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

Abstract

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

Description

[Technical Field]

[0001] The present application relates to YhhS 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 (OPS) using microorganisms, followed by conversion to L-cysteine ​​by reaction with sulfide under the catalytic action of O-phosphoserine sulfhydrylase (OPSS) (U.S. Patent Publication US 8557549 B2).

[0004] In order to produce cysteine ​​in high yield using the method 3), it is necessary to overproduce the precursor OPS. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] European Patent EP0885962B [Patent Document 2] US Registry Bulletin US 8557549 B2 [License 3] Republic of Korea Registration License No. 10-1381048 [License 4] U.S. Open Patent No. 10-2012-0190081 [Patent Document 5] US Registry Permit US 7662943 B2 [License 6] US Registry Permit US 10584338 B2 [License 7] US Registry Permit US 10273491 B2 [License 8] US Registry Bulletin US 8557549 B2 [License 9] U.S. Open Gazette No. 2012-0190081 [License 10] US Registry Bulletin US 9127324 B2 [License 11] U.S. Patent and Trademark No. 2020-0048619 [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)

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Summary of the Invention

[0007] The present inventors have identified mutants of membrane proteins that have the activity of secreting OPS produced by OPS-producing strains outside the cells, and have confirmed that the mutants improve OPS secretion, thereby completing the present application. [Means for solving the problem]

[0008] One object of the present application is to provide a YhhS variant in which the amino acid corresponding to position 129 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 a YhhS variant in which the amino acid isoleucine corresponding to position 241 of the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine.

[0010] Another object of the present application is to provide polynucleotides encoding the variants of the present application.

[0011] Another object of the present application is to provide a microorganism of the genus Escherichia comprising the mutant of the present application or a polynucleotide encoding the mutant.

[0012] Another object of the present application is to provide a method for producing O-phosphoserine, which comprises culturing in a medium a microorganism containing the variant of the present application or a polynucleotide encoding said variant.

[0013] Another object of the present application is to provide a method for producing cysteine ​​or a derivative thereof, the method comprising: a) culturing an O-phosphoserine-producing microorganism comprising a variant of the present application or a polynucleotide encoding the variant in a medium to produce O-phosphoserine or a medium containing O-phosphoserine; and b) contacting O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing O-phosphoserine, the O-phosphoserine produced in step a) or a medium containing O-phosphoserine, and sulfide. [Effects of the Invention]

[0014] The present application discloses that when a microorganism capable of producing OPS is cultured using a novel mutant polypeptide with O-phosphoserine (OPS) export activity, higher yields of OPS can be produced compared to 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 fall within the scope of this application. Furthermore, the specific descriptions described below are not considered to limit the category of this application.

[0016] Furthermore, throughout this specification, numerous papers and patent documents are referenced and citations are provided, the disclosures of which are incorporated herein by reference in their entirety to more clearly explain the state of the art to which this application pertains and the contents of the present invention.

[0017] One aspect of the present application provides a YhhS variant in which the amino acid corresponding to position 129 of the amino acid sequence of SEQ ID NO: 1 has been substituted with another amino acid.

[0018] As used herein, the term "YhhS" refers to a polypeptide exhibiting O-phosphoserine (OPS) export activity, specifically, a membrane protein capable of exporting OPS to the extracellular space. In the present application, the YhhS may be a YhhS MFS (major facilitator superfamily) transporter, which is a membrane protein capable of exporting OPS to the extracellular space. YhhS has been identified as a protein that exhibits OPS export activity in Escherichia coli cells in which growth inhibition is relieved in the presence of excessive amounts of OPS.

[0019] As used herein, the term "O-phosphoserine (OPS)" refers to a phosphoric acid ester of serine, a component of various proteins. OPS is a precursor of L-cysteine, which can be converted to cysteine ​​by reacting with sulfide under the catalytic action of OPS sulfhydrylase (OPSS) (U.S. Patent Publication No. US 8557549 B2), but is not limited thereto.

[0020] Specifically, the term "YhhS" used herein may be used interchangeably with "YhhS MFS transporter." The amino acid sequence of the YhhS in the present application can be obtained from the publicly known database, GenBank, of NCBI. Specifically, the amino acid sequence may be a polypeptide having YhhS activity encoded by the yhhS gene, more specifically, but not limited to, SEQ ID NO: 1.

[0021] The amino acid corresponding to position 129 of the amino acid sequence of SEQ ID NO: 1 may be a polar amino acid, such as serine, threonine, cysteine, tyrosine, asparagine, or glutamine, and more specifically, serine.

[0022] The variants of the present application may be those in which the polar amino acid corresponding to position 129 of the amino acid sequence of SEQ ID NO: 1 is replaced with a nonpolar amino acid. The nonpolar amino acid may be, for example, glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, or proline, specifically glycine or alanine. The variants may include amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homologous or identical to an amino acid sequence in which the amino acid corresponding to position 129 of the amino acid sequence of SEQ ID NO: 1 is glycine or alanine. 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 variants of the present application.

[0023] For example, the amino acid sequence may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally that do not alter the function of the variant of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0024] As used herein, the term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. The variant can, for example, have one or more conservative substitutions while still retaining one or more biological activities. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues. For example, among amino acids having electrically charged side chains, positively charged (basic) amino acids include arginine, lysine, and histidine, and negatively charged (acidic) amino acids include glutamic acid and aspartic acid; among amino acids having uncharged side chains, nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, polar or hydrophilic amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine, and aromatic amino acids include phenylalanine, tryptophan, and tyrosine.

[0025] As used herein, the term "variant" refers to a polypeptide in which one or more amino acids have been conservatively substituted and / or modified, resulting in a polypeptide that differs from the original amino acid sequence but maintains its functions or properties. Such variants can generally be identified by altering one or more amino acids in the amino acid sequence of the polypeptide and evaluating the properties of the variant. That is, the performance of the variant may be increased, unchanged, or decreased compared to the original polypeptide. Some variants may also include variants in which one or more portions, such as an N-terminal leader sequence or a 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 "variant" includes, but is not limited to, terms such as mutant, modified, mutant polypeptide, mutated protein, mutation, and variant (in English, "modification," "modified polypeptide," "modified protein," "mutant," "mutein," "divergent," etc.), which are used interchangeably and refer to a mutated state. For purposes of the present application, the variant may be a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, in which the serine amino acid corresponding to position 129 of the amino acid sequence of SEQ ID NO: 1 is substituted with glycine or alanine.

