O-phosphoserine sulfhydrase variant and method for producing cysteine ​​using the same

Abstract

This application relates to an O-phosphoserine sulfhydrase mutant and a method for producing cysteine ​​using the same.

Description

Technical Field

[0001] This application relates to an O-phosphoserine sulfhydrylase variant and a method for producing cysteine using the same.

Background Art

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

[0003] As methods for producing L-cysteine using microorganisms, 1) a method of biologically converting D,L-ATC (D,L-2-amino-2-thiazoline-4-carboxylate) using microorganisms, 2) a direct fermentation method for producing L-cysteine using Escherichia coli (Patent Document 1), 3) a method of fermentatively producing O-phosphoserine (O-phosphoserine, hereinafter referred to as "OPS") using microorganisms and then reacting it with a sulfide under the catalytic action of O-phosphoserine sulfhydrylase (hereinafter referred to as "OPSS") to convert it to L-cysteine (Patent Document 2) are known.

[0004] Here, in order to produce cysteine in a high yield by the method of 3) above, it was necessary to overproduce the precursor OPS.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

[0006] [Non-licensed Document 1] Mino K and Ishikawa K, FEBSletters, 551:133-138, 2003 [Non-licensed Document 2] Bums KE et al., J. Am. Chem. Soc, 127: 11602-11603, 2005 [Non-licensed Document 3] Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 [Non-licensed Document 4] Rice et al., 2000, Trends Genet. 16: 276-277 [Non-licensed Document 5] Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453 [Non-licensed Document 6] Devereux, J., et al, Nucleic Acids Research 12: 387 (1984) [Non-licensed Document 7] Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990) [Non-licensed Document 8] Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

Non-licensed Document 16

Non-Patent Document 17

Non-Patent Document 18

Non-Patent Document 19

Non-Patent Document 20

Summary of the Invention

Problems to be Solved by the Invention

[0007] The applicants have confirmed that when the O-phosphoserine sulfhydrylase variant of the present application is used in a microorganism producing cysteine, cysteine can be produced in a higher yield compared to a microorganism having a conventional wild-type O-phosphoserine sulfhydrylase, and have thus completed the present application.

Means for Solving the Problems

[0008] This application provides an O-phosphoserine sulfhydrylase mutant in which 0 to 7 amino acid residues are deleted from the C-terminus and the amino acid corresponding to the 77th position is substituted with alanine in the amino acid sequence of SEQ ID NO: 1.

[0009] This application provides a polynucleotide encoding the O-phosphoserine sulfhydrylase mutant of this application.

[0010] This application provides a microorganism containing the O-phosphoserine sulfhydrylase mutant of this application or the polynucleotide encoding the same.

[0011] This application provides a method for producing cysteine or its derivative, which includes the step of adding and reacting the O-phosphoserine sulfhydrylase mutant of this application to a mixture of O-phosphoserine and sulfide of this application.

Effect of the Invention

[0012] When culturing a microorganism that produces cysteine using the O-phosphoserine sulfhydrylase mutant of this application, cysteine can be produced in a higher yield compared to a microorganism having a conventional wild-type O-phosphoserine sulfhydrylase.

Brief Description of the Drawings

[0013] [Figure 1] It is a figure that confirmed K12 / pUC_Ppro-cysM(P77S)(Msm-T-HA2) and K12 / pUC_Ppro-cysM(P77A), which are CysM mutants, by SDS-PAGE.

Modes for Carrying Out the Invention

[0014] These will be explained in detail below. Note that each description and embodiment disclosed in this application applies to other descriptions and embodiments. That is, any combination of the various elements disclosed in this application is included. Furthermore, this application is not limited to the following specific descriptions. In addition, numerous papers and patent documents are referenced throughout this specification, and their citations are indicated. The disclosures of the cited papers and patent documents are incorporated in their entirety as references in this specification, thereby more clearly explaining the level of the art to which this application belongs and the content of this application.

[0015] One aspect of this application provides an O-phosphoserine sulfhydrase mutant in which, in the amino acid sequence of SEQ ID NO: 1, 0 to 7 amino acid residues from the C-terminus are deleted and the amino acid corresponding to the 77th position is substituted with alanine.

[0016] In this application, "O-phosphoserine sulfhydrylase (OPSS)" refers to an enzyme that catalyzes the reaction of converting OPS to cysteine ​​by donating a thiol group (SH group) to OPS. This enzyme was discovered in Aeropymm pernix, Mycobacterium tuberculosis, Mycobacterium smegmatis, and Trichomonas vaginalis (Non-Patent Documents 1, 2).

[0017] The O-phosphoserine sulfhydrase of this application includes all polypeptides or O-phosphoserine sulfhydrases having O-phosphoserine sulfhydrase activity. The aforementioned polypeptides or O-phosphoserine sulfhydrases having O-phosphoserine sulfhydrase activity include all polypeptides having O-phosphoserine sulfhydrase activity or activity to convert O-phosphoserine (OPS) to cysteine ​​as a substrate. For example, any polypeptide having O-phosphoserine sulfhydrase activity of this application is any polypeptide having O-phosphoserine sulfhydrase activity or activity to convert OPS to cysteine ​​as a substrate, derived from microorganisms. Specifically, any polypeptide having O-phosphoserine sulfhydrase activity of this application is derived from prokaryotic or eukaryotic microorganisms, more specifically from microorganisms of the genus Mycobacterium, but is not limited to these. As another example, any polypeptide having O-phosphoserine sulfhydrilase activity according to this application may be cysteine ​​synthase (CysM) derived from microorganisms of the genus Mycobacterium. The amino acid sequence of CysM can be obtained from known databases such as NCBI's Genebank. For example, it may be WP_003896302.1 derived from microorganisms of the genus Mycobacterium, but it goes without saying that it includes proteins of various origins that have O-phosphoserine sulfhydrilase activity or activity that converts OPS to cysteine ​​as a substrate.

[0018] Furthermore, the O-phosphoserine sulfhydrase of this application includes not only the wild-type O-phosphoserine sulfhydrase protein, but also mutant proteins in which a portion of the polynucleotide sequence encoding the O-phosphoserine sulfhydrase is deleted, substituted, or added, and which exhibit activity equivalent to or greater than that of the wild-type O-phosphoserine sulfhydrase protein. This also includes all of the O-phosphoserine sulfhydrase proteins and their mutant proteins disclosed in Patent Documents 2 and 3.

[0019] The aforementioned "O-phosphoserine sulfhydrase" is also known as "OPSS," "cysteine ​​synthase," or "CysM."

[0020] In this application, "O-phosphoserine sulfhydrylase (OPSS) variant" means any polypeptide having O-phosphoserine sulfhydrylase activity or O-phosphoserine sulfhydrylase, in which 0 to 7 amino acid residues are deleted from the C-terminus of the amino acid sequence of SEQ ID NO: 1, and the amino acid corresponding to the 77th position from the N-terminus of SEQ ID NO: 1 is substituted with alanine.

[0021] The aforementioned "O-phosphoserine sulfhydrase variant" is also called "mutant O-phosphoserine sulfhydrase," "OPSS variant," "mutant OPSS," "CysM variant," or "mutant CysM."

