of the present application. The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0049] The polynucleotide is as described above.

[0050] 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 can be integrated into the genome itself.

[0051] 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 pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pSK, pSKH, and pET can be used as plasmid vectors. Specifically, pCL, pDC, pDCM2, pSK, pSKH130, pDZ, pACYC177, pACYC184, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0052] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome via a vector for chromosomal integration in a cell. 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 also be 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. Markers that confer selectable phenotypes, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of surface polypeptides, can be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0053] 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, thereby enabling the expression of the polypeptide encoded by the polynucleotide in the host cell. A transformed polynucleotide may include any polynucleotide, whether it is located intrachromosomally or extrachromosomally, as long as it is expressible in the host cell. The polynucleotide may also include DNA and / or RNA encoding the target polypeptide. 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.

[0054] 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 variant of the present application.

[0055] Another embodiment of the present application is a microorganism of the genus Escherichia comprising an MdtH mutant in which the amino acid corresponding to position 125 in the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid, or a polynucleotide encoding said mutant.

[0056] The strains of the present application can comprise a mutant polypeptide of the present application, a polynucleotide encoding said polypeptide, and / or a vector comprising a polynucleotide of the present application.

[0057] The mutant, polynucleotide, and vector are as described above.

[0058] In this application, the term "microorganism (or strain)" includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, including those in which a specific mechanism has been weakened or enhanced by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and may also be a microorganism that contains a genetic modification for the production of a desired polypeptide, protein, or product.

[0059] The strain of the present application may be, but is not limited to, a strain comprising one or more of a variant of the present application, a polynucleotide of the present application, and a vector comprising a polynucleotide of the present application; a strain that has been modified to express a variant of the present application or a polynucleotide of the present application; a strain (e.g., a recombinant strain) that expresses a variant of the present application or a polynucleotide of the present application; or a strain (e.g., a recombinant strain) that has activity of a variant of the present application.

[0060] The strain of the present application may be a strain capable of excreting OPS.

[0061] The strains of the present application may be, but are not limited to, microorganisms that naturally possess the ability to excrete MdtH or OPS, or parent strains that lack the ability to excrete MdtH or OPS into which the mutants of the present application or polynucleotides encoding them (or vectors containing the polynucleotides) have been introduced and / or which have been conferred the ability to excrete OPS.

[0062] For example, the strains of the present application are cells or microorganisms that have been transformed with a vector containing a polynucleotide encoding the polynucleotide of the present application or a polynucleotide encoding the variant of the present application and express the variant of the present application. For purposes of this application, the strains of the present application can include all microorganisms that can excrete OPS, including the variant of the present application. For example, the strains of the present application can be recombinant strains with increased OPS excretion ability by introducing a polynucleotide encoding the variant of the present application into a naturally occurring wild-type microorganism or a microorganism that excretes OPS, thereby expressing an MdtH variant. The recombinant strains with increased OPS excretion ability can be, but are not limited to, microorganisms that have increased OPS excretion ability compared to naturally occurring wild-type microorganisms or non-MdtH-modified microorganisms (i.e., microorganisms that express wild-type MdtH (SEQ ID NO: 1) or microorganisms that do not express the mutant MdtH protein (SEQ ID NO: 2 or SEQ ID NO: 3)). For example, the non-MdtH-modified microorganism, which is the subject strain for comparing the presence or absence of an increase in OPS excretion ability, may be, but is not limited to, CA07-0012 (KCCM 11121P, European Patent Publication EP 2444481, or U.S. Patent Publication No. 2012-0190081), a strain in which the activity of endogenous phosphoserine phosphatase (serB) has been attenuated.