[0026] 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.

[0027] As used herein, the terms "homology" or "identity" refer to the degree of similarity between two given amino acid or nucleotide sequences, which can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0028] 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.

[0029] 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, for example, 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.

[0030] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that of Needleman et al. (1970), J Mol Biol. 48:443, as known, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAP program defines a match as the total number of symbols in the shorter of the two sequences divided by the number of similar aligned symbols (i.e., nucleotides or amino acids). Default parameters for the GAP program include: (1) a 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.

[0031] 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 particular amino acid in a sequence that references a particular sequence. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related or reference protein.

[0032] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, based on which each amino acid residue in the amino acid sequence can be numbered with reference to the numeric position of the corresponding amino acid residue in SEQ ID NO: 1. For example, a sequence alignment algorithm such as those described herein can identify amino acid positions or positions where mutations such as substitutions, insertions or deletions occur compared to a query sequence (also referred to as a "reference sequence").

[0033] For such alignment, for example, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) or the Needleman 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 thereto. Sequence alignment programs, pairwise sequence comparison algorithms, and the like known in the art can also be used appropriately.

[0034] Another aspect of the present application is to provide a YhhS variant in which the amino acid isoleucine corresponding to position 241 of the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine or threonine.

[0035] The "amino acid sequence of SEQ ID NO: 1," "YhhS," and "mutant" are as described above in other aspects.

[0036] Specifically, the variant of the present application may be one in which the isoleucine amino acid at position 241 in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine or threonine. The variant may include an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% homology or identity to an amino acid sequence in which the amino acid at position 241 in the amino acid sequence of SEQ ID NO: 1 is glutamine or threonine, as described in the other aspects above.

[0037] In addition, the variant of the present application may have the amino acid corresponding to position 129 of the amino acid sequence of SEQ ID NO: 1 substituted with another amino acid, and further have the isoleucine corresponding to position 241 substituted with glutamine or threonine.

[0038] In addition, the variants of the present application may include, in addition to the substitution of the amino acid corresponding to position 129 or position 241 of the amino acid sequence of SEQ ID NO: 1, a further substitution of the amino acid aspartic acid corresponding to position 246 of the amino acid sequence of SEQ ID NO: 1 with valine, and / or a further substitution of the amino acid valine corresponding to position 330 of the amino acid sequence of SEQ ID NO: 1 with isoleucine.

[0039] Additionally, the variant of the present application may have the amino acid corresponding to position 88 of the amino acid sequence of SEQ ID NO: 1 as phenylalanine and the amino acid corresponding to position 207 of the amino acid sequence of SEQ ID NO: 1 as lysine.

[0040] Specifically, the variants of the present application are SEQ ID NO: 2 in which the amino acid serine corresponding to the 129th position in the amino acid sequence of SEQ ID NO: 1 is substituted with glycine; SEQ ID NO: 3 in which the amino acid serine corresponding to the 129th position in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine; SEQ ID NO: 4 in which the amino acid isoleucine corresponding to the 241st position in the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine; SEQ ID NO: 5 in which the amino acid isoleucine corresponding to the 241st position in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine; and SEQ ID NO: 6 in which the amino acid isoleucine corresponding to the 241st position in the amino acid sequence of SEQ ID NO: 1 is substituted with threonine and the amino acid aspartic acid corresponding to the 246th position in the amino acid sequence of SEQ ID NO: 1 is substituted with valine. and the amino acid sequence of SEQ ID NO: 12 in which the amino acid serine corresponding to position 129 of the amino acid sequence of SEQ ID NO: 1 is substituted with glycine, the amino acid isoleucine corresponding to position 241 of the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid aspartic acid corresponding to position 246 of the amino acid sequence of SEQ ID NO: 1 is substituted with valine, and the amino acid valine corresponding to position 330 of the amino acid sequence of SEQ ID NO: 34 in which the amino acid serine corresponding to position 129 of the amino acid sequence of SEQ ID NO: 1 is substituted with glycine, the amino acid isoleucine corresponding to position 241 of the amino acid sequence of SEQ ID NO: 1 is substituted with threonine, the amino acid aspartic acid corresponding to position 246 of the amino acid sequence of SEQ ID NO: 1 is substituted with valine, and the amino acid valine corresponding to position 330 of the amino acid sequence of SEQ ID NO: 36 in which the amino acid serine corresponding to position 129 of the amino acid sequence of SEQ ID NO: 1 is substituted with glycine and the amino acid isoleucine corresponding to position 241 of the amino acid sequence of SEQ ID NO: 1 is substituted with glutamine.

[0041] The variant of the present application may be a polypeptide consisting of or comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:12 or SEQ ID NO:34 or SEQ ID NO:36.

[0042] Furthermore, the variant of the present application may have a sequence identity (homology or identity) of 99% or more to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 12 or SEQ ID NO: 34 or SEQ ID NO: 36, but may also have a sequence identity of at least 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or less than 100% to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 12 or SEQ ID NO: 34 or SEQ ID NO: 36. Furthermore, it is obvious that variants having amino acid sequences in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted or added are also included within the scope of this application, as long as they have such homology or identity and exhibit efficacy corresponding to the variants of this application.

[0043] As an example of the present application, the variants of the present application can have YhhS activity and can have the activity of increasing OPS excretion compared to the wild-type polypeptide.

[0044] The YhhS is as described in the other embodiment.

[0045] Another aspect of the present application is to provide polynucleotides encoding the variants of the present application.

[0046] The "mutant" is as described above.

[0047] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide units are linked in a long chain by covalent bonds, and a DNA or RNA chain of a certain length or more, and more specifically, refers to a polynucleotide fragment encoding the above-mentioned variant.

[0048] A polynucleotide encoding a variant of the present application may comprise a nucleotide sequence encoding the amino acid sequence set forth as SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:12 or SEQ ID NO:34 or SEQ ID NO:36. As an example of the present application, a polynucleotide of the present application may have or comprise the sequence of SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:17 or SEQ ID NO:35 or SEQ ID NO:37. A polynucleotide of the present application may also consist of or consist essentially of the sequence of SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:17 or SEQ ID NO:35 or SEQ ID NO:37.