[0022] The proteins targeted for mutation introduction in this application may be proteins having O-phosphoserine sulfhydrase activity or activity to convert OPS to cysteine ​​as a substrate. Specifically, the protein includes the amino acid sequence of SEQ ID NO: 1 and has O-phosphoserine sulfhydrase activity or activity to convert OPS to cysteine ​​as a substrate, but is not limited to this. This does not exclude meaningless sequence additions before or after the amino acid sequence of SEQ ID NO: 1, naturally occurring mutations, or silent mutations thereof, and any protein having the same or equivalent activity as a protein containing the amino acid sequence of SEQ ID NO: 1 is included as a protein targeted for mutation introduction in this application. For example, the proteins targeted for mutation introduction in this application may be proteins consisting of the amino acid sequence of SEQ ID NO: 1, or amino acid sequences having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more homology or identity with it. Furthermore, it goes without saying that proteins having amino acid sequences in which some sequences are deleted, modified, substituted, or added, as long as they have such homology or identity and exhibit efficacy equivalent to the aforementioned protein, are also included in the proteins subject to mutation in this application. An example of a protein subject to mutation in this application, i.e., a parent sequence, is the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 13.

[0023] The O-phosphoserine sulfhydrase variant of this application may have 0 to 7 amino acid residues deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1, and the amino acid at the 77th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 may be replaced with an amino acid different from the original amino acid. More specifically, the O-phosphoserine sulfhydrase variant of this application may have 3 to 7 amino acid residues deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1, and the amino acid at the 77th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 may be replaced with an amino acid different from the original amino acid. More specifically, the O-phosphoserine sulfhydrase variant of this application may have 5 amino acid residues deleted from the C-terminus in the amino acid sequence of SEQ ID NO: 1, and the amino acid at the 77th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 may be replaced with an amino acid different from the original amino acid. In this application, the pre-substituted amino acid corresponding to position 77 of SEQ ID NO: 1 in the pre-substituted amino acid sequence of the O-phosphoserine sulfhydrylase protein to be mutagenerated may be proline (P).

[0024] As an example, the O-phosphoserine sulfhydrase mutant may have the amino acid at position 77 in the amino acid sequence of SEQ ID NO: 1 replaced with an amino acid other than proline. As another example, the O-phosphoserine sulfhydrase mutant may have the amino acid at position 77 in the amino acid sequence of SEQ ID NO: 1 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, for example, replaced with alanine. Such amino acid substitutions can generally occur 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; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0025] Furthermore, amino acids are classified into those with electrically charged side chains and those with uncharged side chains. Examples of amino acids with electrically charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino acids with uncharged side chains are further classified into nonpolar amino acids and polar amino acids. Examples of nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Examples of polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Conservative substitutions usually have little to no effect on the activity of proteins or polypeptides.

[0026] As an example, the O-phosphoserine sulfhydrase variant of this application may have the amino acid sequence represented by SEQ ID NO: 3, may contain the amino acid sequence, may consist of the amino acid sequence, or may be substantially composed of the amino acid sequence. The O-phosphoserine sulfhydrase variant of this application may contain 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 more homology or identity with the amino acid sequence of SEQ ID NO: 3. Furthermore, it goes without saying that O-phosphoserine sulfhydrase mutants having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added, as long as they have such homology or identity and exhibit efficacy equivalent to the O-phosphoserine sulfhydrase mutants of this application, are also included in this application.

[0027] For example, the O-phosphoserine sulfhydrase variant of this application may have additions or deletions of sequences that do not alter the function of the O-phosphoserine sulfhydrase variant of this application at its N-terminus, C-terminus, and / or within the amino acid sequence, as well as spontaneous mutations, silent mutations, or conservative substitutions.

[0028] The term "conservative substitution" refers to the substitution of one amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions can generally occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little to no effect on the activity of a protein or polypeptide.

[0029] The O-phosphoserine sulfhydrase variant of this application may further be modified in which an amino acid corresponding to a position selected from the group consisting of the 7th, 55th, 218th positions from the N-terminus and combinations thereof in the amino acid sequence of Sequence ID No. 1 is substituted with an amino acid different from the original amino acid.

[0030] In this application, the pre-substituted amino acid corresponding to the 7th position in SEQ ID NO: 1 of the O-phosphoserine sulfhydrase protein to be mutagenerated is leucine (L), the pre-substituted amino acid corresponding to the 55th position is alanine (A), and the pre-substituted amino acid corresponding to the 218th position is also alanine (A).

[0031] As an example, the O-phosphoserine sulfhydrase variant of this application may be one in which the amino acid corresponding to the 7th position in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than leucine. As another example, the O-phosphoserine sulfhydrase variant may be one in which the amino acid corresponding to the 7th position in the amino acid sequence of SEQ ID NO: 1 is replaced with 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, for example, it may be replaced with proline.

[0032] As an example, the O-phosphoserine sulfhydrase variant of this application may be one in which the amino acid corresponding to the 55th position in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than alanine. As another example, the O-phosphoserine sulfhydrase variant may be one in which the amino acid corresponding to the 55th position in the amino acid sequence of SEQ ID NO: 1 is replaced with 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, for example, it may be replaced with valine.

[0033] As an example, the O-phosphoserine sulfhydrase variant of this application may be one in which the amino acid corresponding to position 218 in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid other than alanine. As another example, the O-phosphoserine sulfhydrase variant may be one in which the amino acid corresponding to position 218 in the amino acid sequence of SEQ ID NO: 1 is replaced with 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, for example, it may be replaced with glycine.

[0034] As one example, the O-phosphoserine sulfhydrase mutant of this application may have 0 to 7 amino acid residues, for example 3 to 7, from the C-terminus in the amino acid sequence of SEQ ID NO: 1 described above, with the amino acid substitution at the position corresponding to the 77th position from the N-terminus remaining fixed, and further, it may contain at least one, at least two, or all three amino acid substitutions at the positions corresponding to the 7th, 55th, or 218th positions from the N-terminus in the amino acid sequence of SEQ ID NO: 1.

[0035] As another example, the O-phosphoserine sulfhydrase variant of this application may have at least one amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, may contain the said amino acid sequence, may consist of the said amino acid sequence, or may be substantially composed of the said amino acid sequence. The O-phosphoserine sulfhydrase variant of this application may contain 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 more homology or identity with the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9. Furthermore, it goes without saying that O-phosphoserine sulfhydrase mutants having amino acid sequences in which some sequences are deleted, modified, substituted, conservatively substituted, or added, as long as they have such homology or identity and exhibit efficacy equivalent to the O-phosphoserine sulfhydrase mutants of this application, are also included in this application.

[0036] In this application, "mutant protein" or "variant" refers to a polypeptide in which at least one amino acid differs from the amino acid sequence of the original polypeptide due to conservative substitution and / or modification, but the functions or properties are maintained. Such variants can generally be identified by modifying at least one amino acid in the amino acid sequence of the polypeptide and evaluating the properties of the modified polypeptide. That is, the capabilities of the variant are improved, unchanged, or decreased compared to the original polypeptide. Some variants also include those in which at least one portion, such as the N-terminal leader sequence or transmembrane domain, is removed. Other variants include those in which a portion of the N and / or C-terminus of a mature protein is removed. The term "mutant protein" is used interchangeably with terms such as mutant, modified, mutant polypeptide, mutated protein, mutation, and variant (in English, these include modification, modified polypeptide, modified protein, mutant, mutein, and divergent), but any term that means mutation is acceptable. For the purposes of this application, the variant of this application 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 at the 77th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid different from the original amino acid. Specifically, the variant of this application 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 at the 77th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid different from the original amino acid.As an example, the variant of this application may be one in which the amino acid corresponding to position 77 in the amino acid sequence 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, for example, it may be replaced with alanine. Such amino acid substitutions can generally occur 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; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan.