[0063] For example, a recombinant strain having increased OPS production ability due to an increase in the OPS excretion ability has an OPS production ability that is about 1% or more, specifically about 1% or more, 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 20% or more, about 22.5% or more, about 25% or more, or about 27.5% or more, compared to the OPS production ability of the parent strain or untransformed microorganism before mutation. Above, about 30% or more, about 32.5% or more, about 35% or more, about 37.5% or more, about 40% or more, about 42.5% or more, about 45% or more, about 47.5% or more, about 50% or more, about 52.5% or more, about 55% or more, about 57.5% or more, about 60% or more, about 62.5% or more, about 65% or more, about 67.5% or more, about 70% or more, about 72.5% or more, about 75% or more, about 77.5% or more, about 80% or more, approximately 82.5% or more, approximately 90% or more, approximately 92.5% or more, approximately 95% or more, approximately 97.5% or more, approximately 100% or more, approximately 102.5% or more, approximately 105% or more, approximately 107.5% or more, approximately 110% or more, approximately 112.5% ​​or more, approximately 115% or more, approximately 117.5% or more, approximately 120% or more, approximately 122.5% or more, approximately 125% or more, approximately 127.5% or more, approximately 130% or more, approximately 132.5 The increase may be about 135% or more, about 137% or more (there is no particular upper limit, and it may be, for example, about 300% or less, about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 40% or less, or about 30% or less), but is not limited thereto as long as there is an increase in the + value compared to the productivity of the parent strain or untransformed microorganism before mutation.In other examples, the recombinant strain having increased OPS production ability due to the increased OPS excretion ability has an OPS production ability that is 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 1.10-fold or more, about 1.125-fold or more, about 1.15-fold or more, about 1.175-fold or more, about 1.20-fold or more, about 1.22-fold or more, compared to the parent strain or untransformed microorganism before mutation. 5x or more, approximately 1.25x or more, approximately 1.275x or more, approximately 1.30x or more, approximately 1.325x or more, approximately 1.35x or more, approximately 1.375x or more, approximately 1.50x or more, approximately 1.525x or more, approximately 1.55x or more, approximately 1.60x or more, approximately 1.625x or more, approximately 1.65x or more, approximately 1.675x or more, approximately 1.70x or more, approximately 1.725x or more, approximately 1.75x or more, approximately 1 0.775x or more, approximately 1.8x or more, approximately 1.825x or more, approximately 1.85x or more, approximately 1.875x or more, approximately 1.90x or more, approximately 1.925x or more, approximately 1.95x or more, approximately 1.975x or more, approximately 2.0x or more, approximately 2.025x or more, approximately 2.05x or more, approximately 2.075x or more, approximately 2.10x or more, approximately 2.125x or more, approximately 2.15x or more, approximately 2.175x or more The increase may be, but is not limited to, about 2.20 times or more, about 2.225 times or more, about 2.25 times or more, about 2.275 times or more, about 2.30 times or more, about 2.325 times or more, about 2.35 times or more, or about 2.37 times or more (there is no particular limitation on the upper limit, and it may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, or about 2 times or less). The term "about" refers to a range that includes ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​in a range that is equal to or similar to the numerical value following the term "about," but is not limited thereto.

[0064] In the present application, the term "non-transformed 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 non-transformed microorganism refers to a strain before the MdtH mutant described herein is introduced or has been introduced. The term "non-transformed microorganism" may be used interchangeably with "pre-transformed strain," "pre-transformed microorganism," "non-mutated strain," "non-transformed strain," "non-mutated microorganism," or "reference microorganism."

[0065] In another embodiment of the present application, the microorganism of the present application is not particularly limited in type as long as it can produce OPS, and may be either a prokaryotic or eukaryotic cell, specifically a prokaryotic cell. Examples of prokaryotic cells include microbial strains belonging to the genera Escherichia, Erwinia, Seratia, Providencia, Corynebacterium, and Brevibacterium, and specifically may be, but is not limited to, a microorganism of the genus Escherichia, more specifically, Escherichia coli.

[0066] In particular, the Escherichia microorganism of the present application can produce OPS and L-serine through the enzymes SerA, SerC, and SerB in the L-serine biosynthetic pathway (Ahmed Zahoor, Computational and structural biotechnology journal, vol. 3, 2012 October; Wendisch VF et al., Curr Opin Microbiol. 2006 June;9(3):268-74; Peters-Wendisch P et al., Appl Environ Microbiol. 2005 November;7 1(11):7 139-44.). Because SerB, a phosphoserine phosphatase, has the activity of converting OPS to L-serine, microorganisms mutated to attenuate SerB activity are characterized by OPS accumulation and are useful for OPS production. As an example, the microorganism of the present application may be a recombinant microorganism in which, in addition to the introduction of the mutation of the present application, the activity of SerB is further weakened compared to the endogenous activity.

