O-phosphoserine sulfhydrylase variant and method for producing cysteine using same

Abstract

The present disclosure relates to O-phosphoserine sulfhydrylase variants and methods for producing cysteine using the same.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to O-phosphoserine sulfhydrylase variants and a method for producing cysteine using the same. BACKGROUND

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

[0003] Methods for producing L-cysteine using microorganisms known in the art include: 1) a method for biotransforming D,L-2-aminothiazoline-4-carboxylic acid (D,L-ATC) into L-cysteine using a microorganism, 2) a method for producing L-cysteine by direct fermentation using Escherichia coli (EP 0885962 B), and 3) a method for producing O-phosphoserine (hereinafter referred to as “OPS”) by fermentation using a microorganism, and then converting O-phosphoserine into L-cysteine by reacting O-phosphoserine with sulfide under the catalytic action of O-phosphoserine sulfhydrylase (hereinafter referred to as “OPSS”) (US 8557549 B2), and the like.

[0004] In particular, in order to produce cysteine in a high yield by method 3), OPS, which is a precursor, should be overproduced. SUMMARY

[0005] [PROBLEMS TO BE SOLVED]

[0006] The present inventors confirmed that when the O-phosphoserine sulfhydrylase variant of the present disclosure is used in a microorganism producing cysteine, cysteine can be produced in a high yield compared to when a microorganism having an existing wild-type O-phosphoserine sulfhydrylase variant is used, thereby completing the present disclosure.

[0007] [TECHNICAL SOLUTION]

[0008] An object of the present disclosure is to provide an O-phosphoserine sulfhydrylase variant in which 0 to 7 amino acid residues are deleted from the C-terminal end in the amino acid sequence of SEQ ID NO: 1, and the amino acid corresponding to position 77 is substituted with alanine.

[0009] Another object of the present disclosure is to provide a polynucleotide encoding the O-phosphoserine sulfhydrylase variant of the present disclosure.

[0010] Still another object of the present disclosure is to provide a microorganism comprising the O-phosphoserine sulfhydrylase variant of the present disclosure or a polynucleotide encoding the same.

[0011] It is still another object of the present disclosure to provide a method of producing cysteine or a derivative thereof, which comprises reacting a mixture of O-phosphohomoserine and sulfide with the O-phosphohomoserine sulfhydrylase variant of the present disclosure.

[0012] [Advantages]

[0013] When a microorganism producing cysteine is cultured using the O-phosphohomoserine sulfhydrylase variant of the present disclosure, cysteine can be produced at a high yield compared to when a microorganism having an existing wild-type O-phosphohomoserine sulfhydrylase variant is used. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 K12 / pUC_Ppro-cysM(P77S) (Msm-T-HA2) and K12 / pUC_Ppro-cysM(P77A) are shown, which are confirmed to be CysM variants by SDS-PAGE. DETAILED DESCRIPTION

[0015] The present disclosure will be described in detail below. Also, each description and embodiment disclosed herein can be applied to other descriptions and embodiments, respectively. That is, all combinations of various elements disclosed herein fall within the scope of the present disclosure. In addition, the scope of the present disclosure is not limited by the specific descriptions described below. Furthermore, many papers and patent documents are cited throughout the specification and are noted for their citations. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety, and will more clearly describe the level of the art to which the present disclosure belongs and the contents of the present disclosure.

[0016] One aspect of the present disclosure provides an O-phosphohomoserine sulfhydrylase variant in which 0 to 7 amino acid residues are deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1, and the amino acid corresponding to position 77 is substituted with alanine.

[0017] As used herein, the term "O-phosphoserine sulfhydrylase (OPSS)" refers to an enzyme that catalyzes the reaction of converting OPS to cysteine by providing a thiol group (SH group) to OPS. Such an enzyme can be first discovered in Aeropyrum pernix, Mycobacterium tuberculosis, Mycobacterium smegmatis, and Trichomonas vaginalis (Mino K and Ishikawa K, FEBS Letters, 551: 133-138, 2003; Burns K E et al., J. Am. Chem. Soc., 127: 11602-11603, 2005).

[0018] The O-phosphoserine sulfhydrylase of the present disclosure can include any polypeptide having O-phosphoserine sulfhydrylase activity or any O-phosphoserine sulfhydrylase. The polypeptide having O-phosphoserine sulfhydrylase activity or O-phosphoserine sulfhydrylase can include any polypeptide having O-phosphoserine sulfhydrylase activity or activity of converting O-phosphoserine as a substrate into cysteine. In one example, the polypeptide having O-phosphoserine sulfhydrylase activity can be any polypeptide having O-phosphoserine sulfhydrylase activity derived from a microorganism or activity of converting O-phosphoserine as a substrate into cysteine. Specifically, the polypeptide having O-phosphoserine sulfhydrylase activity of the present disclosure can be derived from a prokaryotic microorganism or a eukaryotic microorganism, more specifically, from a genus Mycobacterium microorganism, etc., but is not limited thereto. In another example, the polypeptide having O-phosphoserine sulfhydrylase activity of the present disclosure can be cysteine synthase (CysM) derived from a genus Mycobacterium microorganism. The amino acid sequence of CysM can be obtained from a known database GenBank of NCBI, etc., and for example, it can be WP_003896302.1 derived from a genus Mycobacterium microorganism, but obviously can include any protein having O-phosphoserine sulfhydrylase activity or activity of converting OPS as a substrate into cysteine from various sources.

[0019] Further, the O-phosphosermme sulfhydrylase can include not only a wild-type O- phosphosermme sulfhydrylase protein, but also a variant protein in which a part of the sequence is deleted, substituted, or added in a polynucleotide sequence encoding the O- phosphosermme sulfhydrylase protein, shows an activity equal to or higher than the biological activity of the wild-type O-phosphosermme sulfhydrylase protein, and can also include all O- phosphosermme sulfhydrylase proteins and variant proteins thereof disclosed in EP 2444481 A1 and US 2012-0190080 A.

[0020] The "O-phosphosermme sulfhydrylase" can be referred to as "OPSS", "cysteine synthase", "CysM", etc.

[0021] As used herein, the term "O-phosphosermme sulfhydrylase (OPSS) variant" can refer to a variant in which 0 to 7 amino acid residues are deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1 and the amino acid corresponding to position 77 is substituted with alanine in any polypeptide having O-phosphosermme sulfhydrylase activity or O- phosphosermme sulfhydrylase.

[0022] The O-phosphosermme sulfhydrylase variant can be referred to as "modified O- phosphosermme sulfhydrylase", "OPSS variant", "modified OPSS", "CysM variant", "modified CysM", etc.

[0023] The protein targeted for introduction of a mutation of the present disclosure can be a protein having O-phosphoseryl thiolohydrolase activity or activity of converting O-phosphoserine as a substrate into cysteine. Specifically, the protein can include the amino acid sequence of SEQ ID NO: 1 and have O-phosphoseryl thiolohydrolase activity or activity of converting O-phosphoserine as a substrate into cysteine, but is not limited thereto. The protein does not exclude addition of a meaningless sequence, a naturally occurring mutation or a silent mutation therein upstream or downstream of the amino acid sequence of SEQ ID NO: 1, and any protein can fall within the scope of the protein targeted for introduction of a mutation of the present disclosure, as long as the protein has the same or corresponding activity to that of the protein including the amino acid sequence of SEQ ID NO: 1. For example, the protein targeted for introduction of a mutation of the present disclosure can be a protein consisting of the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity thereto. Furthermore, obviously, any protein having an amino acid sequence in which a part of the sequence is deleted, modified, substituted, or added can also fall within the scope of the protein targeted for mutation of the present disclosure, as long as the amino acid sequence has such homology or identity and exhibits a corresponding efficacy to that of the protein. An example of the protein targeted for introduction of a mutation of the present disclosure, i.e., a parent sequence, can include the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 13.

[0024] The O-phosphosermme sulfhydrylase variant of the present disclosure can be a variant in which 0 to 7 amino acid residues are deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1, and the amino acid corresponding to the 77th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than the amino acid before the replacement. Specifically, the O-phosphosermme sulfhydrylase variant of the present disclosure can be a variant in which 3 to 7 amino acid residues are deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1, and the amino acid corresponding to the 77th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than the amino acid before the replacement. More specifically, the O-phosphosermme sulfhydrylase variant of the present disclosure can be a variant in which 5 amino acid residues are deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1, and the amino acid corresponding to the 77th amino acid from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than the amino acid before the replacement. Before the replacement, the amino acid at the 77th position in the amino acid sequence of SEQ ID NO: 1 of the O-phosphosermme sulfhydrylase protein of the protein targeted for introduction of a mutation in the present disclosure can be proline (P).

[0025] In one example, the O-phosphosermme sulfhydrylase variant can be a variant in which the amino acid corresponding to the 77th of SEQ ID NO: 1 is replaced with an amino acid other than proline. In another example, the O-phosphosermme sulfhydrylase variant can be a variant in which the amino acid corresponding to the 77th of SEQ ID NO: 1 is replaced with an amino acid selected from the group consisting of alanine, tyrosine, arginine, lysine, aspartic acid, asparagine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, threonine, cysteine, histidine, glycine, glutamic acid, and glutamine, and in one example, alanine. Such an amino acid replacement typically occurs based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0026] Furthermore, amino acids can be classified into amino acids with charged side chains and amino acids without charged side chains. Amino acids with charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine; while amino acids without charged side chains can be further classified into nonpolar amino acids and polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, while polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conserved substitutions have little or no effect on the activity of proteins or peptides.