[0037] Furthermore, amino acids are classified into those with electrically charged side chains and those with uncharged side chains. Examples of amino acids with electrically charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino acids with uncharged side chains are further classified into nonpolar amino acids and polar amino acids. Examples of nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. Examples of polar amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Conservative substitutions usually have little to no effect on the activity of proteins or polypeptides.

[0038] As another example, the variant of this application is a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3, but is not limited thereto.

[0039] The variant of this application is a polypeptide in which five amino acid residues are deleted from the C-terminus of the amino acid sequence of SEQ ID NO: 1, and the amino acid at the 77th position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid different from the original amino acid. Furthermore, the amino acid at a position selected from the group consisting of the 7th, 55th, 218th positions from the N-terminus of the amino acid sequence of SEQ ID NO: 1 and combinations thereof may be replaced with an amino acid different from the original amino acid. For example, in the amino acid sequence of SEQ ID NO: 1, the amino acid at the 7th position may be replaced with 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, for example, it may be replaced with proline. The variant of this application may be one in which the amino acid corresponding to position 55 in the amino acid sequence of SEQ ID NO: 1 is replaced with 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, for example, it may be replaced with valine. Alternatively, the variant of this application may be one in which the amino acid corresponding to position 218 in the amino acid sequence of SEQ ID NO: 1 is replaced with 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, for example, it may be replaced with glycine. Other examples include, but are not limited to, polypeptides comprising the amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9.The aforementioned variant may include the modifications described above, and may also have improved O-phosphoserine sulfhydrase enzyme activity compared to the polypeptide in which the amino acid at the 77th position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is proline.

[0040] Furthermore, the mutants may include the deletion or addition of amino acids that have minimal effect on the polypeptide's properties and secondary structure. For example, the N-terminus of the mutant may be conjugated with a signal (or leader) sequence that is involved in protein translocation co-translationally or post-translationally. The mutants may also be conjugated with other sequences or linkers so that they can be identified, purified, or synthesized.

[0041] In this application, "homology" or "identity" refers to the degree to which two given amino acid sequences or base sequences are similar, and is expressed as a percentage. Homology and identity are often used interchangeably.

[0042] The sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard sequence algorithms, and a default gap penalty established by the program used may also be applied. Substantially, homologous or identical sequences generally hybridize with all or part of the sequence under moderate to high stringent conditions. Needless to say, hybridization includes hybridization with polynucleotides that have common codons or codons considering codon degeneracy in the polynucleotide.

[0043] Whether any two polynucleotide or polypeptide sequences are homologous, similar, or identical can be determined using default parameters, such as those in Non-Patent Document 3, and known computer algorithms such as the "FASTA" program. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Non-Patent Document 5), as performed in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 4) (version 5.0.0 or later) (including the GCG program package (Non-Patent Document 6), BLASTP, BLASTN, and FASTA (Non-Patent Documents 7, 8, and 9)). For example, homology, similarity, or identity can be determined using BLAST or Clustal W from the National Center for Biotechnology Information.

[0044] The homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as Non-Patent Document 5, as disclosed in Non-Patent Document 10, for example. In summary, the GAP program is defined as the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of two sequences. Default parameters for the GAP program include (1) unitary matrices (where identity is 1 and non-identity is 0) and the weighted comparison matrix of Non-Patent Document 12 (or EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix) as disclosed in Non-Patent Document 11, (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap open penalty of 10 and a gap extended penalty of 0.5), and (3) no penalty for terminal gaps.

[0045] The O-phosphoserine sulfhydrase mutant of this application may have improved cysteine ​​conversion activity compared to wild-type O-phosphoserine sulfhydrase.

[0046] For example, the O-phosphoserine sulfhydrase mutant used as a target enzyme for comparing whether or not the cysteine ​​conversion activity has improved is Msm-T-HA2 (Patent Document 4), but it is not limited to this.

[0047] As an example, the mutant of this application with improved cysteine ​​conversion activity is an improvement of approximately 1% or more compared to the cysteine ​​conversion activity of the enzyme before mutation or the unmodified enzyme, specifically approximately 5% or more, approximately 10% or more, approximately 13% or more, approximately 15% or more, approximately 20% or more, approximately 25% or more, approximately 30% or more, approximately 35% or more, approximately 40% or more, approximately 41% or more, approximately 45% or more, approximately 46% or more, approximately 50% or more, approximately 55% or more, approximately 60% or more, approximately 65% ​​or more, approximately 70% or more, approximately 72% or more, or approximately 72.5% or more (there is no particular limit on the upper limit, for example, approximately 200% or less, approximately 150% or less, approximately 100% or less, approximately 50% or less, approximately 40% or less, approximately 30% or less, approximately 20% or less, or approximately 15% or less), but any mutant that shows a positive increase in cysteine ​​conversion activity compared to the enzyme before mutation or the unmodified enzyme is acceptable. As another example, the mutant of this application with improved cysteine ​​conversion activity shows an improvement in cysteine ​​conversion activity of approximately 1.01 times or more, approximately 1.05 times or more, approximately 1.1 times or more, approximately 1.13 times or more, approximately 1.15 times or more, approximately 1.2 times or more, approximately 1.25 times or more, approximately 1.3 times or more, approximately 1.35 times or more, approximately 1.4 times or more, approximately 1.41 times or more, approximately 1.45 times or more, approximately 1.46 times or more, approximately 1.5 times or more, approximately 1.55 times or more, approximately 1.6 times or more, approximately 1.65 times or more, approximately 1.7 times or more, approximately 1.72 times or more, or approximately 1.725 times or more (there is no particular limit on the upper limit, for example, approximately 10 times or less, approximately 5 times or less, approximately 3 times or less, or approximately 2 times or less) compared to the enzyme before mutation or the unmodified enzyme, but is not limited to these.

[0048] Such cysteine ​​conversion ability can be evaluated by measuring the amount of cysteine ​​produced by the cysteine ​​conversion reaction. In this evaluation, the amount of cysteine ​​produced can be measured using suitable methods known in the art. For example, the amount of cysteine ​​produced can be measured using suitable methods known in the art, such as HPLC (High Performance Liquid Chromatography), GC (Gas Chromatography), GC / MS (Gas Chromatography-Mass Spectrometry), LC / MS (Liquid chromatography-mass spectrometry), GPC (Gel Permeation Chromatography), or a combination thereof.

[0049] In this application, "corresponding to" means an amino acid residue at a position listed in the polypeptide, or an amino acid residue that is similar, identical, or equivalent to a residue listed in the polypeptide. Identifying the amino acid at the corresponding position will determine the specific amino acid in the sequence referencing the particular sequence. In this application, "corresponding region" generally means a similar or corresponding position in the related protein or reference protein.