[0067] The SerB of the present application may be, but is not limited to, a protein having or comprising the amino acid sequence set forth in NCBI Accession No. AAC77341.1, or a protein consisting of or essentially consisting of the amino acid sequence set forth in AAC77341.1. Furthermore, the SerB of the present application may have or comprise an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino acid sequence set forth in AAC77341.1, so long as it exhibits the activity of converting OPS to L-serine. Furthermore, the SerB of the present application may be, but is not limited to, a protein consisting of, or essentially consisting of an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino acid sequence set forth in AAC77341.1.

[0068] Furthermore, the polynucleotide encoding SerB of the present application may have or include the nucleotide sequence set forth in NCBI NP_415583.4. The polynucleotide encoding SerB of the present application may have or include a nucleotide sequence that is at least 70%, 80%, 90%, 95%, or 99% or more, and less than 100%, homologous or identical to the nucleotide sequence of NP_415583.4. Furthermore, the polynucleotide encoding SerB of the present application may consist of or essentially consist of a nucleotide sequence that is at least 70%, 80%, 90%, 95%, or 99% or more, and less than 100% homologous or identical to the nucleotide sequence of NP_415583.4, but is not limited thereto.

[0069] As used herein, the term "enhancement" of polypeptide activity means that the activity of a polypeptide is increased compared to its endogenous activity. The term "enhancement" may be used interchangeably with terms such as "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and increase can all encompass the display of an activity not inherently possessed, or the display of an activity that is improved compared to the endogenous activity or the activity prior to transformation. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or an unaltered microorganism prior to transformation, in cases where a trait has been altered by genetic mutation due to natural or artificial factors. This term may be used interchangeably with "activity prior to transformation." "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 prior to transformation.

[0070] 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.

[0071] 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 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.).

[0072] Specifically, the enhancement of the polypeptide activity of the present application is achieved by: 1) an increase in the intracellular copy number of the polynucleotide encoding the polypeptide; 2) Modification of the expression regulatory region of a gene on a chromosome that encodes a polypeptide; 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 a polypeptide and selectively modifying or chemically modifying exposed sites; or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0073] 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 capable of replicating and functioning independently of the host, to which a polynucleotide encoding the polypeptide is operably linked. 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.

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

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

[0076] The 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.

[0077] 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 modification can be achieved by, but is not limited to, inserting the polynucleotide into a chromosome via homologous recombination. The vector used in this case may further contain a selection marker for detecting the presence or absence of insertion into the chromosome. The selection marker is as described above.

[0078] The introduction of an exogenous polynucleotide that exhibits the activity of a polypeptide (6) may be the introduction of an exogenous polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide into a host cell. The exogenous polynucleotide is not limited in its origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The method used for the introduction can be any known transformation method appropriately selected by those skilled in the art. The introduced polynucleotide is expressed in a host cell to produce a polypeptide, and its activity can be increased.

[0079] 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.

[0080] 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 results, and selecting and modifying exposed sites to be modified or chemically modified.

[0081] Such enhancement of 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.

[0082] The term "attenuation" of a polypeptide activity in this application encompasses a reduction in activity or absence of 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.

[0083] The attenuation can also include 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 level of 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." The term "inactivation, deficiency, reduction, downregulation, decrease, or attenuation" of a polypeptide activity compared to the endogenous activity means that the activity of a specific polypeptide is reduced compared to that originally possessed by a parent strain or unaltered microorganism before the trait has been altered.

[0084] 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.).

[0085] Specifically, the attenuation of the activity of the polypeptide of the present application is 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 or 5'-UTR region of a gene transcript encoding a polypeptide; 6) introduction of antisense oligonucleotides (e.g., antisense RNA) that are complementary to and bind to the transcript of the gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure that does not allow ribosome attachment; 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.

[0086] 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 polypeptide of interest in the chromosome, replacement with a polynucleotide lacking some nucleotides, or replacement with a marker gene. Furthermore, the modification of the expression regulatory region (or expression regulatory sequence) in 2) above may be a mutation in the expression regulatory region (or expression regulatory sequence) caused by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or a replacement with a sequence having 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.

[0087] Furthermore, the 5) modification of the base sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, a substitution with a base sequence encoding another start codon that has a lower polypeptide expression rate than the endogenous start codon, but is not limited thereto.