[0027] In one embodiment, the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure may have, include, be composed of, or substantially consist of the amino acid sequence represented by SEQ ID NO: 3. The O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure may include an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or higher homology or identity with the amino acid sequence of SEQ ID NO: 3. Furthermore, it is obvious that any O-phosphoserine hydrogen sulfide hydrolase variant having a portion of its sequence that is deleted, modified, substituted, conservatively substituted, or added may also fall within the scope of this disclosure, provided that the amino acid sequence has such homology or identity and exhibits efficacy corresponding to the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure.

[0028] For example, it could be a sequence addition or deletion, a naturally occurring mutation, a silent mutation thereof, or a conserved substitution within the N-terminus, C-terminus, and / or amino acid sequence that does not alter the function of the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure.

[0029] As used herein, the term "conservative substitution" refers to the replacement of an amino acid with another amino acid having similar structure and / or chemical properties. Such amino acid substitutions typically occur based on the similarity of the residue's polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties. Typically, conserved substitutions have little or no effect on the activity of a protein or peptide.

[0030] The O-phosphoserine hydrogen sulfide hydrolase variant disclosed herein may be a variant in which the amino acid in the amino acid sequence of SEQ ID NO:1 corresponding to the position of the group consisting of 7, 55, 218 and combinations thereof selected from the N-terminus is further replaced by an amino acid different from the amino acid before replacement.

[0031] In the amino acid sequence of the O-phosphoserine hydrogen sulfide hydrolase protein, which is the target of the mutation introduced in this disclosure, the amino acid corresponding to position 7 of SEQ ID NO: 1 before the substitution can be leucine (L), the amino acid corresponding to position 55 before the substitution can be alanine (A), and the amino acid corresponding to position 218 before the substitution can be alanine (A).

[0032] In one example, the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure may be a variant in which the amino acid corresponding to position 7 of SEQ ID NO: 1 may be replaced by an amino acid other than leucine. In another example, the O-phosphoserine hydrogen sulfide hydrolase variant may be a variant in which the amino acid corresponding to position 7 of SEQ ID NO: 1 may be replaced by an amino acid selected from the group consisting of: proline, tyrosine, glycine, alanine, aspartic acid, glutamic acid, histidine, valine, lysine, arginine, isoleucine, methionine, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, and glutamine, and in one example, proline.

[0033] In one example, the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure may be a variant in which the amino acid corresponding to position 55 of SEQ ID NO: 1 may be replaced by an amino acid other than alanine. In another example, the O-phosphoserine hydrogen sulfide hydrolase variant may be a variant in which the amino acid corresponding to position 55 of SEQ ID NO: 1 may be replaced by an amino acid selected from the group consisting of: valine, tyrosine, glycine, leucine, aspartic acid, glutamic acid, histidine, lysine, arginine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, and glutamine, and in one example, valine.

[0034] In one example, the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure may be a variant in which the amino acid corresponding to position 218 of SEQ ID NO: 1 can be replaced by an amino acid other than alanine. In another example, the O-phosphoserine hydrogen sulfide hydrolase variant may be a variant in which the amino acid corresponding to position 218 of SEQ ID NO: 1 can be replaced by an amino acid selected from the group consisting of: glycine, tyrosine, leucine, aspartic acid, glutamic acid, histidine, valine, lysine, arginine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, and glutamine, and in one example, glycine.

[0035] In one embodiment, the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure may be a variant that has 0 to 7, for example 3 to 7, amino acid residues deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1, and further includes one or more, two or more, or all three amino acid substitutions corresponding to the 7th, 5th, or 218th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1, while the amino acid substitution corresponding to the 77th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is fixed.

[0036] In another embodiment, the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure may have, comprise, or consist of any one or more amino acid sequences selected from the group consisting of SEQ ID NO: 5, 7, and 9, or may consist substantially of the aforementioned amino acid sequences. The O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure may include an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% or higher homology or identity with the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. Furthermore, it is obvious that any O-phosphoserine hydrogen sulfide hydrolase variant having a portion of its sequence that is deleted, modified, substituted, conservatively substituted, or added may also fall within the scope of this disclosure, provided that the amino acid sequence has such homology or identity and exhibits efficacy corresponding to the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure.

[0037] As used herein, the term "modified protein" or "variant" refers to a polypeptide having one or more amino acids that differ from the amino acid sequence of the pre-mutant polypeptide, and whose function and properties are preserved through conserved substitutions and / or modifications. Such variants are typically identified by modifying one or more amino acid sequences of the polypeptide and evaluating the properties of the modified polypeptide. That is, the capabilities of the variant may be enhanced, unchanged, or reduced relative to the pre-mutant polypeptide. Furthermore, some variants may include those in which one or more regions, such as the N-terminal leader sequence or transmembrane domain, have been removed. Other variants may include those in which one region has been removed from the N- and / or C-terminus of the mature protein. The term "modified protein" may be used interchangeably with terms such as modification, modified polypeptide, modified protein, mutant, mutant protein, divergent, etc., without limitation, as long as these terms are used to refer to mutation.

[0038] For the purposes of this disclosure, a variant of this disclosure may be a polypeptide in which 0 to 7 amino acid residues are deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1, and the amino acid corresponding to position 77 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced by another amino acid. Specifically, a variant of this disclosure may be a polypeptide in which 5 amino acid residues are deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1, and the amino acid corresponding to position 77 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced by an amino acid other than the amino acid before replacement. In one example, a variant of this disclosure may be a variant in which the amino acid corresponding to position 77 in the amino acid sequence of SEQ ID NO: 1 is replaced by an amino acid selected from the group consisting of: alanine, tyrosine, arginine, lysine, aspartic acid, asparagine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, threonine, cysteine, histidine, glycine, glutamic acid, and glutamine, and in one example, alanine. Such amino acid substitutions typically occur based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0039] Furthermore, amino acids can be classified into amino acids with charged side chains and amino acids without charged side chains. Amino acids with charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine; while amino acids without charged side chains can be further classified into nonpolar amino acids and polar amino acids. Nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline, while polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Typically, conserved substitutions have little or no effect on the activity of proteins or peptides.

[0040] In another instance, a variant of this disclosure may be a polypeptide containing the amino acid sequence represented by SEQ ID NO: 3, but is not limited thereto.

[0041] Variants of this disclosure may be polypeptides in which five amino acid residues are deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1, and the amino acid corresponding to position 77 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced by an amino acid other than the amino acid before the replacement. Variants may also be polypeptides in which the amino acid corresponding to position 7, 55, 218, or a combination thereof from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced by an amino acid other than the amino acid before the replacement. In one example, a variant of this disclosure may be a variant in which the amino acid corresponding to position 7 in the amino acid sequence of SEQ ID NO: 1 is replaced by an amino acid selected from the group consisting of: proline, tyrosine, glycine, alanine, aspartic acid, glutamic acid, histidine, valine, lysine, arginine, isoleucine, methionine, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, and glutamine, and in one example, proline. Variants of this disclosure may be variants in which the amino acid corresponding to position 55 in the amino acid sequence of SEQ ID NO: 1 may be replaced by an amino acid selected from the group consisting of: valine, tyrosine, glycine, leucine, aspartic acid, glutamic acid, histidine, lysine, arginine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, and glutamine, and in one example, valine. Furthermore, variants of this disclosure may be variants in which the amino acid corresponding to position 218 in the amino acid sequence of SEQ ID NO: 1 may be replaced by an amino acid selected from the group consisting of: glycine, tyrosine, leucine, aspartic acid, glutamic acid, histidine, valine, lysine, arginine, isoleucine, methionine, proline, phenylalanine, tryptophan, serine, threonine, cysteine, asparagine, and glutamine, and in one example, glycine. In another instance, a variant of this disclosure may be a polypeptide comprising, but is not limited to, the amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. The variant may be a variant including the modifications described above, wherein the enzymatic activity of O-phosphoserine hydrogen sulfide hydrolase is enhanced compared to a polypeptide whose amino acid position 77 from the N-terminus in the amino acid sequence corresponding to SEQ ID NO: 1 is proline.

[0042] Furthermore, the variants may also include the deletion or addition of amino acids that have minimal impact on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated to an N-terminal signal (or leader) sequence involved in protein co-translation or post-translational transport. Additionally, the polypeptide may be conjugated to another sequence or linker to identify, purify, or synthesize the polypeptide.

[0043] As used herein, the terms “homology” or “identity” refer to the degree of similarity between two given amino acid sequences or nucleotide sequences, and can be expressed as a percentage. The terms homology and identity are often used interchangeably.

[0044] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined using standard alignment algorithms and can be used in conjunction with default gap penalties established by the program being used. Fundamentally homologous or identical sequences are generally expected to hybridize with all or part of the sequence under moderately or highly stringent conditions. This obviously also includes hybridization with polynucleotides containing universal or degenerate codons.