[0050] For example, by aligning any amino acid sequence with Sequence ID No. 1, each amino acid residue in the amino acid sequence can be numbered based on the number and position of amino acid residues corresponding to the amino acid residues in Sequence ID No. 1. For example, the sequence alignment algorithm in this application can be used to identify the positions of amino acids, or the positions where modifications such as substitutions, insertions, or deletions occur, by comparing it with a query sequence ("reference sequence").

[0051] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Document 5) and the Needle program from the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 4) can be used, but are not limited to these. Sequence alignment programs and pairwise sequence comparison algorithms known in the art can be used as appropriate.

[0052] Another aspect of this application provides a polynucleotide encoding an O-phosphoserine sulfhydrylase variant of the present application.

[0053] The O-phosphoserine sulfhydrase protein of this application may be encoded by the cysM gene.

[0054] For example, the cysM gene is a polynucleotide encoding WP_003896302.1 derived from microorganisms of the genus Mycobacterium, but is not limited to this. Another example is the cysM gene derived from microorganisms of the genus Mycobacterium, but is not limited to this; it includes genes from various sources encoding proteins with O-phosphoserine sulfhydrilase activity.

[0055] In this application, "polynucleotide" means a polymer of nucleotides in which nucleotide monomers are covalently linked together in a long chain, and refers to a DNA or RNA chain longer than a predetermined length, and more specifically, refers to a polynucleotide fragment encoding the O-phosphoserine sulfhydrilase variant.

[0056] The polynucleotide encoding the O-phosphoserine sulfhydrilase variant of this application may include a nucleotide sequence encoding an O-phosphoserine sulfhydrilase variant in which five amino acid residues from the C-terminus are deleted in the amino acid sequence of SEQ ID NO: 1, and the amino acid at the 77th position from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid. For example, the polynucleotide of this application may include a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 3. As another more specific example of this application, the polynucleotide of this application may have the nucleotide sequence of SEQ ID NO: 4, or may include the said nucleotide sequence. Furthermore, the polynucleotide of this application may consist of the nucleotide sequence of SEQ ID NO: 4, or may be substantially composed of the said nucleotide sequence.

[0057] The polynucleotides of this application can be modified in various ways in the coding region, within the limits that the amino acid sequence of the O-phosphoserine sulfhydrase variant of this application does not change, by codon degeneracy or by taking into consideration codons preferred in organisms that intend to express the O-phosphoserine sulfhydrase variant of this application. Specifically, the polynucleotides of this application have a base sequence that is homologous or identical to the base sequence of SEQ ID NO: 4 by 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%, or include the said base sequence, consist of the said base sequence, or are substantially composed of the said base sequence. Here, in the homologous or identical sequence, the codon encoding the amino acid corresponding to the 93rd position of SEQ ID NO: 1 may be one of the codons encoding an amino acid other than proline, such as alanine.

[0058] Furthermore, the polynucleotide encoding the O-phosphoserine sulfhydrilase variant of this application may include a nucleotide sequence encoding an O-phosphoserine sulfhydrilase variant in which five amino acid residues are deleted from the C-terminus of the amino acid sequence of SEQ ID NO: 1, and the amino acid at the 77th position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is replaced with an amino acid different from the original amino acid, and further, the amino acid at a position selected from the group consisting of the 7th, 55th, 218th positions from the N-terminus of the amino acid sequence of SEQ ID NO: 1 and combinations thereof is replaced with an amino acid different from the original amino acid. As an example, the polynucleotide of this application may include a nucleotide sequence encoding at least one amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9. As another more specific example of this application, the polynucleotide of this application may have the nucleotide sequence of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or may include the said nucleotide sequence. Furthermore, the polynucleotide of this application may consist of the nucleotide sequence of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or may be substantially composed of the said nucleotide sequence.

[0059] Specifically, the polynucleotides of this application have a base sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, and less than 100% homology or identity with the base sequence of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or include the said base sequence, consist of the said base sequence, or are substantially composed of the said base sequence. Here, in the homologous or identical sequence, the codon encoding the amino acid corresponding to the 7th position of SEQ ID NO: 1 may be one of the codons encoding an amino acid other than leucine, such as proline; the codon encoding the amino acid corresponding to the 55th position of SEQ ID NO: 1 may be one of the codons encoding an amino acid other than alanine, such as valine; and the codon encoding the amino acid corresponding to the 218th position of SEQ ID NO: 1 may be one of the codons encoding an amino acid other than alanine, such as glycine.

[0060] Furthermore, the polynucleotide of this application may be any sequence that hybridizes under stringent conditions with a probe prepared from a known gene sequence, for example, a sequence complementary to all or part of the polynucleotide sequence of this application. The “stringent condition” means a condition that enables specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 13 and 14). For example, this could involve hybridizing polynucleotides with high homology or identity, such as polynucleotides with 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology or identity, while not hybridizing polynucleotides with lower homology or identity. Alternatively, it could involve washing once, specifically two to three times, at a salt concentration and temperature equivalent to the washing conditions of a typical Southern hybridization: 60°C, 1×SSC, 0.1%SDS, more specifically 60°C, 0.1×SSC, 0.1%SDS, or more specifically 68°C, 0.1×SSC, 0.1%SDS.

[0061] Hybridization requires that the two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of the hybridization. "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 application may include not only substantially similar nucleic acid sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0062] Specifically, polynucleotides homologous or identical to the polynucleotide of this application can be detected using hybridization conditions in which the hybridization step is performed at a Tm value of 55°C and the conditions described above. The Tm value may be 60°C, 63°C, or 65°C, but is not limited to these, and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0063] The appropriate stringency for hybridizing the aforementioned polynucleotides depends on the length and degree of complementarity of the polynucleotides, and these variables are known in the art (e.g., Non-Patent Document 13).

[0064] Further embodiments of this application provide vectors comprising the polynucleotides of this application.

[0065] The aforementioned vector is an expression vector for expressing the polynucleotide in a host cell, but is not limited to this.

[0066] The vector of this application means a DNA product comprising a polynucleotide sequence encoding a target polypeptide operably ligated to a suitable regulatory region (or regulatory sequence) so as to enable the expression of the target polypeptide in a suitable host. The regulatory region includes a promoter that initiates transcription, an optional operator sequence for regulating that transcription, a sequence encoding a suitable mRNA-ribosome binding site, and sequences that regulate the termination of transcription and translation. When transformed into a suitable host cell, the vector can replicate and function independently of the host genome and is integrated into the genome itself.

[0067] The vectors used in this application are not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include plasmids, cosmids, viruses, and bacteriophages in their natural or recombinant state. For example, as phage vectors or cosmid vectors, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used, and as plasmid vectors, pDZ series, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, pET series, etc. can be used. Specifically, pUC, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0068] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using an intracellular chromosome introduction vector. The insertion of the polynucleotide into the chromosome can be carried out by any method known in the art, such as homologous recombination, but is not limited thereto. The vector may further include a selection marker to confirm whether or not the polynucleotide has been inserted into the chromosome. The selection marker is used to select cells transformed by the vector, that is, to confirm whether or not the target nucleic acid molecule has been inserted, and markers that confer selectable phenotypes such as drug resistance, nutritional requirements, resistance to cytotoxic agents, and expression of surface polypeptides are used. In an environment treated with a selective agent, only cells expressing the selection marker will survive or exhibit different phenotypes, thus allowing for the selection of transformed cells.