[0088] Furthermore, 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 sequence of the polynucleotide 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, gene expression can be inhibited or attenuated by introducing a mutation into the polynucleotide sequence to form a stop codon, but is not limited to this.

[0089] 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].

[0090] 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.

[0091] 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) may create an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide, thereby attenuating the activity.

[0092] Modification of a portion or all of a polynucleotide in the microorganism of the present application may 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 may 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.

[0093] Furthermore, the microorganism may be a microorganism in which the ability to influx OPS into cells and to decompose OPS has been reduced.

[0094] In addition to the above, the contents of European Patent Publication EP 2444481 and U.S. Patent Publication No. 2012-0190081, etc., regarding the OPS-producing microorganisms described above, are also incorporated herein by reference, but are not limited thereto.

[0095] In the microorganism of the present application, the mutant, polynucleotide, OPS, etc. are as described above in other aspects.

[0096] Another embodiment of the present application is a method for producing O-phosphoserine, comprising the step of culturing in a medium a microorganism containing an MdtH mutant in which the amino acid corresponding to position 125 of the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid, or a polynucleotide encoding the mutant.

[0097] The method for producing an OPS of the present application can include culturing in a medium a microorganism that includes a variant of the present application, a polynucleotide of the present application, or a vector of the present application.

[0098] In the present application, the term "culturing" means growing the microorganism of the present application under appropriately controlled environmental conditions. The culturing process of the present application can be carried out using appropriate media and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing may be, but is not limited to, a batch, continuous, and / or fed-batch culture.

[0099] In the present application, the term "culture medium" refers to a substance containing a mixture of nutrients, primarily as components, 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, and the microorganism of the present application can be cultured under aerobic conditions while controlling the temperature, pH, etc. in a conventional culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins, etc.

[0100] Carbon sources contained in the medium may include, but are not limited to, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid, which may be used individually or as a mixture.

[0101] Nitrogen sources contained in the medium include, but are not limited to, organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn steep liquor, and soybean meal, and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate, which may be used alone or in combination.

[0102] The phosphorus source contained in the medium may include, but is not limited to, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salts.

[0103] The medium may also contain metal salts such as magnesium sulfate or iron sulfate, as well as amino acids, vitamins, and appropriate precursors. These mediums or precursors may be added to the culture in a batch or continuous manner, but are not limited thereto.

[0104] During cultivation, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. Furthermore, foam formation can be suppressed during cultivation using an antifoaming agent such as a fatty acid polyglycol ester. Furthermore, oxygen or an oxygen-containing gas can be injected into the culture medium to maintain an aerobic state, or nitrogen, hydrogen, or carbon dioxide gas can be injected without gas injection or to maintain anaerobic and microaerobic states, but these are not limited thereto.

[0105] In the culture of the present application, the culture temperature can be maintained at 25°C to 40°C, specifically 30°C to 35°C, and the culture period is continued until the desired amount of useful substance is produced, specifically 10 to 100 hours, but is not limited to these examples.

[0106] The OPS produced by the culture of the present application can be secreted into the medium.

[0107] The OPS production method of the present application can further include, for example, before the culturing step, 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).

[0108] The method for producing OPS of the present application may further include a step of recovering OPS from the culture medium (the medium in which the culture was performed) or the microorganism of the present application. The recovery step may be performed after the culturing step.

[0109] The method for recovering the OPS may involve collecting the desired OPS using a suitable method known in the art from the microbial culture method of the present application, such as a batch, continuous, or fed-batch culture method. For example, the desired OPS can be recovered from the medium or the microorganism using a suitable method known in the art, such as 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.

[0110] The OPS production method of the present application may further include a purification step. The purification can be performed using any suitable method known in the art. For example, when the OPS production method 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.

[0111] In the methods of the present application, the mutants, polynucleotides, vectors, strains, etc. are as described above in other aspects.

[0112] Another aspect of the present application is a method for producing cysteine ​​or a derivative thereof.

[0113] Specifically, the method may include the steps of: a) culturing in a medium an MdtH mutant in which the amino acid corresponding to position 125 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid, or a microorganism containing a polynucleotide encoding the mutant, to produce O-phosphoserine or a medium containing the same; and b) reacting O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing the same and the O-phosphoserine produced in step a) or a medium containing the same with sulfide.