[0049] The polynucleotides of the present application may have various modifications in the coding region, taking into account codon degeneracy or codons preferred in the organism in which the variants of the present application are to be expressed, as long as the amino acid sequence of the variants of the present application is not changed. Specifically, the polynucleotide of the present application may have or comprise 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, and less than 100% homologous or identical to the sequence of SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:17 or SEQ ID NO:35 or SEQ ID NO:37, 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, and less than 100% homologous or identical to the sequence of SEQ ID NO:6 or SEQ ID NO:7 or SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:17 or SEQ ID NO:35 or SEQ ID NO:37, but is not limited thereto. In this case, in the sequence having the homology or identity, the codon encoding the amino acid corresponding to the 129th position of SEQ ID NO: 1 may be one of the codons encoding glycine or alanine, the codon encoding the amino acid corresponding to the 241st position may be one of the codons encoding glutamine or threonine, the codon encoding the amino acid corresponding to the 246th position may be one of the codons encoding valine, and the codon encoding the amino acid corresponding to the 330th position may be one of the codons encoding isoleucine.

[0050] Furthermore, the polynucleotides of the present application may include, without limitation, probes that can be prepared from known gene sequences, for example, sequences that can hybridize under stringent conditions to a complementary sequence to the entire or partial 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, the conditions include conditions under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 70% or more, 75% or more, 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; and conditions for washing once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions for conventional Southern hybridization, such as 60°C, 1X SSC, and 0.1% SDS, specifically 60°C, 0.1X SSC, and 0.1% SDS, more specifically 68°C, 0.1X SSC, and 0.1% SDS.

[0051] Hybridization requires that two nucleic acids have complementary sequences, even though mismatches between bases are possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize to one another. For example, for DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Thus, the polynucleotides of the present application can include isolated nucleic acid fragments that are complementary to entire sequences, as well as substantially similar nucleic acid sequences.

[0052] 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 may be appropriately adjusted by those skilled in the art depending on the purpose.

[0053] The appropriate stringency for hybridizing such polynucleotides depends on the length and degree of complementation of the polynucleotides, variables well known in the art (eg, J. Sambrook et al., supra).

[0054] Another aspect of the present application is to provide a vector comprising a polynucleotide of the present application.

[0055] The vector may be, but is not limited to, an expression vector for expressing the polynucleotide in a host cell.

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

[0057] The vectors used in this application are not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pSK, pSKH, and pET can be used as plasmid vectors. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pSK, pSKH130, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0058] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome of a cell using a vector for chromosomal integration. The polynucleotide can be inserted into a chromosome by any method known in the art, including, but not limited to, homologous recombination. A selection marker for confirming the presence or absence of insertion into the chromosome may also be included. The selection marker is used to select cells transformed with the vector, i.e., to confirm the presence or absence of insertion of the target nucleic acid molecule. A marker that confers a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface polypeptide, may be used. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0059] The term "transformation" as used herein 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 may typically include a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of an autonomously replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form, operably linked to sequences necessary for expression in the host cell, but is not limited thereto.

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

[0061] Another aspect of the present application is to provide a microorganism of the genus Escherichia comprising a variant of the present application or a polynucleotide encoding said variant.

[0062] The microorganism of the present application can comprise a mutant polypeptide of the present application, a polynucleotide encoding said polypeptide, or a vector comprising a polynucleotide of the present application.

[0063] In this application, the term "strain (or microorganism)" includes all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially, and may be microorganisms in which a specific mechanism has been weakened or enhanced by inserting an exogenous gene or by enhancing or inactivating the activity of an endogenous gene, and may also be microorganisms that contain genetic modifications for the production of a desired polypeptide, protein, or product.

[0064] The microorganism of the present application may be, but is not limited to, a microorganism comprising one or more of the variants of the present application, the polynucleotides of the present application, and the vectors comprising the polynucleotides of the present application; a microorganism that has been modified to express the variants of the present application or the polynucleotides of the present application; a microorganism (e.g., a recombinant microorganism) that expresses the variants of the present application or the polynucleotides of the present application; or a microorganism (e.g., a recombinant microorganism) that has the activity of the variants of the present application.

[0065] The microorganism of the present application may be a microorganism capable of producing O-phosphoserine.

[0066] The microorganism of the present application may be, but is not limited to, a microorganism that naturally has the ability to produce YhhS or O-phosphoserine, or a microorganism into which a mutant of the present application or a polynucleotide encoding the mutant (or a vector containing the polynucleotide) has been introduced into a parent strain that does not have the ability to produce YhhS or O-phosphoserine, and / or which has been conferred the ability to produce O-phosphoserine.

[0067] For example, the strains of the present application are cells or microorganisms 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 expressing the variant of the present application. For purposes of this application, the strains of the present application can include all microorganisms capable of producing O-phosphoserine, including the variant of the present application. For example, the strains of the present application can be recombinant strains with increased O-phosphoserine production ability due to the introduction of a polynucleotide encoding the variant of the present application into a naturally occurring wild-type microorganism or a microorganism that produces O-phosphoserine, thereby expressing a YhhS variant. The recombinant strain with increased O-phosphoserine production ability can be, but is not limited to, a naturally occurring wild-type microorganism or a non-YhhS mutant microorganism (i.e., a microorganism that expresses wild-type YhhS (SEQ ID NO: 1) or a microorganism that does not express mutant YhhS (SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 12 or SEQ ID NO: 34 or SEQ ID NO: 36)). For example, the microorganism into which wild-type YhhS has been introduced, which is the target strain for comparing the increase in O-phosphoserine production ability, may be, but is not limited to, CA07-0012 (KCCM 11121P, Korean Patent Registration No. 10-1381048 and U.S. Patent Publication No. 10-2012-0190081), a microorganism in which SerB activity is weakened compared to its endogenous activity.