[0069] In this application, "transformation" means introducing a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism to express the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any form that is expressed in the host cell, regardless of whether it is inserted into or outside the host cell's chromosome. The polynucleotide also contains DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced into the host cell in any form that is expressed therein. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a gene structure containing all the elements necessary for its expression. Typically, the expression cassette includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may also be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to the sequence necessary for expression in the host cell, but is not limited to this.

[0070] Furthermore, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates the transcription of the polynucleotide encoding the target O-phosphoserine sulfhydrilase variant of this application.

[0071] Further embodiments of this application provide microorganisms comprising the O-phosphoserine sulfhydrylase variant of this application or the polynucleotide encoding it.

[0072] The strain of this application may include the O-phosphoserine sulfhydrase variant of this application, a polynucleotide encoding the polypeptide, or a vector containing the polynucleotide of this application.

[0073] In this application, "microorganism (or strain)" includes all wild-type microorganisms and microorganisms that have been genetically modified naturally or artificially, and may also include microorganisms in which a specific mechanism has been weakened or strengthened due to causes such as the insertion of an external gene or the enhancement or inactivation of the activity of an endogenous gene, and which have been genetically modified for the production of a target polypeptide, protein, or product.

[0074] The bacterial strains of this application may be strains that naturally possess cysteine ​​production ability, or they may be microorganisms in which cysteine ​​production ability has been conferred to strains that do not originally possess cysteine ​​production ability. For example, the microorganisms may have improved cysteine ​​production ability due to the introduction of the O-phosphoserine sulfhydrilase mutant of this application or the polynucleotide encoding it, but are not limited to these.

[0075] The strain of this application may be a microorganism with improved cysteine ​​production capacity compared to a parental strain that does not contain the mutant of this application, or to a wild-type Mycobacterium strain. The microorganism may have improved cysteine ​​production capacity due to the introduction of the O-phosphoserine sulfhydrase mutant of this application, which has improved cysteine ​​conversion activity compared to wild-type O-phosphoserine sulfhydrase, or the polynucleotide encoding it. Specifically, the strain of this application may have improved cysteine ​​production capacity compared to a Mycobacterium microorganism containing wild-type O-phosphoserine sulfhydrase having the amino acid sequence of SEQ ID NO: 1, or the polynucleotide encoding it.

[0076] In this application, "unmodified microorganism" does not exclude strains containing naturally occurring mutations in microorganisms, but rather refers to wild-type strains or natural strains themselves, or strains before genetic mutation and changes in phenotype due to natural or artificial factors. Furthermore, in this application, "O-phosphoserine sulfhydrilase unmodified microorganism" refers to strains in which the O-phosphoserine sulfhydrilase variants described herein have not been introduced, or before they have been introduced. In this application, O-phosphoserine sulfhydrilase unmodified microorganisms do not exclude strains in which other proteins or genes have been modified, other than O-phosphoserine sulfhydrilase or the polynucleotide encoding it.

[0077] In this application, "unmodified microorganism" is used interchangeably with "pre-modification strain," "pre-modification microorganism," "non-mutant strain," "unmodified strain," "non-mutant microorganism," or "reference microorganism."

[0078] The microorganisms of this application are, but are not limited to, microorganisms containing an O-phosphoserine sulfhydrase mutant or a polynucleotide encoding it, or microorganisms genetically modified to contain an O-phosphoserine sulfhydrase mutant or a polynucleotide encoding it (e.g., recombinant microorganisms). The term "endogenous activity" refers to the activity of a specific polypeptide that was originally present in the parent strain, wild type, or unmodified microorganism before the trait change due to genetic mutation caused by natural or artificial factors. This is used interchangeably with "activity before modification."

[0079] The microorganisms of this application are not particularly limited in type, as long as they are capable of producing cysteine, and may be either prokaryotic or eukaryotic cells, specifically prokaryotic cells. Examples include microbial strains belonging to the genera Escherichia, Erwinia, Serratia, Providencia, Corynebacterium, and Brevibacterium, and more specifically Escherichia microorganisms, and more specifically Escherichia coli (Escherichia coli), but are not limited to these.

[0080] As yet another example of this application, the recombinant microorganisms of this application may be microorganisms whose cysteine ​​production capacity is further enhanced by enhancing the activity of some proteins in the cysteine ​​biosynthesis pathway or weakening the activity of some proteins in the cysteine ​​degradation pathway.

[0081] In this application, "enhancement" of polypeptide activity means improving the polypeptide activity compared to its endogenous activity. This enhancement is used interchangeably with activation, upregulation, overexpression, and increase. Here, activation, enhancement, upregulation, overexpression, and increase all include exhibiting activity that was not originally present, or improving activity compared to endogenous activity or the activity before modification. When polypeptide activity is "enhanced," "upregulated," "overexpressed," or "improved" compared to endogenous activity, it means that it is improved compared to the activity and / or concentration (expression level) of a specific polypeptide that was originally present in the parent strain or unmodified microorganism before the trait change.

[0082] The enhancement may be carried out by introducing an exogenous polypeptide, or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether or not the activity of the polypeptide has been enhanced can be confirmed by an increase in the degree of the polypeptide's activity, its expression level, or the amount of product produced from the polypeptide.

[0083] Various methods known in the field can be applied to enhance the activity of the polypeptide, and any method that can enhance the activity of the target polypeptide compared to the microorganism before modification is acceptable. Specifically, this includes, but is not limited to, conventional methods in molecular biology, including genetic engineering and / or protein engineering known to those with ordinary skill in the field (e.g., Non-Patent Documents 13, 15, etc.).

[0084] Specifically, the enhancement of polypeptide activity in this application is carried out by: 1) increasing the intracellular copy number of the polynucleotide encoding the polypeptide; 2) replacing the expression regulatory region of the gene on the chromosome encoding the polypeptide with a highly active sequence; 3) modifying the start codon or the base sequence encoding the 5'UTR region of the gene transcript encoding the polypeptide; 4) modifying the amino acid sequence of the polypeptide so as to enhance polypeptide activity; 5) modifying the polynucleotide sequence encoding the polypeptide so as to enhance polypeptide activity (for example, modifying the polynucleotide sequence of the polypeptide gene to encode a polypeptide modified to enhance polypeptide activity); 6) introducing an exogenous polypeptide exhibiting polypeptide activity or an exogenous polynucleotide encoding it; 7) optimizing the codon of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide and selectively modifying or chemically modifying exposed regions; 9) regulating the cellular localization of the protein (polypeptide); or 10) a combination of two or more selected from 1) to 9) above, but is not limited to these.