[0114] In this application, the term "derivative" refers to a similar compound obtained by chemically modifying a part of a compound, usually a compound in which a hydrogen atom or a specific atomic group of the compound is replaced by a different atom or atomic group.

[0115] In this application, the term "cysteine ​​derivative" refers to a compound in which a hydrogen atom or a specific atomic group of cysteine ​​is replaced by another atom or atomic group. Examples of such cysteine ​​include those in which another atom or atomic group is attached to the nitrogen atom of the amine group (-NH2) or the sulfur atom of the thiol group (-SH) of cysteine. Examples include, but are not limited to, NAC (N-acetylcysteine), SCMC (S-Carboxymetylcysteine), BOC-CYS(ME)-OH, (R)-S-(2-Amino-2-carboxyethyl)-L-homocysteine, (R)-2-Amino-3-sulfopropionic acid, D-2-Amino-4-(ethylthio)butyric acid, 3-sulfino-L-alanine, Fmoc-Cys(Boc-methyl)-OH, Seleno-L-cystine, S-(2-Thiazolyl)-L-cysteine, S-(2-Thienyl)-L-cysteine, and S-(4-Tolyl)-L-cysteine.

[0116] Once cysteine ​​is produced by the method of the present application, it may be easily converted into various cysteine ​​derivatives by methods widely known in the art.

[0117] Specifically, the method for producing a cysteine ​​derivative may further include a step of converting the cysteine ​​produced in step b) into a cysteine ​​derivative. For example, N-acetylcysteine ​​(NAC) can be synthesized by reacting cysteine ​​with an acetylation agent, or S-carboxymethylcysteine ​​(SCMC) can be synthesized by reacting cysteine ​​with haloacetic acid under basic conditions, but this is not limitative.

[0118] The cysteine ​​derivatives can be used as pharmaceutical raw materials, primarily as antitussives, cough relievers, and therapeutic agents for bronchitis, bronchial asthma, sore throat, etc., but are not limited thereto.

[0119] As used herein, the term "O-phosphoserine sulfhydrylase (OPSS)" refers to an enzyme that catalyzes the reaction of converting OPS to cysteine ​​by donating a thiol group (SH group) to OPS. The enzyme may have been first identified in Aeropyrum pernix, Mycobacterium tuberculosis, Mycobacterium smegmatis, and Trichomonas vaginalis (Mino K and Ishikawa K, FEBS Letters, 551:133-138, 2003; Bums KE et al., J. Am. Chem. Soc, 127:11602-11603, 2005). Furthermore, the OPSS includes not only wild-type OPSS protein but also mutant proteins in which a portion of the sequence in the polynucleotide sequence encoding the OPSS is deleted, substituted, or added, and which exhibit biological activity equivalent to or greater than that of the wild-type OPSS protein, including all of the OPSS proteins and their mutant proteins disclosed in European Patent Publication EP 2444481 and U.S. Patent Registration US 9127324.

[0120] The sulfide is not only a solid substance commonly used in the art, but also a liquid or gaseous substance depending on the pH, pressure, and solubility. 2- ), thiosulfate (thiosulfate,S2O3 2-Any sulfide that can be converted to a thiol group (SH group) in the form of, for example, thiol group (SH group) can be used without limitation. Specifically, Na2S, NaSH, H2S, (NH4)2S, NaSH, and Na2S2O3 that provide a thiol group to OPS can be used, but are not limited to these. The reaction is a reaction in which one thiol group is provided to one OPS reactive group to produce one cysteine ​​or cysteine ​​derivative. The amount of sulfide added during the reaction may be 0.1 to 3 times, specifically 1 to 2 times, the molar concentration of OPS, but is not limited to these.

[0121] The present invention may further include a step of recovering the cysteine ​​produced through the reaction step, in which the desired cysteine ​​can be separated, purified, and collected from the reaction mixture using an appropriate reaction known in the art.

[0122] Another embodiment of the present application is a composition for producing OPS, comprising a microorganism containing an MdtH mutant in which the amino acid corresponding to position 125 in the amino acid sequence of SEQ ID NO:1 is substituted with another amino acid, or a polynucleotide encoding the mutant; a culture medium in which the same is cultured; or a combination of two or more of them.