[0045] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined by known computer algorithms, such as the “FASTA” program with default parameters as described in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444. Alternatively, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine this. This algorithm uses the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (version 5.0.0 or later) (GCG package (Devereux, J. et at, Nucleic Acids Research 12:387(1984)), BLASTP, BLASTN, FASTA (Atschul, SF et al., J MOLEC BIOL 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and CARILLO et al. (1988) SIAM J (Applied Math 48:1073) can be used. For example, BLAST or ClustalW from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0046] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by aligning sequence information using, for example, a GAP computer program (such as Needleman et al. (1970), J Mol Biol. 48:443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482). In summary, the GAP program defines homology, similarity, or identity as a value obtained by dividing the number of similarly arranged symbols (i.e., nucleotides or amino acids) by the total number of symbols in the shorter sequence of the two sequences. The default parameters of the GAP procedure may include (1) a binary comparison matrix (with a value of 1 indicating identity and a value of 0 indicating non-identity) and a weighted comparison matrix (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) replacement matrix) as disclosed in Atlas Of Protein Sequence And Structure, edited by Schwartz and Dayhoff, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each vacancy and an additional penalty of 0.10 for each symbol in each vacancy (or a penalty of 10 for vacancy opening and 0.5 for vacancy expansion); and (3) no penalty for terminal vacancy.

[0047] Compared with wild-type O-phosphoserine hydrogen sulfide hydrolase, the O-phosphoserine hydrogen sulfide hydrolase variant disclosed herein may have increased cysteine ​​conversion activity.

[0048] For example, the O-phosphoserine hydrogen sulfide hydrolase variant that can be used as a target enzyme for comparing increased cysteine ​​conversion activity could be Msm-T-HA2 (EP 2444486 B1), but is not limited to this.

[0049] In one instance, the variants of this disclosure with increased cysteine ​​conversion activity compared to the unmodified or unmodified enzyme may have about 1% or more, specifically, about 5% or more, about 10% or more, about 13% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 41% or more, about 45% or more, about 46% or more, about 50% or more, about 55% or more. More, about 60% or more, about 65% or more, about 70% or more, about 72% or more, or about 72.5% or more (no particular upper limit, e.g., about 200% or less, about 150% or less, about 100% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, or about 15% or less) of increased cysteine ​​conversion activity, but not limited thereto, as long as it has an increased (+) value compared to the unmodified enzyme or the unmodified enzyme. In another instance, the variants of this disclosure with increased cysteine ​​conversion activity compared to the unmodified or unmodified enzyme may have an increased cysteine ​​conversion activity of about 1.01 times or more, about 1.05 times or more, about 1.1 times or more, about 1.13 times or more, about 1.15 times or more, about 1.2 times or more, about 1.25 times or more, about 1.3 times or more, about 1.35 times or more, about 1.4 times or more, about 1.41 times or more, about 1.45 times or more, about 1.46 times or more, about 1.5 times or more, about 1.55 times or more, about 1.6 times or more, about 1.65 times or more, about 1.7 times or more, about 1.72 times or more, or about 1.725 times or more (no particular upper limit is specified, but may be, for example, about 10 times or less, about 5 times or less, about 3 times or less, about 2 times or less), but are not limited thereto.

[0050] Cysteine ​​conversion capacity can be evaluated by measuring the amount of cysteine ​​produced through the cysteine ​​conversion reaction. This evaluation can be performed using suitable methods known in the art to measure cysteine ​​yield. For example, HPLC (High Performance Liquid Chromatography), GC (Gas Chromatography), GC / MS (Gas Chromatography-Mass Spectrometry), LC / MS (Liquid Chromatography-Mass Spectrometry), GPC (Gel Permeation Chromatography), or combinations thereof can be used, and suitable methods known in the art can be used to measure cysteine ​​yield.

[0051] As used herein, the term "corresponding to" refers to an amino acid residue at the position described in the peptide, or an amino acid residue that is similar to, identical to, or homologous to the residue described in the peptide. Identification of the amino acid at the corresponding position can be a specific amino acid within a defined sequence (referring to a specific sequence). As used herein, "corresponding region" typically refers to a similar or corresponding position in a related protein or a reference protein.

[0052] For example, any amino acid sequence can be aligned with SEQ ID NO: 1, and based on the alignment, each amino acid residue in the amino acid sequence can be numbered with reference to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, sequence alignment algorithms such as those described herein can identify the position of an amino acid or the position where modifications such as substitutions, insertions, or deletions have occurred compared to the query sequence (also known as the “reference sequence”).

[0053] Examples of alignment can be performed using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol. Biol. 48: 443-453) and the Needleman program in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), but are not limited thereto, and sequence alignment programs known in the art, such as pairwise sequence comparison algorithms, may be used as appropriate.

[0054] Another aspect of this disclosure provides a polynucleotide encoding the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure.

[0055] The O-phosphoserine hydrogen sulfide hydrolase protein disclosed herein can be encoded by the cysM gene.

[0056] In one instance, the cysM gene may be, but is not limited to, a polynucleotide encoding WP_003896302.1 derived from a Mycobacterium genus. In another instance, the cysM gene may be, but is not limited to, a cysM gene derived from a Mycobacterium genus, and obviously includes genes from various sources encoding proteins having O-phosphoserine hydrogen sulfide hydrolase activity.

[0057] As used herein, the term "polynucleotide" is a polymer of nucleotides comprising nucleotide monomers linked together by covalent bonds in a long chain, and is a DNA or RNA chain of at least a certain length. More specifically, it can refer to a polynucleotide fragment encoding a variant of O-phosphoserine hydrogen sulfide hydrolase.

[0058] The polynucleotide encoding the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure may include a nucleotide sequence encoding the O-phosphoserine hydrogen sulfide hydrolase variant, wherein five amino acid residues are deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1, and the amino acid corresponding to position 77 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted by another amino acid. In one example, the polynucleotide of this disclosure may include a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 3. In a more specific embodiment of this disclosure, the polynucleotide of this disclosure may have or include the nucleotide sequence of SEQ ID NO: 4. Furthermore, the polynucleotide of this disclosure may consist of or substantially consist of the nucleotide sequence of SEQ ID NO: 4.

[0059] Due to codon degeneracy or taking into account the codons preferred in organisms to express the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure, the polynucleotides of this disclosure can be modified in various ways in the coding region without altering the amino acid sequence of the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure. Specifically, the polynucleotides of this disclosure may have or include nucleotide sequences having 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% homology or identity with the nucleotide sequence of SEQ ID NO: 4, or may consist of or substantially consist of nucleotide sequences having 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% homology or identity with the nucleotide sequence of SEQ ID NO: 4, but are not limited thereto. In particular, in sequences having the above-mentioned homology or identity, the codon encoding the amino acid corresponding to position 93 of SEQ ID NO: 1 may be one of the codons encoding an amino acid other than proline, such as alanine.

[0060] Furthermore, the polynucleotide encoding the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure may include a nucleotide sequence encoding an O-phosphoserine hydrogen sulfide hydrolase variant, said variant being a polypeptide in which five amino acid residues are deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1, and the amino acid corresponding to position 77 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced by an amino acid other than the amino acid, and furthermore, the amino acid in the amino acid sequence of SEQ ID NO: 1 corresponding to positions selected from the group consisting of 7, 55, 218 and combinations thereof from the N-terminus is replaced by an amino acid other than the amino acid before replacement. In one example, the polynucleotide of this disclosure may include a nucleotide sequence encoding any one or more amino acid sequences selected from the group consisting of SEQ ID NO: 5, 7 and 9. In a more specific embodiment of this disclosure, the polynucleotide of this disclosure may have or include the nucleotide sequences of SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10. Furthermore, the polynucleotides disclosed herein may consist of or substantially consist of the nucleotide sequences of SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10.

[0061] Specifically, the polynucleotides of this disclosure may have or include nucleotide sequences having 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% homology or identity with the nucleotide sequences of SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, or may be composed of or substantially composed of nucleotide sequences having 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% homology or identity with the nucleotide sequences of SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10, but are not limited thereto. Specifically, in sequences having the above homology or identity, the codon encoding the amino acid corresponding to position 7 of SEQ ID NO: 1 can be one of the codons encoding an amino acid other than leucine, such as proline; the codon encoding the amino acid corresponding to position 55 of SEQ ID NO: 1 can be one of the codons encoding an amino acid other than alanine, such as valine; and the codon encoding the amino acid corresponding to position 218 of SEQ ID NO: 1 can be one of the codons encoding an amino acid other than alanine, such as glycine.

[0062] Furthermore, the polynucleotides disclosed herein may include probes, which may be prepared from known gene sequences, such as any polynucleotide sequence that can hybridize with all or part of the complementary sequence of the polynucleotide sequence disclosed under stringent conditions, without limitation. “Stringent conditions” refers to conditions that allow specific hybridization between polynucleotides. Such conditions are disclosed in detail in the literature (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold SpringHarbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, 9.50-9.51, 11.7-11.8). For example, the stringent conditions may include conditions under which polynucleotides with high homology or identity hybridize with each other, i.e., polynucleotides with 70% or higher, 75% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher hybridize with each other, while polynucleotides with homology or identity lower than the above-mentioned homology or identity will not hybridize with each other; or may include ordinary washing conditions for Southern hybridization, i.e., washing once, specifically twice or three times, at salt concentrations and temperatures corresponding to 60°C, 1×SSC, 0.1% SDS, specifically 60°C, 0.1×SSC, 0.1% SDS, and more specifically 68°C, 0.1×SSC, 0.1% SDS.

[0063] Hybridization requires two nucleic acids to have complementary sequences, although mismatches between bases are possible depending on the strictness of hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Therefore, the polynucleotides of this disclosure may also include isolated nucleic acid fragments complementary to the whole sequence as well as nucleic acid sequences substantially similar to it.