[0085] More specifically, increasing the intracellular copy number of the polynucleotide encoding the polypeptide (as described in 1) above may be carried out by introducing a vector into a host cell that is operablely linked to the polynucleotide encoding the polypeptide and replicates and functions independently of the host. Alternatively, it may be carried out by introducing one or more copies of the polynucleotide encoding the polypeptide (protein), operablely linked to a suitable regulatory sequence, into the chromosomes within a host cell (microorganism). The introduction into the chromosome is carried out by introducing a vector into the host cell (microorganism) that can insert the polynucleotide into the chromosomes within the host cell (microorganism), but is not limited to this. The vector is as described above. The regulatory sequence may be a sequence that is natural (of the same origin) as the encoding polynucleotide sequence, a foreign (derived from another gene) sequence, a variant sequence thereof, or another artificial sequence, and may also induce the expression of the polynucleotide in the host cell. As one example, the regulatory sequence of the gene (cysM) encoding the O-phosphoserine sulfhydrylase (OPSS) protein derived from Mycobacterium smegmatics in this application is the PcysK promoter, but is not limited to this.

[0086] 2) The substitution of a gene expression regulatory region (or expression regulatory sequence) on a chromosome encoding a polypeptide with a more potent sequence may be carried out, for example, by generating a sequence mutation through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, so as to further enhance the activity of the expression regulatory region, or by substituting it with a sequence having higher activity. The expression regulatory region includes, but is not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences regulating transcription and translation termination. For example, this may be carried out by substituting the original promoter with a potent promoter, but is not limited to this.

[0087] Examples of known strong promoters include, but are not limited to, the CJ1-CJ7 promoters (Patent Document 5), the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, the tet promoter, the gapA promoter, the SPL7 promoter, the SPL13 (sm3) promoter (Patent Document 6), the O2 promoter (Patent Document 7), the tkt promoter, and the yccA promoter.

[0088] 3) Modifying the start codon or the nucleotide sequence encoding the 5'UTR region of a gene transcript encoding a polypeptide is performed, for example, by substituting it 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 to this.

[0089] Modifying the amino acid sequence or polynucleotide sequence described in 4) and 5) above is carried out by causing a sequence mutation through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, in order to enhance the activity of the polypeptide, or by substituting it with an improved amino acid sequence or polynucleotide sequence that has higher activity, or an improved amino acid sequence or polynucleotide sequence that has improved activity. Specifically, the substitution is carried out by inserting the polynucleotide into the chromosome by homologous recombination, but is not limited to this. The vector used here may further include a selection marker to confirm whether or not it has been inserted into the chromosome.

[0090] The introduction of a foreign polynucleotide exhibiting polypeptide activity (6) above may be carried out by introducing a foreign polynucleotide encoding a polypeptide exhibiting identical or similar activity to the polypeptide into the host cell. The foreign polynucleotide may have any origin or sequence, as long as it exhibits identical or similar activity to the polypeptide. The introduction can be carried out by a person skilled in the art using a known transformation method as appropriate, and as described above, the introduction of the polynucleotide into the host cell results in the production of the polypeptide and improvement of its activity.

[0091] The optimization of codons of polynucleotides encoding polypeptides described in 7) above may be performed by optimizing endogenous polynucleotide codons so as to increase transcription or translation within the host cell, or by optimizing exogenous polynucleotide codons so as to perform optimized transcription or translation within the host cell.

[0092] The 8) analysis of the tertiary structure of the polypeptide, and the selection and modification or chemical modification of exposed portions may be carried out, for example, by comparing the sequence information of the polypeptide to be analyzed with a database in which sequence information of known proteins is stored, determining candidate template proteins according to the degree of sequence similarity, confirming the structure based on these, and selecting and modifying or chemically modifying exposed portions.

[0093] 9) The regulation of the intracellular location of a protein (polypeptide) may be carried out by targeting the protein (polypeptide) to a specific intracellular organelle or specific intracellular space. For example, this can be done by adding or removing a leader sequence that functions to target the protein (polypeptide), thereby targeting the periplasm or cytoplasm, but is not limited to these.

[0094] As one example, the enhancement of the activity of the O-phosphoserine sulfhydrase protein of this application may be carried out by modifying the expression regulatory region of the gene on the chromosome encoding the polypeptide of 2) above, by modifying the amino acid sequence or polynucleotide sequence of 4) and 5) above, or by a combination thereof.

[0095] In any of the embodiments described above, the activity enhancement of the O-phosphoserine sulfhydrase protein of this application may be carried out by including an enhanced gene expression regulatory sequence upstream of the cysM gene encoding it. For example, the gene expression regulatory sequence is a promoter, but is not limited to this. In any other embodiment described above, the activity enhancement of the O-phosphoserine sulfhydrase protein of this application may be carried out by modifying the amino acid sequence of the O-phosphoserine sulfhydrase protein or the polynucleotide sequence encoding it. Modification of the amino acid sequence or polynucleotide sequence includes deletions, substitutions, and insertions. The modifications to the sequence are as described above.

[0096] Such enhancement of polypeptide activity is achieved by increasing the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in the wild-type or pre-modification microbial strain, or by increasing the amount of product produced from the polypeptide, but is not limited to these methods.

[0097] In the microorganisms of this application, modification of part or all of the polynucleotides can be induced by (a) homologous recombination using a chromosome introduction vector in the microorganism, or genome editing using an engineered nuclease (e.g., CRISPR-Cas9), and / or (b) light and / or chemical treatment such as ultraviolet light or radiation. The methods for modifying part or all of the genes include methods using DNA recombination technology. For example, deletion of part or all of the gene can be achieved by introducing a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism to induce homologous recombination. The introduced nucleotide sequence or vector contains, but is not limited to, a dominant selection marker.

[0098] In this application, "weakening" of the activity of polypeptides (including proteins identified by the names of each enzyme) is a concept that encompasses all cases where the activity is reduced compared to endogenous activity or where the activity is eliminated. The term "weakening" is used interchangeably with terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0099] The aforementioned weakening includes at least one of the following: the activity of the polypeptide itself is reduced or eliminated compared to the original polypeptide activity of the microorganism due to mutations in the polynucleotide encoding the polypeptide; the overall degree and / or concentration (expression level) of polypeptide activity within the cell is reduced compared to the natural strain due to inhibition of the expression of the gene encoding the polynucleotide or inhibition of translation into the polypeptide; there is no expression of the polynucleotide at all; and even if the polynucleotide is expressed, there is no polypeptide activity. "Inactivation," "deficiency," "reduction," "downregulation," "decrease," or "attenuation" of polypeptide activity compared to endogenous activity means that it is reduced compared to the activity of the specific polypeptide that was originally present in the parent strain or unmodified microorganism before the trait change.

[0100] Such weakening of polypeptide activity is not limited to these methods and can be achieved by applying various methods well known in the field (e.g., Non-Patent Documents 16, 17, etc.).

[0101] Specifically, weakening the activity of a polypeptide in this application involves: 1) deleting all or part of the gene encoding the polypeptide; 2) modifying the expression regulatory region (or expression regulatory sequence) so that the expression of the gene encoding the polypeptide is reduced; 3) modifying the amino acid sequence constituting the polypeptide so that the activity of the polypeptide is deleted or weakened (for example, deleting / substituting / adding one or more amino acids to the amino acid sequence); 4) modifying the gene sequence encoding the polypeptide so that the activity of the polypeptide is deleted or weakened (for example, modifying the gene sequence to encode a polypeptide that has been modified so that the activity of the polypeptide is deleted or weakened). 1) Deleting / substituting / adding one or more nucleic acid bases in the nucleic acid base sequence of the polypeptide gene; 5) Modifying the base sequence encoding the start codon or 5'UTR region of the polypeptide-encoding gene transcript; 6) Introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementaryly to the polypeptide-encoding gene transcript; 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of the polypeptide-encoding gene so that a secondary structure is formed that prevents ribosome attachment; 8) Adding a promoter to the 3' end of the open reading frame (ORF) of the polypeptide-encoding gene sequence to reverse transcription (Reverse transcription engineering, RTE); 9) Regulating the cellular localization of the protein (polypeptide); or 10) Combining two or more of the above 1) to 9), but not being particularly limited thereto.