[0123] The compositions of the present application may further comprise any suitable excipient commonly used in compositions for producing OPS, and such excipients may be, for example, but are not limited to, a preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer, or isotonic agent.

[0124] In the composition of the present application, the variant, polynucleotide, vector, strain, medium, OPS, etc. are as described above in other aspects.

[0125] Another aspect of the present application is the use of a mutant polypeptide of the present application that exhibits OPS-efflux activity in the production of OPS, cysteine, or a cysteine ​​derivative.

[0126] Another aspect of the present application is use of a polypeptide of the present application having a mutant polypeptide that exhibits OPS-efflux activity to excrete OPS from a microorganism.

[0127] As used herein, the variants, polynucleotides, vectors, strains, media, OPS, etc. are as described above in other aspects.

[0128] The present application will be described in more detail below through examples. However, these examples are for illustrative purposes only and the scope of the present application is not limited to these examples.

[0129] Example 1: mdtH library construction and screening To select MdtH mutants with increased OPS efflux activity, a library of mdtH gene mutant plasmids was constructed as follows.

[0130] Using the genomic DNA of E. coli K12 W3110 (Genbank: NC_007779.1) as a template, random mutagenesis PCR was performed using the primer pair (primers 3 and 8) of SEQ ID NOs: 9 and 10 (Takara Diversify PCR random mutagenesis kit, Cat. no. 630703).

[0131] PCR was performed by denaturing at 94°C for 5 minutes, repeating 20 cycles of denaturing at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerizing at 72°C for 1 minute, followed by polymerization at 72°C for 5 minutes.

[0132] First, pCL_Ptrc was constructed to insert the resulting mutant gene fragment into the pCL1920 vector (GenBank No. AB236930), which contains the trc promoter. PCR was performed using a primer pair (primer 1 and 2) of SEQ ID NOs: 7 and 8 to isolate the trc promoter fragment. PCR consisted of denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, followed by polymerization at 72°C for 5 minutes.

[0133] The primer sequences used here are as shown in Table 1 below.

[0134] [Table 1]

[0135] The trc promoter fragment was cloned into the pCL1920 vector cleaved with EcoRI and SalI using an in-fusion cloning kit (Clontech Laboratories, Inc.) to obtain pCL_Ptrc. The obtained pCL_Ptrc vector was cleaved with PstI and EcoRV, and the mutated gene fragments obtained through PCR were cloned using the in-fusion cloning kit. Cloning was carried out at 50°C for 60 minutes, and a pCL_Ptrc-mdtH gene mutant plasmid library was constructed.

[0136] The constructed pCL_Ptrc-mdtH gene mutant plasmid library was transformed into CA07-0012 (KCCM 11121P, European Patent Publication EP 2444481 or US Publication No. 2012-0190081) by electroporation. Two strains containing mutants were selected, and the plasmids were isolated and analyzed by sequencing.

[0137] Analysis of the base sequences confirmed that the two selected mutants were a mutant in which the 125th amino acid residue, valine, was replaced with isoleucine in the amino acid sequence of wild-type MdtH [mdtH(V125I)], and a mutant in which the 60th amino acid residue, glutamine, was replaced with arginine, the 125th amino acid residue, valine, was replaced with isoleucine, the 180th amino acid residue, phenylalanine, was replaced with leucine, and the 398th amino acid residue, leucine, was replaced with proline [mdtH(Q60R, V125I, F180L, L398P)]. The strain CA07-0012 / pCL_Ptrc-mdtH(V125I) transformed with the mutant mdtH(V125I) was named E. coli CA07-0379, and the strain CA07-0012 / pCL_Ptrc-mdtH(Q60R, V125I, F180L, L398P) transformed with the mutant mdtH(Q60R, V125I, F180L, L398P) was named E. coli CA07-0380.

[0138] Example 2: Evaluation of OPS production ability of strains into which MdtH mutants have been introduced The OPS-producing ability of the strains into which the MdtH mutants were introduced was evaluated using the following medium (Table 2).

[0139] [Table 2]

[0140] Specifically, each strain was smeared on LB solid medium and then cultured overnight in an incubator at 33°C. The strains cultured overnight on LB solid medium were inoculated into 25 mL of the titer medium shown in Table 2, which was then cultured in an incubator at 200 rpm at 33°C for 48 hours, and the OPS production ability was evaluated. The results are shown in Table 3 below.