[0064] Specifically, polynucleotides homologous to or identical to the polynucleotides disclosed herein can be used for detection under hybridization conditions, said hybridization conditions including a T50°C environment. m The value, and the hybridization steps under the above conditions. Furthermore, the T... m The value can be 60°C, 63°C or 65°C, but is not limited thereto, and can be appropriately adjusted by those skilled in the art for their purposes.

[0065] The appropriate stringency of polynucleotide hybridization depends on the length of the polynucleotide and the degree of complementarity of the polynucleotides, and these variables are well known in the art (see Sambrook et al., above).

[0066] Another aspect of this disclosure provides a vector containing the polynucleotides of this disclosure.

[0067] The vector can be an expression vector used to express polynucleotides in host cells, but is not limited to this.

[0068] The vector disclosed herein may comprise a DNA construct containing a nucleotide sequence encoding a target polypeptide, the polynucleotide being operatively linked to a suitable expression regulatory region (expression regulatory sequence) to express the target polypeptide in a suitable host cell. The expression regulatory sequence may include a promoter capable of initiating transcription, any operon sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a termination sequence for regulating transcription and translation. Once transformed into a suitable host cell, the vector may replicate or function independently of the host genome, or it may integrate into the host genome.

[0069] There are no particular limitations on the vectors used in this disclosure, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, granules, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or granule vectors; and those based on pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc., can be used as plasmid vectors. Specifically, pUC, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors may be used.

[0070] In one example, a polynucleotide encoding a target protein can be inserted into a chromosome using a vector for intracellular chromosome insertion. The polynucleotide can be inserted into the chromosome by any method known in the art, such as homologous recombination, but is not limited thereto. The vector may also include a selection marker to confirm insertion into the chromosome. The selection marker is used to select cells transformed with the vector, i.e., to confirm whether the target nucleic acid molecule has been inserted, and may use markers that provide selectable phenotypes, such as drug resistance, auxotrophic phenotypes, resistance to cytotoxic agents, or expression of surface peptides. Only cells expressing the selection marker are able to survive or exhibit a different phenotype under conditions treated with a selection agent, thus allowing for the selection of transformed cells.

[0071] As used herein, the term "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism so that the polypeptide encoded by the polynucleotide can be expressed in the host cell. It is not important whether the transformed polynucleotide is integrated into and located within or outside the chromosome of the microorganism, as long as it can be expressed in the host cell, and both are acceptable. Furthermore, the polynucleotide may comprise DNA and / or RNA encoding the target polypeptide. The polynucleotide can be introduced in any form, as long as it can be introduced into 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 gene construct containing all the elements required for its autonomous expression. The expression cassette typically includes a promoter, transcription termination codon, ribosome binding site, and / or translation terminator operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicable expression vector. Furthermore, the polynucleotide may be introduced into the host cell on its own and operably linked to a sequence required for expression in the host cell, but is not limited thereto.

[0072] Furthermore, the term "operably linked" refers to the functional linking of a polynucleotide sequence to a promoter sequence that initiates and mediates the transcription of a polynucleotide encoding the target O-phosphoserine hydrogen sulfide hydrolase variant disclosed herein.

[0073] Another aspect of this disclosure provides microorganisms comprising the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure or the polynucleotide encoding thereof.

[0074] The strains disclosed herein may include the O-phosphoserine hydrogen sulfide hydrolase variants disclosed herein, polynucleotides encoding the polypeptides thereon, or vectors containing the polynucleotides disclosed herein.

[0075] As used herein, the term “microorganism (or strain)” includes all wild-type microorganisms, or naturally or artificially genetically modified microorganisms, and can refer to microorganisms that are weakened or enhanced by specific mechanisms such as the insertion of exogenous genes or the enhancement or inactivation of endogenous gene activity, and can contain genetically modified microorganisms that produce desired polypeptides, proteins or products.

[0076] The strains disclosed herein may be microorganisms naturally possessing cysteine ​​production capacity; or microorganisms in which cysteine ​​production capacity has been introduced into strains that do not possess cysteine ​​production capacity. In one instance, it may be a microorganism in which an O-phosphoserine hydrogen sulfide hydrolase variant or a polynucleotide encoding it has been introduced, thus possessing increased cysteine ​​production capacity, but is not limited thereto.

[0077] The strains disclosed herein may be microorganisms with increased cysteine ​​production capacity compared to parental strains or wild-type strains of the genus *Mycobacterium* that do not contain variants of this disclosure. The microorganisms may be those in which a variant of the O-phosphoserine sulfonate enzyme of this disclosure has been introduced, exhibiting increased cysteine ​​conversion activity compared to wild-type O-phosphoserine sulfonate hydrolase or the polynucleotide encoding it, and thus possessing improved cysteine ​​production capacity. Specifically, the strains of this disclosure may have increased cysteine ​​production capacity compared to wild-type O-phosphoserine sulfonate hydrolase or the polynucleotide encoding it, which contains the amino acid sequence of SEQ ID NO: 1.

[0078] As used herein, the term "unmodified microorganism" does not exclude strains containing mutations that can occur naturally in microorganisms, and can refer to the wild-type strain or the naturally occurring strain itself, or the strain before its traits are altered due to genetic modifications caused by natural or artificial factors. Furthermore, as used herein, the term "O-phosphoserine hydrogen sulfide unmodified microorganism" can refer to strains that have not been introduced with the O-phosphoserine hydrogen sulfide variant disclosed herein, or strains prior to its introduction. In addition to modifications of O-phosphoserine hydrogen sulfide or the polynucleotide encoding it, the O-phosphoserine hydrogen sulfide unmodified microorganisms of this disclosure do not exclude strains containing modifications of other proteins or genes.

[0079] As used herein, “unmodified microorganism” may be used interchangeably with “unmodified strain”, “unmodified microorganism”, “unmutated strain”, “unmodified strain”, “unmutated microorganism”, or “reference microorganism”.

[0080] The microorganisms disclosed herein may be microorganisms containing a variant of O-phosphoserine hydrogen sulfide hydrolase or a polynucleotide encoding thereof; or microorganisms that have been genetically modified to include a variant of O-phosphoserine hydrogen sulfide hydrolase or a polynucleotide encoding thereof (e.g., recombinant microorganisms), but are not limited thereto. "Endogenous activity" refers to the activity of a specific polypeptide originally possessed by the parental strain before transformation, or by the wild-type unmodified microorganism, when the trait is altered due to genetic variation caused by natural or artificial factors. Endogenous activity may be used interchangeably with "unmodified activity".

[0081] The microorganisms disclosed herein are not limited by type, as long as they can produce cysteine, and can be any prokaryotic or eukaryotic microorganism, and specifically, prokaryotic microorganisms. In one instance, it may include strains of microorganisms belonging to the genera *Escherichia*, *Erwinia*, *Serratia*, *Providencia*, *Corynebacterium*, and *Brevibacterium*, and specifically, microorganisms belonging to the genus *Escherichia*, and more specifically, *Escherichia coli*, but are not limited thereto.

[0082] In another embodiment of this disclosure, the recombinant microorganisms of this disclosure may be microorganisms with enhanced cysteine ​​production capacity, due to increased activity of the protein portions involved in the cysteine ​​biosynthesis pathway or decreased activity of the protein portions involved in the cysteine ​​degradation pathway.

[0083] As used herein, the term “enhancement” of peptide activity refers to an increase in the activity of a peptide compared to its intrinsic activity. Enhancement can be used interchangeably with terms such as activation, upregulation, overexpression, and increase. Specifically, activation, enhancement, upregulation, overexpression, and increase can include two scenarios: those exhibiting activity not initially present, or those showing increased activity compared to intrinsic or unmodified activity.

[0084] Compared to endogenous activity, “enhanced,” “upregulated,” “overexpressed,” or “increased” peptide activity refers to an increase in the activity and / or concentration (expression level) of a specific peptide compared to that originally possessed by the parental strain or unmodified microorganism before transformation.

[0085] This enhancement can be achieved by introducing exogenous peptides or by increasing the activity and / or concentration (expression level) of endogenous peptides. Enhanced peptide activity can be confirmed by increases in peptide activity levels, expression levels, or the amount of peptide secreted products.

[0086] Enhancement of peptide activity can be achieved through a variety of methods well known in the art, and such methods are not limited, as long as they enhance the activity of the target peptide compared to the unmodified microorganism. Specifically, genetic engineering and / or protein engineering, which are well known to those skilled in the art and are commonly used in molecular biology, can be used, but the methods are 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.).

[0087] Specifically, the enhanced activity of the peptides disclosed herein can be achieved in the following ways:

[0088] 1) Increase the intracellular copy number of the polynucleotide encoding the polypeptide;

[0089] 2) Replace the expression regulatory region of the gene encoding the polypeptide on the chromosome with a sequence that has stronger activity;

[0090] 3) Modify the nucleotide sequence of the start codon or 5'-UTR of the transcript of the gene encoding the polypeptide;

[0091] 4) Modify the amino acid sequence of the polypeptide to enhance its activity;

[0092] 5) Modify the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide (e.g., modify the polynucleotide sequence of the polypeptide gene to encode a polypeptide that has been modified to enhance the activity of the polypeptide).