[0068] For example, the recombinant strain with increased production ability has an O-phosphoserine production ability of about 1% or more, specifically about 1% or more, about 10% or more, about 20% or more, about 29% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 100% or more, about 110% or more, about 120% or more, about 130% or more, about 140% or more, about 144% or more, about 150% or more, about 160% or more, about 170% or more, about 180% or more, about 190% or more, about 200% or more, about 210% or more, about 220% or more, or about 230% or more, compared to the O-phosphoserine production ability of the parent strain or unmodified microorganism before mutation. , about 233% or more, about 240% or more, about 250% or more, about 252% or more, about 260% or more, about 267% or more, about 270% or more, about 280% or more, about 290% or more, about 300% or more, about 310% or more, about 320% or more, about 330% or more, or about 338% or more (there is no particular limitation on the upper limit, and it may be, for example, about 300% or less, about 200% 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), but is not limited thereto as long as there is an increase in the productivity compared to the parent strain or unmodified microorganism before mutation. In another example, the recombinant strain with increased production ability has an IMP production ability that is about 1.01-fold or more, about 1.1-fold or more, about 1.2-fold or more, about 1.29-fold or more, about 1.3-fold or more, about 1.4-fold or more, about 1.5-fold or more, about 1.6-fold or more, about 1.7-fold or more, about 1.8-fold or more, about 1.9-fold or more, about 2.0-fold or more, about 2.1-fold or more, about 2.2-fold or more, about 2.3-fold or more, about 2.4-fold or more, about 2.44-fold or more, about 2.5-fold or more, about 2.6-fold or more, about 2.7-fold or more, about 2.8-fold or more, about 2.9-fold or more, compared to the parent strain or unmodified microorganism before mutation. fold or more, about 3.0 times or more, about 3.1 times or more, about 3.2 times or more, about 3.3 times or more, about 3.33 times or more, about 3.4 times or more, about 3.5 times or more, about 3.52 times or more, about 3.6 times or more, about 3.67 times or more, about 3.7 times or more, about 3.8 times or more, about 3.9 times or more, about 4.0 times or more, about 4.1 times or more, about 4.2 times or more, about 4.3 times or more, or about 4.38 times or more (there is no particular restriction 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), but is not limited thereto.

[0069] In the present application, the term "unmodified microorganism" does not exclude strains containing mutations that may occur naturally in microorganisms, but refers to a wild-type or naturally occurring strain itself, or a strain before its traits are changed due to genetic mutations caused by natural or artificial factors. For example, the unmodified microorganism refers to a strain before or before the introduction of a YhhS mutant described herein. The term "unmodified microorganism" may be used interchangeably with "strain before modification," "microorganism before modification," "non-mutated strain," "unmodified strain," "non-mutated microorganism," or "reference microorganism."

[0070] In another embodiment of the present application, the microorganism of the present application may be a microorganism capable of producing O-phosphoserine, and the type thereof is not particularly limited. The microorganism of the present application may be either a prokaryotic cell or a eukaryotic cell, and specifically may be a prokaryotic cell. Examples of the prokaryotic cell 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. 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).

[0071] The O-phosphoserine-producing microorganism of the present application may further have a weakened activity of phosphoserine phosphatase (SerB) compared to the endogenous activity.

[0072] Because SerB of the present application has the activity of converting O-phosphoserine to L-serine, microorganisms mutated to attenuate the SerB activity are characterized by accumulating O-phosphoserine and are useful for producing O-phosphoserine. SerB of the present application may be, but is not limited to, a protein having or including the amino acid sequence set forth in SEQ ID NO: 10, or a protein consisting of or essentially consisting of the amino acid sequence set forth in SEQ ID NO: 10. Furthermore, SerB of the present application may have or include an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 10, as long as it exhibits SerB activity. Furthermore, SerB of the present application may consist of, or essentially consist of, an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 10, but is not limited to this. Furthermore, the polynucleotide encoding SerB may have or include a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 10. Furthermore, the polynucleotide encoding SerB may consist of, or essentially consist of, a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 10. The polynucleotide encoding SerB of the present application may have various modifications in the coding region within a range that does not change the amino acid sequence of the SerB protein, taking into account codon degeneracy or codons preferred in the organism in which the SerB protein is to be expressed. 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% homologous or identical to the nucleotide sequence of SEQ ID NO: 11, but less than 100% identical. Furthermore, the polynucleotide encoding SerB of the present application consists of or essentially consists of a base sequence that has at least 70%, 80%, 90%, 95%, or 99% or more, but less than 100%, homology or identity to the base sequence of SEQ ID NO: 11, but is not limited thereto.

[0073] The term "attenuation" of a polypeptide in this application encompasses a reduction in activity or absence of activity compared to the intrinsic activity. The term "attenuation" may be used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0074] 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 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 non-mutated 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 mutation." 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 non-mutated microorganism before the trait has been altered.

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

[0076] Specifically, the attenuation 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) Mutation of the amino acid sequence constituting the polypeptide (e.g., removal / substitution / addition of one or more amino acids in the amino acid sequence) so that the activity of the polypeptide is eliminated or attenuated; 4) Mutation of the gene sequence encoding the polypeptide so that the activity of the polypeptide is eliminated or attenuated (e.g., removal / substitution / addition of one or more nucleic acid bases on the nucleic acid base sequence of the polypeptide gene so as to encode a modified polypeptide so that the activity of the polypeptide is eliminated or attenuated); 5) a mutation in the nucleotide sequence encoding the initiation codon or 5'-UTR region of a gene transcript encoding a polypeptide; 6) introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the polypeptide; 7) Addition of a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide to form a secondary structure 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.

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

[0078] The mutation of the expression regulatory region (or expression regulatory sequence) may be a deletion, insertion, non-conservative or conservative substitution, or a combination thereof, which may result in a mutation in the expression regulatory region (or expression regulatory sequence), or may be replaced with a sequence having a weaker activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

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

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

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

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

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

[0084] 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" can 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 an improved activity compared to the endogenous activity or activity prior to mutation. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or non-mutated microorganism prior to mutation, in cases where a trait has been altered by genetic mutation due to natural or artificial factors. This term can be used interchangeably with "activity prior to mutation." "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 non-mutated microorganism prior to mutation.

[0085] 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.

[0086] 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 method may utilize 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, 1-16; Sambrook et al., Molecular Cloning 2012, etc.).

[0087] Specifically, the enhancement of the polypeptide of the present application is 1) an increase in the intracellular copy number of the polynucleotide encoding the polypeptide; 2) Mutation of the expression regulatory region of the gene on the chromosome encoding the polypeptide (e.g., occurrence of a mutation within the expression regulatory region, replacement with a sequence having stronger activity, or insertion of a sequence having stronger activity); 3) a mutation in the nucleotide sequence encoding the initiation codon or 5'-UTR region of the gene transcript encoding the polypeptide; 4) mutating the amino acid sequence of the polypeptide so that the polypeptide activity is enhanced; 5) Mutation of the polynucleotide sequence encoding the polypeptide so as to enhance the polypeptide activity (e.g., mutation of the polynucleotide sequence of the polypeptide gene so as to encode a modified polypeptide so as to enhance the polypeptide activity); 6) introduction of a foreign polypeptide exhibiting the activity of the polypeptide or a foreign polynucleotide encoding the same; 7) codon optimization of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide and selectively deforming or chemically modifying exposed sites; or 9) It may be a combination of two or more selected from the above 1) to 8), but is not particularly limited thereto.