[0141] [Table 3]

[0142] As a result, the strain CA07-0012 / pCL_Ptrc-mdtH(V125I) containing mdtH(V125I) showed approximately 180% of the productivity of the strain CA07-0012 / pCL_Ptrc-mdtH containing wild-type mdtH, and the strain CA07-0012 / pCL_Ptrc-mdtH(Q60R, V125I, F180L, L398P) containing mdtH(Q60R, V125I, F180L, L398P) showed approximately 237% of the productivity of the strain CA07-0012 / pCL_Ptrc-mdtH.

[0143] The CA07-0012 / pCL_Ptrc-mdtH(V125I) was named CA07-0379, and the CA07-0012 / pCL_Ptrc-mdtH(Q60R, V125I, F180L, L398P) was named CA07-0380.

[0144] 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. An MdtH mutant in which the amino acid corresponding to position 125 of the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine, The variant has a sequence identity of 90% or more and less than 100% with the amino acid sequence set forth in SEQ ID NO: 1, An MdtH mutant, wherein the MdtH mutant has increased OPS-producing ability compared to a wild-type MdtH mutant.

2. The MdtH mutant of claim 1 , wherein the amino acid corresponding to position 125 is valine.

3. The MdtH mutant according to claim 1, wherein the amino acid corresponding to the 60th position in the amino acid sequence of SEQ ID NO: 1 is glutamine or arginine.

4. The MdtH mutant according to claim 1, wherein the amino acid corresponding to position 180 in the amino acid sequence of SEQ ID NO: 1 is phenylalanine or leucine.

5. The MdtH mutant according to claim 1, wherein the amino acid corresponding to position 398 in the amino acid sequence of SEQ ID NO: 1 is leucine or proline.

6. The MdtH mutant of claim 1 , wherein the mutant consists of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO:

3.

7. A polynucleotide encoding the variant of claim 1.

8. A recombinant Escherichia microorganism comprising an MdtH mutant in which the amino acid corresponding to position 125 of the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine, or a polynucleotide encoding said mutant, The variant has a sequence identity of 90% or more and less than 100% with the amino acid sequence set forth in SEQ ID NO: 1, A recombinant Escherichia microorganism, wherein the MdtH mutant has increased OPS-producing ability compared to the wild-type.

9. The microorganism according to claim 8, wherein the recombinant microorganism further has an attenuated activity of phosphoserine phosphatase (SerB) compared to its endogenous activity.

10. 1. A method for producing O-phosphoserine, comprising the step of culturing in a medium a microorganism containing an MdtH mutant in which the amino acid corresponding to position 125 of the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine, or a polynucleotide encoding said mutant, The variant has a sequence identity of 90% or more and less than 100% with the amino acid sequence set forth in SEQ ID NO: 1, The method, wherein the MdtH mutant has increased OPS production ability compared to the wild-type.

11. a) producing O-phosphoserine or a medium containing the same by culturing in a medium an MdtH mutant in which the amino acid corresponding to position 125 of the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine or a microorganism containing a polynucleotide encoding the mutant, The variant has a sequence identity of 90% or more and less than 100% with the amino acid sequence set forth in SEQ ID NO: 1, the MdtH mutant has increased OPS production ability compared to a wild-type MdtH mutant; b) reacting O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing the same and the O-phosphoserine produced in step a) or a medium containing the same with sulfide;

12. Use of an MdtH mutant in which the amino acid corresponding to position 125 of the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine for the production of OPS, cysteine, or a cysteine ​​derivative, comprising: The variant has a sequence identity of 90% or more and less than 100% with the amino acid sequence set forth in SEQ ID NO: 1, The MdtH mutant has increased OPS production ability compared to the wild type.

13. 1. Use of an MdtH mutant, in which the amino acid corresponding to position 125 of the amino acid sequence of SEQ ID NO: 1 is substituted with isoleucine, for improving OPS production ability from a microorganism compared to a wild-type MdtH mutant, The variant has a sequence identity of 90% or more and less than 100% with the amino acid sequence set forth in SEQ ID NO: 1, The MdtH mutant has increased OPS production ability compared to the wild type.

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