[0093] 6) Introduce an exogenous polypeptide exhibiting the activity of the stated polypeptide or an exogenous polynucleotide encoding it;

[0094] 7) Codon optimization for the polynucleotides encoding the polypeptide;

[0095] 8) Analyze the tertiary structure of the polypeptide to select and modify exposed sites, or to chemically modify them;

[0096] 9) Regulating the cellular localization of the protein (peptide); or

[0097] 10) Selected from two or more of the above 1 to 9), but not specifically limited thereto.

[0098] More specifically,

[0099] 1) The method of increasing the intracellular copy number of the polynucleotide encoding the polypeptide can be achieved by introducing a vector into a host cell, the vector being operatively linked to the polynucleotide encoding the polypeptide and capable of replication and functioning independently of the host cell. Alternatively, the method can be achieved by introducing one or two copies of the polynucleotide encoding the polypeptide (protein) into the chromosome of a host cell (microorganism), the polynucleotide being operatively linked to a suitable regulatory sequence. Chromosomal introduction can be performed by introducing a vector capable of inserting the polynucleotide into the host cell (microorganism) chromosome, but is not limited thereto. The vector is as described above. The regulatory sequence for the polynucleotide encoding sequence can be natural (from the same source) or exogenous (from a different gene), or it can be a mutant sequence or other artificial sequence, and can induce the expression of the polynucleotide in the host cell (microorganism). In one embodiment, the regulatory sequence of the cysM gene encoding the O-phosphoserine hydrogen sulfide hydrolase (OPSS) of Mycobacterium smegma disclosed herein can be the PcysK promoter, but is not limited thereto.

[0100] 2) The method of replacing the expression regulatory region (or expression regulatory sequence) of the gene encoding the polypeptide on the chromosome with a highly active sequence can be achieved, for example, by inducing sequence modifications to further enhance the activity of the expression regulatory region through deletion, insertion, non-conserved or conserved substitution, or a combination thereof, or by replacing the sequence with a more active sequence. The expression regulatory region may include, but is not particularly limited to, promoters, operon sequences, sequences encoding ribosome binding sites, and sequences regulating transcription and translation termination. In one example, the method may include replacing the original promoter with a strong promoter, but is not limited thereto.

[0101] Examples of known strong promoters include CJ1 to CJ7 promoters (US 7662943 B2), lac promoter, trp promoter, trc promoter, tac promoter, λ phage PR promoter, PL promoter, tet promoter, gapA promoter, SPL7 promoter, SPL13 (sm3) promoter (US 10584338 B2), O2 promoter (US 10273491 B2), tkt promoter, yccA promoter, etc., but strong promoters are not limited to these.

[0102] 3) Modifying the nucleotide sequence of the start codon or 5'-UTR of the gene transcript encoding the polypeptide can be achieved, for example, by replacing the nucleotide sequence with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate compared to the endogenous start codon, but is not limited thereto.

[0103] 4) and 5) The methods of modifying amino acid sequences or polynucleotide sequences can be achieved by inducing sequence modification to enhance the activity of the polypeptide or polynucleotide sequence encoding the polypeptide through deletion, insertion, non-conserved or conserved substitution, or a combination thereof, of the amino acid sequence, or by replacing the sequence with a modified amino acid sequence or polynucleotide sequence that has stronger activity, or a modified amino acid sequence or polynucleotide sequence that enhances activity, but are not limited thereto. Specifically, substitution can be achieved by inserting polynucleotides into the chromosome through homologous recombination, but is not limited thereto. The vectors used herein may also include selection markers to confirm insertion into the chromosome.

[0104] 6) The method for introducing a foreign polynucleotide exhibiting the activity of the said polypeptide can be achieved by introducing a foreign polynucleotide encoding a polypeptide exhibiting the same / similar activity as the said polypeptide into a host cell. The foreign polynucleotide can be used without restriction, regardless of its source or sequence, as long as it exhibits the same / similar activity as the said polypeptide. Those skilled in the art can perform the introduction by appropriately selecting transformation methods known in the art, and the expression of the introduced polynucleotide in the host cell can produce the said polypeptide, thereby increasing its activity.

[0105] 7) Codon optimization of the polynucleotide encoding the polypeptide can be achieved by codon optimization of endogenous polynucleotides to increase transcription or translation in the host cell, or by optimizing their codons so that optimized transcription and translation of exogenous polynucleotides can be achieved in the host cell.

[0106] 8) The method of analyzing the tertiary structure of the polypeptide and thereby selecting and modifying the exposed sites or chemically modifying them can be, for example, by comparing the sequence information of the polypeptide to be analyzed with a database (which stores the sequence information of known proteins) to determine template protein candidates based on sequence similarity, and thus confirming the structure based on this information, thereby selecting and transforming or modifying the exposed sites to be modified or chemically modified.

[0107] 9) The method of regulating the cellular localization of the protein (peptide) can be achieved by targeting the protein (peptide) to a specific organelle or a specific space within the cell. For example, it can be achieved by adding or removing a leader sequence that functions in the target protein (peptide) to target the protein (peptide) to the periplasm or cytoplasm, but is not limited thereto.

[0108] In one implementation, the enhancement of O-phosphoserine hydrogen sulfide hydrolase activity can be achieved by modifying the expression regulatory region of the gene encoding the polypeptide on the chromosome in 2), by modifying the amino acid sequence or polynucleotide sequence or a combination thereof in 4) and 5).

[0109] In any of the above embodiments, enhancement of O-phosphoserine hydrogen sulfide hydrolase activity may include a gene expression regulatory sequence having enhancing activity upstream of the cysM gene encoding it. For example, the gene expression regulatory sequence may be a promoter, but is not limited thereto. In another embodiment of the above embodiments, enhancement of O-phosphoserine hydrogen sulfide hydrolase activity may be achieved by modifying the amino acid sequence of O-phosphoserine hydrogen sulfide hydrolase or the polynucleotide sequence encoding it. Modification of the amino acid sequence or polynucleotide sequence may include all deletions, substitutions, insertions, etc. Sequence modifications are as described above.

[0110] This enhancement of peptide activity may mean, but is not limited to, an increase in the activity or concentration of the peptide relative to the activity or concentration of the peptide expressed in the unmodified wild-type strain or microbial strain, or an increase in the amount of product produced by the peptide.

[0111] The modification of part or all of the polynucleotides in the microorganisms disclosed herein can be achieved (a) by homologous recombination involving chromosomal insertion in the microorganism using a vector or by genome editing using engineered nucleases (e.g., CRISPR-Cas9), and / or (b) by, but is not limited to, induction by light, such as ultraviolet light and radiation, and / or chemical treatment. Methods for modifying part or all of the genes can include, but are not limited to, methods using DNA recombination technology. For example, part or all of the genes can be deleted by injecting a nucleotide sequence or a vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination. The injected nucleotide sequence or vector can include, but is not limited to, a dominant selection marker.

[0112] As used herein, the term “attenuation” of peptide activity (e.g., protein specified by the name of each enzyme) is a comprehensive concept, encompassing reduced or absent activity compared to its endogenous activity. Attenuation may be used interchangeably with terms such as inactivation, lack, downregulation, reduction, decrease, and weakening.

[0113] The attenuation may also include situations where the polypeptide activity itself is reduced or eliminated compared to the polypeptide activity initially possessed by the microorganism due to mutations in the polynucleotide encoding the polypeptide; situations where the overall level of intracellular polypeptide activity and / or concentration (expression level) is reduced compared to the natural strain due to inhibition of gene expression encoding the polypeptide polynucleotide, or inhibition of translation into the polypeptide, etc.; situations where the polynucleotide is not expressed at all; and / or situations where no polypeptide activity is observed even if the polynucleotide is expressed. The expression "inactivation, lack, reduction, downregulation, decrease, or weakening" of polypeptide activity compared to endogenous activity refers to a decrease in polypeptide activity compared to the activity of a specific polypeptide initially possessed by the parental strain or unmodified microorganism before transformation.

[0114] The reduction of peptide activity can be achieved by any method known in the art, but is not limited thereto, and can be achieved by applying a variety of methods well known in the art (e.g., Nakashima N et al., Bacterialcellular engineering by genome editing and gene silencing. Int J Mol Sci. 2014;15(2):2773-2793; Sambrook et al., Molecular Cloning 2012, etc.).

[0115] Specifically, the reduction of peptide activity disclosed herein can be achieved in the following ways:

[0116] 1) The gene encoding the polypeptide is missing, either partially or completely;

[0117] 2) Modify the expression regulatory region (expression regulatory sequence) to reduce the expression of the gene encoding the polypeptide;

[0118] 3) Modify the amino acid sequence constituting the polypeptide to remove or reduce the polypeptide activity (e.g., deletion / substitution / addition of one or more amino acids in the amino acid sequence).

[0119] 4) Modify the gene sequence encoding the polypeptide so that the polypeptide activity is removed or weakened (e.g., deletion / substitution / addition of one or more nucleotides in the nucleotide sequence of the polypeptide gene to encode a polypeptide that has been modified to remove or weaken the polypeptide activity).

[0120] 5) Modify the nucleotide sequence of the start codon or 5'-UTR of the transcript of the gene encoding the polypeptide;

[0121] 6) Introduce an antisense oligonucleotide (e.g., antisense RNA) that binds complementary to the transcript of the gene encoding the polypeptide.