[0088] 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.

[0089] The 2) replacement of the expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide with a sequence with stronger activity may be, for example, by introducing a mutation in the sequence through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the expression regulatory region, or by replacing it with a sequence with stronger activity. The expression regulatory region may include, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation. For example, the original promoter may be replaced with a strong promoter, but this is not limited thereto.

[0090] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (U.S. Patent No. 7,662,943 B2), lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (U.S. Patent No. 10,584,338 B2), O2 promoter (U.S. Patent No. 10,273,491 B2), tkt promoter, and yccA promoter.

[0091] The modification of the nucleotide sequence encoding the start codon or 5'-UTR region of the gene transcript encoding the polypeptide may be, for example, but is not limited to, substituting a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon.

[0092] 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.

[0093] 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, and the introduced polynucleotide may be expressed in a host cell to produce a polypeptide, and its activity may be increased.

[0094] 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.

[0095] 8) Analyzing the tertiary structure of a polypeptide and selecting and mutating or chemically modifying exposed sites may involve, for example, comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and mutating or modifying exposed sites to be deformed or chemically modified.

[0096] Such an 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 unmutated microbial strain, or an increase in the amount of a product produced from the polypeptide.

[0097] Mutation 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 mutating a portion or all of the gene include methods using DNA recombination technology. 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.

[0098] Furthermore, the microorganism may be a microorganism in which the ability to influx OPS into cells and to decompose OPS has been reduced.

[0099] In addition to the above, the disclosures of U.S. Patent Publication No. US 8557549 B2 and U.S. Publication No. 2012-0190081, etc., regarding the OPS-producing microorganisms described above are incorporated herein by reference, but are not limited thereto.

[0100] In the microorganisms of the present application, the terms "mutant," "polynucleotide," and "O-phosphoserine" are as defined above in other aspects.

[0101] Another aspect of the present application is to provide a method for producing O-phosphoserine, comprising culturing in a medium a microorganism comprising a variant of the present application or a polynucleotide encoding said variant.

[0102] The method for producing O-phosphoserine of the present application can include culturing a microorganism containing a variant of the present application or a polynucleotide encoding the variant in a medium.

[0103] As used herein, the term "culturing" refers to 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, or fed-batch culture.

[0104] In the present application, the term "culture medium" refers to a substance containing a mixture of nutrients, primarily those required for culturing the microorganism of the present application, and provides nutrients such as water essential for survival and growth, as well as growth factors, etc. Specifically, the culture medium and other culture conditions used for culturing the microorganism of the present application can be any medium used for culturing ordinary microorganisms without any particular limitations. The microorganism of the present application can be cultured in an ordinary culture medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids, and / or vitamins, etc., under aerobic conditions while controlling the temperature, pH, etc.

[0105] In the present application, examples of the carbon source include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. Natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn steeping liquid can also be used. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) can be used. Various other carbon sources can also be used in appropriate amounts without limitation. These carbon sources can be used alone or in combination, and are not limited thereto.

[0106] Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources may be used alone or in combination of two or more, and are not limited thereto.

[0107] The phosphorus source may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts. Inorganic compounds include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Other compounds may include amino acids, vitamins, and / or appropriate precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, they are not limited to these.

[0108] The medium may contain metal salts such as magnesium sulfate or iron sulfate, and may also contain amino acids, vitamins, and appropriate precursors. These medium or precursors may be added to the culture in a batch or continuous manner, but are not limited thereto.

[0109] For example, when culturing a recombinant microorganism in which SerB activity is weakened compared to endogenous activity, serine auxotrophy of the microorganism is induced, and the culture medium may further contain glycine or serine. Glycine may be provided in the form of purified glycine, a yeast extract containing glycine, or tryptone, and its concentration in the culture medium may typically be 0.1 to 10 g / L, specifically 0.5 to 3 g / L. Serine may also be provided in the form of purified serine, a yeast extract containing serine, or tryptone, and its concentration in the culture medium may typically be 0.1 to 5 g / L, specifically 0.1 to 1 g / L.

[0110] During the cultivation of the microorganism of the present application, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the medium in an appropriate manner. During cultivation, foam formation can be suppressed using an antifoaming agent such as a fatty acid polyglycol ester. To maintain an aerobic state in the medium, oxygen or an oxygen-containing gas can be injected into the medium, or to maintain an anaerobic or microaerobic state, no gas can be injected or nitrogen, hydrogen, or carbon dioxide gas can be injected, but this is not limited to these.

[0111] In the culture of the present application, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and the culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0112] The O-phosphoserine produced by the culture of the present application is either secreted into the medium or remains intracellularly.

[0113] The method for producing O-phosphoserine of the present application may further include a step of preparing the microorganism of the present application, a step of preparing a medium for culturing the microorganism, or a combination thereof (in any order), for example, before the culturing step.

[0114] The method for producing O-phosphoserine of the present application may further include a step of recovering O-phosphoserine from the culture medium (the medium in which the culture was performed) or the microorganism. The recovery step may be further included after the culture step.

[0115] The recovery may involve collecting the target O-phosphoserine using an appropriate method known in the art based on the microbial culture method of the present application, such as a batch, continuous, or fed-batch culture method. For example, centrifugation, filtration, treatment with a crystallized protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various types of chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination of these methods can be used, and the target O-phosphoserine can be recovered from the medium or the microorganism using an appropriate method known in the art.

[0116] Furthermore, the method for producing O-phosphoserine of the present application may further include a purification step. The purification can be performed using an appropriate method known in the art. In one example, when the method for producing O-phosphoserine 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, simultaneously, or integrated into one step, regardless of the order, but are not limited thereto.

[0117] In the methods of the present application, the terms "mutant", "polynucleotide", "vector" and "microorganism" are as defined above in other aspects.

[0118] Another aspect of the present application is to provide a method for producing cysteine ​​or a derivative thereof, the method comprising: a) culturing an O-phosphoserine-producing microorganism comprising a variant of the present application or a polynucleotide encoding the variant in a medium to produce O-phosphoserine or a medium containing O-phosphoserine; and b) contacting O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing O-phosphoserine, the O-phosphoserine produced in step a) or a medium containing O-phosphoserine, and sulfide.