[0102] For example, the deletion of part or all of the gene encoding the polypeptide described in 1) above may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide within the chromosome, or by substituting it with a polynucleotide or marker gene in which some nucleotides are deleted.

[0103] Furthermore, modifying the regulatory expression region (or regulatory expression sequence) described in 2) above may be carried out by causing a mutation in the regulatory expression region (or regulatory expression sequence) through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting it with a sequence having lower activity. The regulatory expression region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence that regulates the termination of transcription and translation.

[0104] Modifying the amino acid sequence or polynucleotide sequence described in 3) and 4) above is carried out by introducing a sequence mutation through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide, so as to weaken the activity of the polypeptide, or by substituting it with an amino acid sequence or polynucleotide sequence modified to have lower activity, or an amino acid sequence or polynucleotide sequence modified to eliminate activity, but is not limited to these methods. For example, gene expression can be inhibited or weakened by introducing a mutation into a polynucleotide sequence to form a stop codon, but is not limited to these methods.

[0105] Modification of the start codon or 5'UTR region of the gene transcript encoding the polypeptide (as described in 5) above is performed, for example, by substituting it with a nucleotide sequence encoding another start codon that has a lower polypeptide expression rate compared to the endogenous start codon, but is not limited to this.

[0106] The introduction of an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding the polypeptide (6) above may be carried out, for example, as described in Non-Patent Document 18.

[0107] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence in the polypeptide-coding gene so that a secondary structure is formed that makes ribosome attachment impossible may be done by making mRNA translation impossible or slowing down the rate of mRNA translation.

[0108] Furthermore, the process of adding a promoter to the 3' end of the ORF (open reading frame) of the gene sequence encoding the polypeptide (reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the gene transcript encoding the polypeptide and reducing its activity.

[0109] 9) The regulation of the intracellular location of a protein (polypeptide) may be carried out by targeting the protein (polypeptide) to a specific intracellular organelle or specific intracellular space. For example, this can be done by adding or removing a leader sequence that functions to target the protein (polypeptide), thereby targeting the periplasm or cytoplasm, but is not limited to these.

[0110] Such weakening of polypeptide activity is achieved by reducing the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in the wild-type or pre-modification microbial strain, or by reducing the amount of product produced from the polypeptide, but is not limited to these methods.

[0111] As previously mentioned, the O-phosphoserine sulfhydrase variant, polynucleotides, cysteine, and other components in the microorganism of this application are as described above.

[0112] A further aspect of this application provides a method for producing cysteine ​​and its derivatives, comprising the step of reacting a mixture of O-phosphoserine and a sulfide with the O-phosphoserine sulfhydrylase variant of this application.

[0113] The method for producing cysteine ​​and its derivatives according to this application may include, prior to the above method, a step of culturing a microorganism containing the O-phosphoserine sulfhydrase variant, the polynucleotide, or the vector in a culture medium.

[0114] In this application, "cultivation" means growing the microorganisms of this application under appropriately adjusted environmental conditions. The cultivation process of this application can be carried out using suitable culture media and cultivation conditions known in the art. Such a cultivation process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the cultivation is batch, continuous, and / or fed-batch culture, but is not limited to these.

[0115] In this application, "culture medium" refers to a substance that is a mixture mainly composed of nutrients necessary for culturing the microorganisms of this application, and supplies nutrients and growth factors, including water, which are essential for survival and growth. Specifically, the culture medium and other culture conditions used for culturing the microorganisms of this application may be any that are normally used for culturing microorganisms, and the microorganisms of this application can be cultured in a normal culture medium containing a suitable carbon source, nitrogen source, phosphorus source, inorganic compounds, amino acids and / or vitamins, under aerobic conditions, with the temperature, pH, etc. adjusted.

[0116] In this application, the carbon source can be carbohydrates such as glucose, sucrose, lactose, fructose, sucrose, and maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, and citric acid; and amino acids such as glutamic acid, methionine, and lysine. In addition, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, and corn maceration liquid can be used. Specifically, carbohydrates such as glucose and sterilized pre-treated molasses (i.e., molasses converted to reducing sugars) can be used, and any other carbon source in an appropriate amount may be used. These carbon sources can be used individually or in combination of two or more, but are not limited to these uses.

[0117] As the nitrogen source, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate can be used, as well as organic nitrogen sources such as amino acids like glutamic acid, methionine, and glutamine, peptone, NZ-amine, meat extracts, yeast extracts, malt extracts, corn maceration liquid, casein hydrolysates, fish or their decomposition products, defatted soybean cake or its decomposition products. These nitrogen sources can be used individually or in combination of two or more, but are not limited to these uses.

[0118] As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or equivalent sodium-containing salts can be used. As inorganic compounds, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc., can be used, and in addition, amino acids, vitamins, and / or suitable precursors can be used. These components or precursors can be added to the culture medium in batches or continuously, but are not limited to these.

[0119] Furthermore, the pH of the culture medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the culture medium in a suitable manner during the cultivation of the microorganisms of this application. In addition, during cultivation, the formation of bubbles can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, oxygen or oxygen-containing gas may be injected into the culture medium to maintain an aerobic state, and to maintain an anaerobic and microaerobic state, it is not necessary to inject gas, but nitrogen, hydrogen, or carbon dioxide gas may be injected, but the invention is not limited to these.

[0120] In the culture described in this application, the culture temperature is maintained at 20-45°C, specifically 25-40°C, and the culture is performed for approximately 10-160 hours, but is not limited to these values.

[0121] The O-phosphoserine sulfhydrase mutant produced by the culture described in this application remains in the cells.

[0122] The cysteine ​​production method of this application may further include, for example, the steps of preparing the microorganism of this application, preparing a culture medium for culturing the strain, or a combination thereof (in any order) before the culturing step.

[0123] The cysteine ​​production method of this application may further include the step of recovering the O-phosphoserine sulfhydrase mutant from the culture medium used for the culture (the culture medium in which the culture was performed) or from the microorganism of this application.

[0124] The aforementioned recovery may involve collecting the target mutant using a suitable method known in the art, depending on the microorganism culture method of this application, such as batch, continuous, or fed-batch culture. For example, various chromatography methods such as centrifugation, filtration, crystallization, treatment with protein precipitants (salting-out method), extraction, sonication, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof can be used to recover the target mutant from the microorganism using a suitable method known in the art.

[0125] Furthermore, a purification step may be included after the recovery step. The purification can be carried out by a preferred method known in the art. For example, if both the recovery step and the purification step are included, the recovery step and the purification step may be carried out sequentially or discontinuously, regardless of the order, simultaneously, or integrated as a single step, but are not limited thereto.