[0122] 7) Add a sequence complementary to the SD sequence to the front of the Shine-Dalgarno (SD) sequence of the gene encoding the polypeptide to form a secondary structure, thereby inhibiting ribosome attachment;

[0123] 8) Reverse transcription engineering (RTE), which adds a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide;

[0124] 9) Regulating the cellular localization of the protein (peptide); or

[0125] 10) Selected from two or more of the above 1 to 9), but not specifically limited thereto.

[0126] For example,

[0127] 1) The method of deleting part or all of the gene encoding the polypeptide can be achieved by deleting all polynucleotides encoding the endogenous target polypeptide within the chromosome, or by replacing the polynucleotide with a polynucleotide that has partially deleted nucleotides, or by replacing the polynucleotide with a marker gene.

[0128] 2) Modification of expression regulatory regions (expression regulatory sequences) can be achieved through deletion, insertion, non-conservative substitution, or conserved substitution, or a combination thereof; or by replacing the sequence with a less active sequence. Expression regulatory regions may include, but are not limited to, promoters, operon sequences, sequences encoding ribosome binding sites, and sequences regulating the termination of transcription and translation.

[0129] 3) and 4) The methods of modifying amino acid sequences or polynucleotide sequences can be achieved by inducing sequence modification by deleting, inserting, non-conserved or conserved substitutions or combinations thereof in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, to weaken the activity of the polypeptide, or by replacing the sequence with a modified amino acid sequence or polynucleotide sequence that has weaker activity, or a modified amino acid sequence or polynucleotide sequence that is inactive, but are not limited thereto. For example, gene expression can be suppressed or weakened by introducing mutations into the polynucleotide sequence to form a stop codon, but are not limited thereto.

[0130] 5) The method of modifying the nucleotide sequence of the start codon or 5'-UTR of the gene transcript encoding the polypeptide can be achieved, for example, by replacing the nucleotide sequence with a nucleotide sequence encoding another start codon that has a lower expression rate of the polypeptide than the endogenous start codon, but is not limited thereto.

[0131] 6) Methods for introducing antisense oligonucleotides (e.g., antisense RNA) that are complementary to the transcript of the gene encoding the polypeptide can be found in the literature [Weintraub, H. et al., Antisense-RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986].

[0132] 7) A method of adding a sequence complementary to the SD sequence to the front of the Shine-Dalgarno (SD) sequence of the gene encoding the polypeptide to form a secondary structure, thereby inhibiting ribosome attachment by inhibiting mRNA translation or reducing its rate;

[0133] 8) Reverse transcription engineering (RTE), which involves adding a promoter to be reverse transcribed to the 3' end of the open reading frame (ORF) of the gene sequence encoding the polypeptide. This can be achieved by forming an antisense nucleotide complementary to the transcript of the gene encoding the polypeptide to reduce its activity.

[0134] Furthermore, 9) the method of regulating the cellular localization of the protein (peptide) can be achieved by targeting the protein (peptide) to a specific organelle or a specific space within the cell. For example, it can be achieved by adding or removing a leader sequence that functions in the target protein (peptide) to target the protein (peptide) to the periplasm or cytoplasm, but is not limited thereto.

[0135] This reduction in peptide activity may mean, but is not limited to, a decrease in the activity or concentration of the corresponding peptide relative to the activity or concentration of the peptide expressed in the unmodified wild-type strain or microbial strain, or a reduction in the amount of product produced by the corresponding peptide.

[0136] In the microorganisms disclosed herein, O-phosphoserine hydrogen sulfide hydrolase variants, polynucleotides, and cysteine, etc., are as described in other aspects above.

[0137] Another aspect of this disclosure provides a method for producing cysteine ​​or a derivative thereof, comprising reacting a mixture of O-phosphoserine and a sulfide with a variant of the O-phosphoserine hydrogen sulfide hydrolase disclosed herein.

[0138] The method of producing cysteine ​​or cysteine ​​derivatives disclosed herein may include culturing microorganisms in a culture medium prior to carrying out the method, said microorganisms including the O-phosphoserine hydrogen sulfide hydrolase variant of the present disclosure, the polynucleotide of the present disclosure, or the vector of the present disclosure.

[0139] As used herein, the term "culture" refers to the growth of microorganisms under appropriately controlled environmental conditions. The culture process of this disclosure can be carried out in suitable culture media and culture conditions known in the art. Those skilled in the art can readily adapt this culture method for use according to the strain to be selected. Specifically, the culture can be a batch culture, a continuous culture, or a fed-batch culture, but is not limited thereto.

[0140] As used herein, the term "culture medium" refers to a mixture of substances containing, as a major component, the nutrients required for culturing the microorganisms of this disclosure, providing the nutrients and growth factors necessary for survival and growth, as well as water. Specifically, the culture medium and other culture conditions used to culture the microorganisms of this disclosure can be any culture medium used for routine microbial culture, without any particular limitation. However, the microorganisms of this disclosure can be cultured under aerobic conditions in a conventional culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while adjusting temperature, pH, etc.

[0141] In this disclosure, carbon sources may include carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; and amino acids such as glutamic acid, methionine, lysine, etc. Furthermore, carbon sources may include natural organic nutrients such as starch hydrolysates, molasses, molasses, rice bran, cassava, cane molasses, and corn steep liquor. Specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted to reducing sugars) may be used. In addition, various other carbon sources may be used in unlimited quantities. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0142] Nitrogen sources can include inorganic nitrogen sources, such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; amino acids, such as glutamic acid, methionine, and glutamine; and organic nitrogen sources, such as peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysate, fish or its decomposition products, and defatted soybean meal or its decomposition products. These nitrogen sources can be used alone or in combination of two or more, but are not limited to these.

[0143] Phosphorus sources may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or corresponding sodium-containing salts. Examples of inorganic compounds may include sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the culture medium in batches or continuously, but these phosphorus sources are not limited thereto.

[0144] Furthermore, the pH of the culture medium can be adjusted appropriately during the cultivation of the microorganisms disclosed herein by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid. Additionally, the use of antifoaming agents (such as polyethylene glycol esters of fatty acids) during cultivation can prevent bubble formation. Furthermore, oxygen or oxygen-containing gases can be injected into the culture medium to maintain aerobic conditions; alternatively, no gas injection or the injection of nitrogen, hydrogen, or carbon dioxide can maintain anaerobic or microaerobic conditions, but the gas is not limited to these.

[0145] The temperature during the cultivation process disclosed herein can be in the range of 20°C to 45°C, specifically from 25°C to 40°C, and the cultivation can last from about 10 hours to 160 hours, but is not limited thereto.

[0146] The O-phosphoserine hydrogen sulfide hydrolase variant produced by culture according to this disclosure can be retained in cells.

[0147] The method for producing cysteine ​​disclosed herein may also include the steps of preparing the microorganisms of the present disclosure, preparing a culture medium for culturing the microorganisms, or a combination thereof (in any order, for example, prior to the culturing step).

[0148] The method for producing cysteine ​​disclosed herein may further include the step of recovering an O-phosphoserine hydrogen sulfide hydrolase variant from a culture medium (on which the culture is carried) or from a microorganism of the present disclosure.

[0149] In the recovery step, the target variant can be collected using the methods for culturing microorganisms disclosed herein, for example, using suitable methods known in the art, such as batch culture, continuous culture, or fed-batch culture. For example, methods such as centrifugation, filtration, treatment with a protein crystallizing precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographic methods (such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc.), HPLC, or combinations thereof can be used, and the target variant can be recovered from the microorganism using suitable methods known in the art.

[0150] Furthermore, the method of this disclosure may further include a purification step after the recovery step, which may be performed using suitable methods known in the art. In one instance, when the method of this disclosure includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or intermittently, simultaneously, or integrated into a single step, but the method is not limited thereto.

[0151] In the methods disclosed herein, O-phosphoserine hydrogen sulfide hydrolase, polynucleotides, vectors, and strains are as described in other aspects above.

[0152] Sulfides can be any sulfide, provided not only in the solid form commonly used in the art, but also in liquid or gaseous form due to differences in pH, pressure, and solubility, and thus can be converted into sulfides (S). 2− ), thiosulfate (S2O3) 2− The sulfide can be in the form of a thiol (SH) group, without limitation. Specifically, the sulfide may include Na₂S, NaSH, (NH₄)₂S, H₂S, or Na₂S₂O₃, which can provide a thiol group to the OPS, but is not limited thereto. In the reaction, a single thiol group is provided to a single reactive OPS group to produce a single cysteine ​​or a derivative thereof. Here, based on the molar concentration of the OPS, the sulfide may be added in an amount of 0.1 to 3 molar equivalents, and specifically 1 to 2 molar equivalents, but is not limited thereto.

[0153] Furthermore, in this disclosure, the method for producing cysteine ​​may also include a step of recovering the cysteine ​​produced by the above-described reaction steps. Here, the desired cysteine ​​can be collected by separating and purifying the cysteine ​​from the reaction solution using suitable reactions known in the art.

[0154] As used herein, the term "derivative" refers to a similar compound obtained by chemically modifying a portion of any compound. The term generally refers to a compound in which one hydrogen atom or a particular group of atoms is replaced by another atom or group of atoms.