[0119] Specifically, the method includes providing a polypeptide having O-phosphoserine export activity and comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:12 or SEQ ID NO:34 or SEQ ID NO:36; a polypeptide having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.7%, or 99.9% or more homology or identity thereto; or a polypeptide having at least one of the following amino acid sequences in the amino acid sequence of SEQ ID NO:1: i) positions 129; ii) positions 241; iii) positions 129 and 241; iv) positions 241, 246, and 330; and v) positions 129, 241, 246, and 330. The method may also include a step of culturing an O-phosphoserine-producing microorganism containing one or more selected from the following in a medium to produce O-phosphoserine or a medium containing O-phosphoserine; and a step of reacting O-phosphoserine sulfhydrylase or a microorganism expressing O-phosphoserine, or the O-phosphoserine produced in the step or a medium containing O-phosphoserine, with a sulfide.

[0120] In this application, the term "derivative" refers to a similar compound obtained by chemically changing a part of a compound, and usually refers to a compound in which a hydrogen atom or a specific atomic group in the compound is replaced with another atom or atomic group.

[0121] 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 a cysteine ​​include a form 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, and examples thereof include, but are not limited to, NAC (N-acetylcysteine), SCMC (S-carboxymethylcysteine), 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.

[0122] Once cysteine ​​is produced by the method of the present application, it can be easily converted into various cysteine ​​derivatives by methods well known in the art.

[0123] 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, by reacting cysteine ​​with an acetylation agent to synthesize N-acetylcysteine ​​(NAC), or by reacting cysteine ​​with haloacetic acid under basic conditions to synthesize S-carboxymethylcysteine ​​(SCMC), but is not limited thereto.

[0124] 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.

[0125] As used herein, the term "O-phosphoserine sulfhydrylase (OPSS)" refers to an enzyme that catalyzes the conversion of O-phosphoserine to cysteine ​​by donating a thiol group (SH group) to the O-phosphoserine. This enzyme was 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 O-phosphoserine sulfhydrylase includes not only wild-type O-phosphoserine sulfhydrylase but also mutants which have a sequence in which a portion of the sequence in the polynucleotide sequence encoding the O-phosphoserine sulfhydrylase is deleted, substituted, or added, and which exhibit biological activity equivalent to or greater than that of the wild-type O-phosphoserine sulfhydrylase, and may include all of the O-phosphoserine sulfhydrylases and mutants thereof disclosed in U.S. Registered Publications US 8557549 B2 and US 9127324 B2.

[0126] 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 into a thiol group (SH group) in the form of, for example, thiol group (SH group) can be used without limitation. Specifically, NaS, NaSH, HS, (NH)S, and NaSO can be used, providing a thiol group to O-phosphoserine, but are not limited to these. The reaction is a reaction in which one thiol group is provided to one O-phosphoserine 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 O-phosphoserine, but is not limited thereto.

[0127] 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.

[0128] Another aspect of the present application is to provide a composition for producing O-phosphoserine, comprising a variant of the present application, a polynucleotide encoding the variant, a vector comprising the polynucleotide, a microorganism comprising the variant of the present application or a polynucleotide encoding the variant, or a combination of two or more thereof.

[0129] The composition of the present application may further contain any suitable excipient commonly used in compositions for producing O-phosphoserine, and such excipients may be, for example, but are not limited to, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, or an isotonic agent.

[0130] In the compositions of the present application, the terms "variant," "polynucleotide," "vector," "microorganism," "medium," and "O-phosphoserine" are as defined above in other aspects.

[0131] Another aspect of the present application is to provide use of a microorganism comprising a variant of the present application, a polynucleotide encoding the variant, a vector comprising the polynucleotide, or a variant of the present application or a polynucleotide encoding the variant, for producing O-phosphoserine, cysteine, or a cysteine ​​derivative.

[0132] Another aspect of the present application is to provide a use of the mutant of the present application for excreting O-phosphoserine from a microorganism.

[0133] The present application will be described in more detail through experimental examples below. However, the following examples are merely preferred embodiments for illustrating the present application, and are not intended to limit the scope of the present application. Meanwhile, technical matters not described in this specification can be fully understood and easily performed by those of ordinary skill in the technical field of the present application or a similar technical field.

[0134] Example 1: Selection of YhhS mutants To select YhhS mutants with increased OPS efflux activity, a yhhS gene mutant plasmid library was constructed as follows.

[0135] Random mutagenesis PCR was performed using the genomic DNA of Escherichia coli (E. coli) K12 W3110 as a template and a primer pair with the base sequences of SEQ ID NOs: 13 and 14 shown in Table 1. PCR was performed using a Diversity PCR Random Mutation Kit (Takara). The PCR was performed by denaturing at 94°C for 5 minutes, followed by 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.

[0136] To insert the mutated gene fragments prepared in this process into the pCL1920 vector containing the rhtB promoter, pCL_PrhtB was first prepared.

[0137] To isolate the rhtB promoter fragment, PCR was performed using SEQ ID NOs: 15 and 16 with the genomic DNA of E. coli K12 W3110 as a template. The 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. The rhtB promoter fragment was cloned into pCL1920 vector (GeneBank No. AB236930) digested with EcoRI and SalI using an infusion cloning kit to isolate pCL_PrhtB. The isolated pCL_PrhtB vector was digested with ScaI, and the mutated gene fragment obtained through PCR was cloned using an infusion cloning kit. Cloning was performed at 50°C for 60 minutes, resulting in the construction of a pCL_PrhtB-yhhS gene mutant plasmid library. Then, CA07-0012 (KCCM 11121P, Korean Patent Registration No. 10-1381048 and US Patent Publication No. 10-2012-0190081) was transformed by electroporation.

[0138] Three strains containing mutants were selected, and plasmids were isolated from them and analyzed by sequencing. The results of the analysis confirmed that the selected mutants were: a mutant in which the serine at position 129 of wild-type YhhS was replaced with glycine; a mutant in which the isoleucine at position 241 was replaced with glutamine; a mutant in which the isoleucine at position 241 was replaced with threonine; a mutant in which the aspartic acid at position 246 was replaced with valine; and a mutant in which the valine at position 330 was replaced with isoleucine. The three strains were designated CA07-0012 / pCL_PrhtB-yhhS(S129G), CA07-0012 / pCL_PrhtB-yhhS(I241Q), and CA07-0012 / pCL_PrhtB-yhhS(I241T / D246V / V330I), respectively. The CA07-0012 / pCL_PrhtB-yhhS(I241T / D246V / V330I) strain, also known as Escherichia coli CA07-0352, was deposited with the Korean Culture Center of Microorganisms (KCCM) under the Budapest Treaty on May 14, 2020, and was assigned the deposit number KCCM 12720P.