[0155] As used herein, the term "cysteine ​​derivative" refers to a compound in which a hydrogen atom or a specific group of atoms in cysteine ​​is replaced by another atom or group of atoms. For example, cysteine ​​derivatives can have a form in which the nitrogen atom of the amino group (-NH2) or the sulfur atom of the thiol group (-SH) in cysteine ​​has another atom or group attached thereto, and examples of cysteine ​​derivatives may include, but are not limited to, N-acetylcysteine ​​(NAC), S-carboxymethylcysteine ​​(SCMC), BOC-CYS(ME)-OH, (R)-S-(2-amino-2-carboxyethyl)-L-homocysteine, (R)-2-amino-3-sulfonylpropionic acid, D-2-amino-4-(ethylthio)butyric acid, 3-sulfinyl-L-alanine, Fmoc-Cys(Boc-methyl)-OH, seleno-L-cysteine, S-(2-thiazolyl)-L-cysteine, S-(2-thienyl)-L-cysteine, S-(4-tolyl)-L-cysteine, etc.

[0156] Provided that cysteine ​​is produced according to the method of this disclosure, the conversion to cysteine ​​derivatives can be readily carried out by methods well known in the art to convert to various cysteine ​​derivatives.

[0157] In this disclosure, the method for producing cysteine ​​derivatives may further include converting the cysteine ​​produced above into cysteine ​​derivatives.

[0158] Specifically, in this disclosure, a method for producing cysteine ​​derivatives may include the following steps: producing cysteine ​​according to the method of this disclosure described above; and converting the produced cysteine ​​into cysteine ​​derivatives.

[0159] The conversion of produced cysteine ​​into cysteine ​​derivatives can be carried out by methods known in the art. For example, according to methods known in the art, cysteine ​​can be synthesized into N-acetylcysteine ​​(NAC) by reacting with an acetylation agent, or into S-carboxymethylcysteine ​​(SCMC) by reacting with haloacetic acid under alkaline conditions, but examples are not limited thereto.

[0160] These cysteine ​​derivatives are mainly used as pharmaceutical materials for antitussives, cough suppressants, and treatments for bronchitis, bronchial asthma, pharyngitis, etc., but are not limited to these applications.

[0161] Another aspect of this disclosure provides a composition for producing cysteine ​​or cysteine ​​derivatives comprising: an O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure; a polynucleotide encoding the variant; a vector containing the polynucleotide; or a microorganism containing the polynucleotide of this disclosure; a culture medium for culturing the microorganism; or a combination of two or more of these.

[0162] The compositions disclosed herein may also include any suitable excipient commonly used in compositions for the production of cysteine ​​or cysteine ​​derivatives, and such excipients may include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers or isotonic agents.

[0163] Another aspect of this disclosure provides a method for producing recombinant microorganisms for producing O-phosphoserine, the method comprising: introducing an O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure; a polynucleotide of this disclosure; or a vector of this disclosure into the microorganism.

[0164] Another aspect of this disclosure provides the use of the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure for the production of cysteine ​​or cysteine ​​derivatives.

[0165] Another aspect of this disclosure provides the use of recombinant microorganisms for the production of O-phosphoserine, said recombinant microorganisms comprising the O-phosphoserine hydrogen sulfide hydrolase variant of this disclosure for the production of cysteine ​​or cysteine ​​derivatives; the polynucleotide of this disclosure; or the vector of this disclosure.

[0166] The above-mentioned O-phosphoserine hydrogen sulfide hydrolase variants, polynucleotides, carriers, cysteine, cysteine ​​derivatives, and microorganisms are as described above.

[0167] [Modes for Implementing the Invention]

[0168] The present disclosure will be described in detail below by way of examples. However, these examples are merely preferred examples given for illustrative purposes, and therefore the scope of the present disclosure is not intended to be limited to or restricted by these examples. Furthermore, those skilled in the art within the scope of this disclosure or similar technical fields will fully understand and readily implement technical features not described herein.

[0169] Example 1: Selection of CysM variants

[0170] Using the error-prone PCR kit (Diversity PCR Random Mutagenesis Kit, Clontech) based on Msm-T-HA2 (US 9127324 B2, SEQ ID NO: 11), a variant library encoding the cysM gene of the CysM variant was prepared by random mutagenesis. Msm-T-HA2 is a variant that is missing 5 amino acid residues from the C-terminus and has its proline residue 77 from the N-terminus replaced by serine. In CysM (Msm-OPSS, SEQ ID NO: 1) derived from wild-type Mycobacterium smegmatis with O-phosphoserine hydrogen sulfide hydrolase (OPSS) activity, it exhibits the activity of converting O-phosphoserine (OPS) to cysteine.

[0171] Specifically, random mutagenesis PCR was performed using primer pairs SEQ ID NO:16 and 17, based on the nucleotide sequence encoding Msm-T-HA2 (SEQ ID NO: 12) as a template. Diversify was used. TM The PCR random mutagenesis kit was used, and PCR was performed under the following conditions: denaturation at 94°C for 5 minutes, followed by 20 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and polymerization at 72°C for 1 minute, and then polymerization at 72°C for 5 minutes.

[0172] The amplified mutant gene fragments were then introduced into pUC vectors containing the cysK promoter (Yanisch-Perron et al., Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. 1985, Gene 33 (1), 103-119) (SEQ ID NO: 15, pUC_PcysK). To prepare the pUC_PcysK vector, PCR was performed using primer pairs SEQ ID NO: 18 and 19, based on E. coli ATCC27325 chromosomal DNA as a template, to first obtain the cysK promoter fragment. PCR was performed under the following conditions: 30 cycles of denaturation at 94°C for 5 minutes, followed by 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 pUC_PcysK vector was prepared by cloning the amplified cysK promoter fragment in the pUC vector, digested with NheI and HindIII restriction enzymes, using the in-fusion cloning kit (Seamless Cloning Kit, Clontech Laboratories, Inc.). The previously amplified mutant gene fragment was then cloned in the pUC_PcysK vector, digested with EcoRV restriction enzymes, using the in-fusion cloning kit. Cloning was performed by incubation at 50°C for 60 minutes, thereby preparing a pUC_PcysK-cysM gene variant plasmid library.

[0173] The primer sequences used in this paper are shown in Table 1 below.

[0174]

[0175] The enzyme expression vector thus prepared was transformed into *Escherichia coli* strain K12 by electroporation (Appl. Microbiol. Biotechnol. (1999) 52:541-545), and homologous recombination was introduced into the chromosome (van der Rest et al., *Appl Microbiol Biotechnol* 52:541-545, 1999). Strains that inserted the vector into the chromosome through homologous sequence recombination were selected from a medium containing 25 mg / L kanamycin.

[0176] DNA was obtained from three selected *E. coli* transformants using the DNA-spin plasmid DNA purification kit (Intron) according to the manufacturer's protocol, and the nucleotide sequence was analyzed using sequencing. The sequencing results confirmed that the CysM variants derived from the three *E. coli* transformants consisted of the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, respectively. In the case of the CysM variant consisting of the amino acid sequence of SEQ ID NO: 5, it was confirmed that in the amino acid sequence of the CysM variant derived from *Mycobacterium smegmatis* (which is the parental sequence), amino acid residue 77 (proline) was replaced by alanine, and amino acid residue 218 (alanine) was replaced by glycine. In the case of the CysM variant consisting of the amino acid sequence of SEQ ID NO: 7, it was confirmed that in the amino acid sequence of the CysM variant derived from *Mycobacterium smegmatis* (which is the parental sequence), amino acid residue 55 (alanine) was replaced by valine, amino acid residue 77 (proline) was replaced by alanine, and amino acid residue 218 (alanine) was replaced by glycine. In the case of the CysM variant consisting of the amino acid sequence of SEQ ID NO: 9, it was confirmed that in the amino acid sequence of the CysM variant derived from Mycobacterium smegmatis (which is the parental sequence), the 7th amino acid residue leucine was replaced by proline, the 77th amino acid residue proline was replaced by alanine, and the 218th amino acid residue alanine was replaced by glycine.

[0177] Example 2: Preparation of CysM variant expression vector and expression strain

[0178] 2-1. Preparation of CysM variant (P77A or P77S) expression vector and expression strain

[0179] To prepare strains expressing only the CysM variant containing the mutations confirmed in Example 1, CysM variant expression vectors were first prepared.

[0180] Specifically, to prepare the CysM(P77A) expression vector, primer pairs SEQ ID NO: 16 and 20 were used, based on the nucleotide sequence encoding Msm-T (US 9127324 B2) (SEQ ID NO: 13) (SEQ ID NO: 14) as a template. Msm-T is a variant of CysM (Msm-OPSS) derived from wild-type Mycobacterium smegmatis that has 5 amino acid residues deleted from its C-terminus. The upstream fragment of the CysM(P77A) gene was obtained by PCR, and the downstream fragment of the CysM(P77A) gene was obtained by PCR using primer pairs SEQ ID NO: 17 and 21 based on the same template. To prepare the CysM(P77S) (Msm-T-HA2) expression vector as a control, primer pairs SEQ ID NO: 16 and 17 were used, based on Msm-T-HA2 as a template, to obtain the cysM(P77S) gene fragment by PCR. PCR was performed under the following conditions: 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, and then polymerization at 72°C for 5 minutes.

[0181] The gene fragments thus obtained were each introduced into the pUC vector (pUC_PcysK) containing the cysK promoter. The vector prepared in Example 1 was used as the pUC_PcysK vector. The previously obtained cysM(P77A) and cysM(P77S) gene fragments were cloned using the seamless cloning kit with EcoRV restriction enzyme-digested pUC_PcysK vector. Cloning was performed by incubation at 50°C for 60 minutes.

[0182] The primer sequences used in this paper are shown in Table 2 below.

[0183]

[0184] Based on this, pUC_PcysK-cysM(P77A) and pUC_PcysK-cysM(P77S) (Msm-T-HA2) vectors were obtained, and the vectors were transformed into Escherichia coli K12 strain in the same manner as in Example 1 to obtain CysM variant (P77A or P77S) expression strains.

[0185] 2-2. Preparation of CysM variant (P77A / A218G) expression vector and expression strain

[0186] To prepare strains expressing CysM variants containing the two combinations of mutations confirmed in Example 1, CysM variant expression vectors were first prepared.

[0187] Specifically, to prepare the cysM(P77A / A218G) expression vector, primer pairs SEQ ID NO:16 and 22 were used, based on cysM-4 (SEQ ID NO: 3) as a template, to obtain the upstream fragment of the cysM(P77A / A218G) gene by PCR. The downstream fragment of the cysM(P77A / A218G) gene was obtained by PCR using primer pairs SEQ ID NO:17 and 23 based on the same template. PCR amplification conditions were as follows: 30 cycles of denaturation at 94℃ for 5 minutes, followed by denaturation at 94℃ for 30 seconds, annealing at 55℃ for 30 seconds, and polymerization at 72℃ for 1 minute, followed by polymerization at 72℃ for 5 minutes.

[0188] The primer sequences used in this paper are shown in Table 3 below.

[0189]

[0190] The gene fragment thus obtained was cloned using a seamless cloning kit with the pUC_PcysK vector of Example 1, which was digested with EcoRV restriction enzyme of Example 1. Cloning was performed by incubation at 50°C for 60 minutes.

[0191] Thus, the pUC_PcysK-cysM(P77A / A218G) vector was obtained, and the vector was transformed into Escherichia coli K12 strain in the same manner as in Example 1 to obtain CysM variant (P77A / A218G) expression strain.

[0192] Example 3. Evaluation of cysteine ​​conversion ability of CysM variant expression strains

[0193] 3-1. Assessment of cysteine ​​conversion capacity of CysM variant (P77A or P77S) expression strains

[0194] Enzyme expression was performed according to the pET system (Novagen) manual. Specifically, single colonies of each strain from Example 2-1 were selected from LB plates containing kanamycin and inoculated into 5 mL LB broth, incubated at 37°C and 200 rpm for 16 hours. Then, the strains were inoculated into LB broth containing kanamycin and incubated at 33°C and 200 rpm for 18 hours. Enzymes were obtained by adding xylene (2% of the broth) to the culture broth. The enzymes obtained by the above method were each analyzed by 14% SDS-PAGE. Figure 1 The results were confirmed, and a soluble CysM (Msm-OPSS) variant was obtained.

[0195] The soluble CysM variants K12 / pUC_Ppro-cysM(P77S) (Msm-T-HA2) and K12 / pUC_Ppro-cysM(P77A) obtained above were used to compare and analyze the enzyme activity in converting OPS, a substrate, to cysteine. The conditions for cysteine ​​synthesis activity assay (CysM enzyme assay) are shown in Table 4 below.

[0196]

[0197] In Table 1, the remaining solutions, excluding the enzyme, were mixed and incubated at 37°C for 5 minutes. Then, 25 μL of xylene-treated soluble CysM variant was added, and the reaction was carried out at 37°C for 10 minutes. After the reaction was complete, 100 μL of the reaction solution was taken and reacted with 900 μL of a mixture containing 100 mM Tris-HCl (pH 8.5) and 10 mM DTT (dithiothreitol) for 30 minutes. The reaction was then terminated by adding 0.1 M HCl. The concentration of cysteine ​​in the reaction solution was quantified by HPLC, and the cysteine ​​synthesis titer was evaluated by comparing the cysteine ​​conversion rate and specific activity (cysteine ​​concentration / time / enzyme amount) during the 10-minute reaction. The results are shown in Table 5 below.

[0198]

[0199] As shown in Table 5 above, compared with the control cysM(P77S) (Msm-T-HA2) expression strain, the cysM(P77A) expression strain showed increased cysteine ​​synthesis activity, with a specific activity increase of 46%.

[0200] 3-2. Assessment of cysteine ​​conversion ability of CysM variant (P77A / A218G) expression strains

[0201] K12 / pUC_PcysK-cysM (P77A / A218G), a soluble CysM variant obtained by the method in Example 2-2, was used to compare and analyze the enzyme activity in converting OPS, a substrate, to cysteine. Experiments were conducted in the same manner as in Example 3-1. The concentration of cysteine ​​in the reaction solution was quantified by HPLC, and the cysteine ​​synthesis titer was evaluated by comparing the cysteine ​​conversion rate and specific activity (cysteine ​​concentration / time / enzyme amount) over a 10-minute reaction period. The results are shown in Table 6 below.

[0202]

[0203] As shown in Table 6 above, compared with the control cysM(P77S) expression strain, the cysM(P77A / A218G) expression strain showed increased cysteine ​​synthesis activity, with a specific activity increase of 13%.

[0204] 3-3. Assessment of cysteine ​​conversion capacity of CysM variant (L7P / P77A / A218G or A55V / P77A / A218G) expression strains

[0205] Regarding the CysM variants, K12 / pUC_PcysK-cysM(P77A / A218G), K12 / pUC_PcysK-cysM(L7P / P77A / A218G), and K12 / pUC_PcysK-cysM(A55V / P77A / A218G), which were obtained by screening from the library prepared as a result of Example 1, the enzyme activities for converting OPS, as a substrate, to cysteine ​​were compared and analyzed. Experiments were performed in the same manner as in Examples 3-1. The concentration of cysteine ​​in the reaction solution was quantified by HPLC, and the cysteine ​​synthesis titer was evaluated by comparing the cysteine ​​conversion rate and specific activity (cysteine ​​concentration / time / enzyme amount) over a 10-minute reaction period. The results are shown in Table 7 below.

[0206]

[0207] As shown in Table 7 above, compared with the control cysM(P77A / A218G) expression strain, the cysM(L7P / P77A / A218G) expression strain and the cysM(A55V / P77A / A218G) expression vector showed increased cysteine ​​synthesis activity, with specific activities increasing by 41% and 72.5%, respectively.

[0208] Based on the foregoing, those skilled in the art will understand that this disclosure can be implemented in other specific forms without modifying the technical concept or essential characteristics of this disclosure. In this regard, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of this disclosure. Rather, this disclosure is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of this disclosure as defined by the appended claims.

Claims

1. An O-phosphoserine sulfhydrylase variant, wherein 0 to 7 amino acid residues are deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1 and the amino acid corresponding to position 77 is substituted with alanine.

2. The variant of claim 1, wherein 5 amino acid residues are deleted from the C- terminus in the amino acid sequence of SEQ ID NO:

1.

3. The variant of claim 1, having at least 80% homology or identity to SEQ ID NO:

1.

4. The variant of claim 1, consisting of the amino acid sequence of SEQ ID NO:

3.

5. The variant of claim 1, wherein the amino acid corresponding to a position selected from the group consisting of 7, 55, 218, and combinations thereof, in the amino acid sequence of SEQ ID NO: 1 is further substituted with another amino acid.

6. The variant of claim 5, wherein the amino acid corresponding to position 7 in the amino acid sequence of SEQ ID NO: 1 is substituted with proline.

7. The variant of claim 5, wherein the amino acid corresponding to position 55 in the amino acid sequence of SEQ ID NO: 1 is substituted with valine.

8. The variant of claim 5, wherein the amino acid corresponding to position 218 in the amino acid sequence of SEQ ID NO: 1 is substituted with glycine.

9. The variant of claim 5, consisting of any one or more of the amino acid sequences selected from the group consisting of SEQ ID NO: 5, 7, and 9.

10. A polynucleotide encoding the O-phosphoserine sulfhydrylase variant of any one of claims 1 to 9.

11. A microorganism comprising the O-phosphoserine sulfhydrylase variant of any one of claims 1 to 9 or a polynucleotide encoding the same.

12. The microorganism of claim 11, having increased cysteine production capacity compared to a microorganism comprising a wild-type O-phosphoserine sulfhydrylase having the amino acid sequence of SEQ ID NO: 1 or a polynucleotide encoding the same.

13. The microorganism of claim 12, wherein the microorganism is of the genus Escherichia.

14. The microorganism of claim 13, wherein the microorganism of the genus Escherichia is Escherichia coli.

15. A method of producing cysteine or a derivative thereof, comprising reacting a mixture of O-phosphoserine and sulfide with the O-phosphoserine sulfhydrylase variant of any one of claims 1 to 9.

16. The method of claim 15, wherein the O-phosphoserine is purified O-phosphoserine or a microbial fermentation broth comprising O-phosphoserine.

17. The method of claim 15, wherein the sulfide is one or more selected from the group consisting of Na2S, NaSH, (NH4)2S, H2S, S2O3, and Na2S2O3.

18. Use of an O-phosphoserine sulfhydrylase variant for the production of cysteine or a derivative thereof, wherein 0 to 7 amino acid residues are deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1 and the amino acid corresponding to position 77 is replaced by alanine.

19. Use of a microorganism for the production of cysteine or a derivative thereof, said microorganism comprising a O-phosphoserine sulfhydrylase variant according to any one of claims 1 to 9; a polynucleotide encoding said variant; or a vector comprising said polynucleotide.

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