[0139] [Table 1]

[0140] Example 2: Construction of vectors for expression of additional YhhS variants 2-1. Construction of YhhS(S129A) mutant expression vector and expression strain To obtain the yhhS(S129A) fragment, a yhhS(S129A) upper fragment was obtained by PCR using SEQ ID NOs: 13 and 18 and E. coli K12 W3110 genomic DNA as a template, and a yhhS(S129A) lower fragment was obtained by PCR using SEQ ID NOs: 19 and 14 shown in Table 2 below and E. coli K12 W3110 genomic DNA as a template. PCR was performed by denaturing at 94°C for 5 minutes, followed by 30 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.

[0141] The pCL_PrhtB vector was digested with ScaI, and the isolated yhhS(S129A) upper fragment and yhhS(S129A) lower fragment were cloned using an in-fusion cloning kit (Clontech Laboratories, Inc.). Cloning was performed at 50°C for 60 minutes to obtain pCL_PrhtB-yhhS(S129A). The isolated plasmid was transformed into CA07-0012 by electroporation to obtain strain CA07-0012 / pCL_PrhtB-yhhS(S129A).

[0142] [Table 2]

[0143] 2-2. Construction of YhhS(I241Q) mutant expression vector, YhhS(I241T) mutant expression vector, and mutant expression strain A strain was constructed and evaluated to confirm the OPS-producing ability when the 241st amino acid residue of yhhS isolated from the library, isoleucine, was replaced with glutamine or threonine.

[0144] To insert the 241st amino acid mutation of yhhS into the CA07-0012 chromosome, trc was used as the promoter and the mgsA gene position was used as the insertion site.

[0145] Specifically, the pSKH130 vector (US Patent Publication No. 2020-0048619, SEQ ID NO: 38) was used for chromosomal insertion. The vector contains the R6K replicon, SacB (levansucrase) gene, and kanamycin resistance gene, which are dependent on the PI protein (pir gene). The desired strain was isolated using R6K and kanamycin in a primary crossover using the vector, and then the antibiotic was removed from the sucrose-containing medium to create the strain.

[0146] To introduce a form in which the 241st amino acid residue of yhhS is substituted with another amino acid into the mgsA gene locus of the CA07-0012 strain, we sought to obtain the pSKH130△mgsA plasmid. pSKH130△mgsA is a vector for deleting the mgsA ORF (open reading frame), and contains both 5' and 3' nucleotide sequences outside the mgsA ORF.

[0147] The 5' and 3' fragments were isolated using the primer pairs shown in Table 3 below, and the pSKH130 vector was digested with BamHI, after which the plasmid was isolated using an infusion cloning kit.

[0148] [Table 3]

[0149] To isolate the trc promoter fragment, PCR was performed using SEQ ID NOs: 24 and 25 shown in Table 4 below. To isolate the two mutant yhhS ORFs, the primer pairs shown in Table 5 were used to isolate the upper and lower fragments of the two mutants, respectively. The isolated upper and lower fragments were then combined with the trc promoter fragment and a vector prepared by cleaving pSKH130ΔmgsA with ScaI to create two plasmids using an infusion cloning kit. Additionally, to isolate a control strain in which wild-type yhhS had been introduced at the mgsA site, the pSKH130ΔmgsA::Ptrc-yhhS plasmid was constructed. To isolate the yhhS ORF, PCR was performed using an oligonucleotide pair shown in SEQ ID NOs: 26 and 27, and the trc promoter fragment and a vector prepared by cleaving pSKH130ΔmgsA with ScaI were combined with an infusion cloning kit to create plasmids.

[0150] [Table 4]

[0151] [Table 5]

[0152] The isolated plasmid was transformed into the CA07-0012 strain by electroporation. Strains with the mutation inserted into the chromosome through recombination (crossover) were selected on LB solid medium containing kanamycin, and then a secondary recombination (exchange) was carried out on medium containing sucrose to excise the plasmid site from the chromosome. Two strains with the yhhS mutation inserted into the mgsA site of the chromosome (CA07-0012△mgsA::Ptrc-yhhS(I241T) and CA07-0012△mgsA::Ptrc-yhhS(I241Q)) were isolated through PCR and sequence analysis using SEQ ID NOs: 20 and 27, and a control strain (CA07-0012△mgsA::Ptrc-yhhS) was also isolated through the same method.

[0153] 2-3. Construction of YhhS(S129G / I241Q) mutant expression vector and expression strain Based on the yhhS(I241Q) mutant, a strain was constructed in which the serine at amino acid residue 129 was replaced with glycine, using the rhtB promoter.

[0154] Specifically, PCR was performed using SEQ ID NOs: 32 and 33 (as of PCR_129G for the entire plasmid) shown in Table 6 below, with pCL_PrhtB-yhhS(I241Q) as a template. The obtained PCR fragment was cloned using an infusion cloning kit.

[0155] The obtained plasmid was transformed into CA07-0012 by electroporation to obtain strain CA07-0012 / pCL_PrhtB-yhhS(S129G / I241Q).

[0156] [Table 6]

[0157] 2-4. Construction of YhhS (S129G / I241T / D246V / V330I) mutant expression vector and expression strain The promoter used was trc, and the mgsA gene was deleted to insert the S129G / I241T / D246V / V330I mutations.

[0158] Specifically, the pSKH130ΔmgsA plasmid constructed in Example 2-2 was used to introduce the I241T, D246V, and V330I mutations into the mgsA gene locus of the CA07-0012 strain.

[0159] The trc promoter fragment isolated in Example 2-2 was used to construct pSKH130ΔmgsA::Ptrc-yhhS(I241T / D246V / V330I). The yhhS(I241T / D246V / V330I) ORF fragment was isolated by PCR using the pCL_Ptrc-yhhS(I241T / D246V / V330I) plasmid as a template and SEQ ID NOs: 26 and 27. The trc promoter fragment and yhhS453 fragment, along with pSKH130ΔmgsA digested with ScaI, were then cloned into the plasmid pSKH130ΔmgsA::Ptrc-yhhS(I241T / D246V / V330I) using an infusion cloning kit.

[0160] Subsequently, PCR was performed using SEQ ID NOs: 32 and 33 (PCR_129G for the entire plasmid) and the pSKH130ΔmgsA::Ptrc-yhhS (I241T / D246V / V330I) as a template. 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 7 minutes, followed by polymerization at 72°C for 5 minutes. The isolated fragment was used to isolate the plasmid pSKH130ΔmgsA::Ptrc-yhhS (129G / I241T / D246V / V330I) using an infusion cloning kit.

[0161] The isolated plasmid was transformed into the CA07-0012 strain by electroporation. The transformed strains were selected for those in which the plasmid had been introduced into the chromosome by recombination (crossover) on LB solid medium containing kanamycin, and then the plasmid site was excised from the chromosome through a secondary recombination (exchange) on medium containing sucrose.

[0162] The strains in which the secondary recombination was completed were subjected to PCR and sequence analysis using SEQ ID NOs: 20 and 27 to identify two strains (CA07-0012 / pSKH130△mgsA::Ptrc-yhhS(I241T / D246V / V330I) and CA07-0012 / pSKH130△mgsA::Ptrc-yhhS(129G / I241T / D246V / V330I)) in which yhhS(I241T / D246V / V330I) and yhhS(129G / I241T / D246V / V330I) were inserted at the mgsA site of the chromosome.

[0163] Example 3: Evaluation of OPS production ability of strains into which YhhS mutants have been introduced To evaluate the ability of the strains into which the YhhS mutants were introduced to produce phosphoserine (O-phosphoserine), the following medium (Table 7) was used.

[0164] 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 7 below, which was then cultured in an incubator at 33°C and 200 rpm for 48 hours, and the results are shown in Tables 8 to 11.

[0165] [Table 7]

[0166] 3-1: Evaluation of OPS production ability of strains with YhhS 129th amino acid substitution mutants In the case of CA07-0012 / pCL_PrhtB-yhhS(S129G), a strain into which the YhhS(S129G) mutant was introduced, the increase rate was approximately 252%, and in the case of CA07-0012 / pCL_PrhtB-yhhS(S129A), a strain into which the YhhS(S129A) mutant was introduced, the increase rate was approximately 29% (Table 8).

[0167] [Table 8]

[0168] 3-2: Evaluation of OPS production ability of strains with yhhS 241 amino acid substitution mutants The increase in OPS efflux capacity was compared with that of the wild-type yhhS strain CA07-0012△mgsA::Ptrc-yhhS. The increase was approximately 425% for the strain CA07-0012△mgsA::Ptrc-yhhS(I241Q) containing the YhhS(I241Q), approximately 233% for the strain CA07-0012△mgsA::Ptrc-yhhS(I241T), and approximately 267% for the strain CA07-0012△mgsA::Ptrc-yhhS(I241T / D246V / V330I) containing the YhhS(I241T / D246V / V330I) (Table 9).

[0169] [Table 9]

[0170] 3-3: Evaluation of OPS production ability of strains with substitution of the 129th amino acid residue of YhhS and the 241st amino acid residue of yhhS The increase in OPS efflux capacity was confirmed by comparing it with the wild-type yhhS strain CA07-0012 / pCL_PrhtB-yhhS. The increase was approximately 338% for the YhhS(I241Q) mutant strain CA07-0012 / pCL_PrhtB-yhhS(S129G / I241Q) (Table 10).

[0171] [Table 10]

[0172] In addition, we confirmed the increase in OPS efflux capacity compared to CA07-0012△mgsA::Ptrc-yhhS(I241T / D246V / V330I). We confirmed that the increase in OPS efflux capacity was approximately 144% in the case of the strain CA07-0012△mgsA::Ptrc-yhhS(129G / I241T / D246V / V330I) containing the YhhS(129G / I241T / D246V / V330I) (Table 11).

[0173] [Table 11]

[0174] 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 all modifications and variations derived from the meaning and scope of the claims below, and equivalent concepts thereof, rather than the above detailed description.

[0175] TIFF0007773641000012.tif186160

Claims

1. A YhhS mutant having O-phosphoserine (OPS) efflux activity and having a sequence identity of 90% or more with the amino acid sequence of SEQ ID NO: 1, in which the amino acid corresponding to position 129 of the amino acid sequence of SEQ ID NO: 1 is replaced with glycine or alanine.

2. A YhhS mutant having O-phosphoserine (OPS) efflux activity and having a sequence identity of 90% or more with the amino acid sequence of SEQ ID NO: 1, in which the amino acid isoleucine corresponding to the 241st position in the amino acid sequence of SEQ ID NO: 1 is replaced with glutamine.

3. 2. The YhhS variant according to claim 1, wherein the isoleucine amino acid corresponding to position 241 of the amino acid sequence of SEQ ID NO: 1 is further substituted with glutamine or threonine.

4. A YhhS mutant described in any one of claims 1 to 3, wherein the mutant further replaces the aspartic acid, an amino acid corresponding to position 246 of the amino acid sequence of SEQ ID NO: 1, with valine, and / or the valine, an amino acid corresponding to position 330 of the amino acid sequence of SEQ ID NO: 1, with isoleucine.

5. 3. The YhhS variant according to claim 1, wherein the variant has a sequence identity of 90% or more with an amino acid sequence selected from SEQ ID NOs: 2 to 5, 34 and 36.

6. A polynucleotide encoding the variant of claim 1 or 2.

7. A microorganism of the genus Escherichia, comprising the mutant according to claim 1 or 2 or a polynucleotide encoding said mutant.

8. The microorganism according to claim 7, further comprising a weakened activity of phosphoserine phosphatase (SerB) compared to the endogenous activity of the microorganism.

9. A method for producing O-phosphoserine, comprising the step of culturing in a medium a microorganism containing the mutant of claim 1 or 2 or a polynucleotide encoding said mutant.

10. a) culturing an O-phosphoserine-producing microorganism containing the mutant according to claim 1 or 2 or a polynucleotide encoding said mutant in a medium to produce O-phosphoserine or a medium containing O-phosphoserine; b) contacting O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing the same with the O-phosphoserine produced in step a) or a medium containing the same, and sulfide.

11. A composition for producing O-phosphoserine, comprising: a mutant according to claim 1 or 2; a polynucleotide encoding the mutant; a vector comprising the polynucleotide; a microorganism comprising the mutant or a polynucleotide encoding the mutant; or a combination of two or more thereof.

12. Use of the mutant according to claim 1 or 2; a polynucleotide encoding said mutant; a vector comprising said polynucleotide; or a microorganism comprising said mutant or a polynucleotide encoding said mutant for the production of O-phosphoserine, cysteine ​​or a cysteine ​​derivative.

13. Use of the mutant described in claim 1 or 2 for excreting O-phosphoserine from a microorganism.

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