[0126] In the method of this application, the O-phosphoserine sulfhydrase variant, polynucleotide, vector, bacterial strain, etc., are as described above.

[0127] The sulfide may be supplied not only as a solid commonly used in the art, but also in liquid or gaseous form depending on differences in pH, pressure, and solubility, and may be any sulfide that can be converted to a thiol group (SH group) in the form of sulfide (S2-), thiosulfate (S2032-), etc. Specifically, examples include, but are not limited to, Na2S, NaSH, (NH4)2S, H2S, and Na2S2O3, which donate a thiol group to OPS. The reaction is a reaction that produces one cysteine ​​or cysteine ​​derivative by donating one thiol group to one OPS functional group, and the amount of sulfide added in the reaction is 0.1 to 3 times the molar concentration of OPS, specifically 1 to 2 times, but is not limited to these amounts.

[0128] Furthermore, this application may further include a step of recovering the cysteine ​​produced in the reaction step. Here, the target cysteine ​​can be separated and purified and recovered from the reaction solution using a suitable reaction known in the art.

[0129] In this application, "derivative" means a similar compound obtained by chemically altering a part of a compound, and generally refers to a compound in which a hydrogen atom or a specific group of atoms in the compound is substituted with another atom or group of atoms.

[0130] In this application, "cysteine ​​derivative" means a compound in which a hydrogen atom or a specific group of atoms of cysteine ​​is substituted with another atom or group of atoms. Examples include forms in which other atoms or groups of atoms are bonded to the nitrogen atom of the amino group (-NH2) or the sulfur atom of the thiol group (-SH) of cysteine. Examples include, but are not limited to, NAC (N-acetylcysteine), SCMC (S-Carboxymetylcysteine), BOC-CYS(ME)-OH, (R)-S-(2-Amino-2-carboxyethyl)-L-homocysteine, (R)-2-Amino-3-sulfopropionic acid, D-2-Amino-4-(ethylthio)butyric acid, 3-sulfino-L-alanine, Fmoc-Cys(Boc-methyl)-OH, Seleno-L-cystine, S-(2-Thiazolyl)-L-cysteine, S-(2-Thienyl)-L-cysteine, and S-(4-Tolyl)-L-cysteine.

[0131] If cysteine ​​is produced by the method of this application, the conversion to a cysteine ​​derivative may be easily carried out by methods well known in the art, and may be converted to various cysteine ​​derivatives.

[0132] In this application, the method for producing cysteine ​​derivatives may further include the step of converting the generated cysteine ​​into a cysteine ​​derivative, as described above.

[0133] Specifically, in this application, the method for producing a cysteine ​​derivative may include the steps of producing cysteine ​​by the method of this application described above, and converting the cysteine ​​produced as described above into a cysteine ​​derivative.

[0134] As mentioned above, the step of converting the produced cysteine ​​into a cysteine ​​derivative can be carried out by methods well known in the art. For example, cysteine ​​can be reacted with an acetylation agent to synthesize NAC (N-acetylcysteine) by methods known in the art, or cysteine ​​can be reacted with haloacetic acid under basic conditions to synthesize SCMC (S-Carboxymetylcysteine), but is not limited to these methods.

[0135] The aforementioned cysteine ​​derivatives are primarily used as pharmaceutical raw materials in cough suppressants, antitussives, and treatments for bronchitis, bronchial asthma, and pharyngitis, but are not limited to these uses.

[0136] Further embodiments of this application provide a composition for the production of cysteine ​​or a cysteine ​​derivative, comprising the O-phosphoserine sulfhydrase variant of this application, a polynucleotide encoding the same, a vector containing the polynucleotide, or a microorganism containing the polynucleotide of this application, a culture medium in which the microorganism is cultured, or a combination of at least two of these.

[0137] The composition of this application may further contain any suitable excipients commonly used in compositions for the production of cysteine ​​or cysteine ​​derivatives. Examples of such excipients include, but are not limited to, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0138] Further embodiments of this application provide a method for producing an O-phosphoserine-producing recombinant microorganism, comprising the step of introducing the O-phosphoserine sulfhydrase variant of this application, the polynucleotide of this application, or the vector of this application into a microorganism.

[0139] Further embodiments of this application provide the use of the O-phosphoserine sulfhydrase variant of this application for the production of cysteine ​​or cysteine ​​derivatives.

[0140] Further embodiments of this application provide the use of O-phosphoserine-producing recombinant microorganisms comprising the O-phosphoserine sulfhydrase variant of this application, the polynucleotide of this application, or the vector of this application for the production of cysteine ​​or cysteine ​​derivatives.

[0141] The O-phosphoserine sulfhydrase variant, polynucleotide, vector, cysteine, cysteine ​​derivative, and microorganism of the above embodiment are as described above. [Examples]

[0142] The present application will be described in more detail below with reference to examples. However, these examples are merely preferred embodiments illustrating the present application, and the application is not limited thereto. Technical matters not described herein can be fully understood and readily implemented by a skilled technician in the art of this application or a similar art. [Examples]

[0143] Selection of CysM variants Based on CysM (Msm-OPSS) (SEQ ID NO: 1), derived from wild-type Mycobacterium smegmatics, which exhibits activity to convert O-phosphoserine (OPS) to cysteine ​​and possesses O-phosphoserine sulfhydrylase (OPSS) activity, we performed random mutagenesis using an error-prone PCR kit (Diversity PCR random mutagenesis kit, Clontech) to create a cysM gene mutant library encoding CysM mutants. This mutant is Msm-T-HA2 (Patent Document 8) (SEQ ID NO: 11), which is a mutant of CysM (Msm-OPSS) (SEQ ID NO: 1) derived from wild-type Mycobacterium smegmatics, in which five amino acid residues are deleted from the C-terminus and proline, the 77th amino acid residue from the N-terminus, is replaced with serine.

[0144] Specifically, random mutagenesis PCR was performed using the base sequence encoding Msm-T-HA2 (SEQ ID NO: 12) as a template and primer pairs of SEQ ID NOs: 16 and 17. A diversity PCR random mutagenesis kit was used, and the PCR conditions were as follows: denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 1 minute, repeated 20 cycles, followed by polymerization at 72°C for 5 minutes.

[0145] As described above, the amplified mutant gene fragments were introduced into pUC (Non-Patent Literature 19) vectors (SEQ ID NO: 15) (pUC_PcysK) containing the cysK promoter. To prepare the pUC_PcysK vector, PCR was performed using E. coli ATCC27325 chromosomal DNA as a template and primer pairs of SEQ ID NOs: 18 and 19 to obtain the cysK promoter fragment. The PCR conditions were denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 1 minute, for 30 cycles, followed by polymerization at 72°C for 5 minutes. As described above, the amplified cysK promoter fragments were cloned into pUC vectors cleaved with restriction enzymes NheI and HindIII using an infusion cloning kit (Clontech Laboratories, Inc.) to prepare the pUC_PcysK vector. As described above, the amplified mutant gene fragment was cloned into a pUC_PcysK vector, which had been cleaved with the restriction enzyme EcoRV, using an infusion cloning kit. Cloning was performed by reacting the vectors at 50°C for 60 minutes. This process created a pUC_PcysK-cysM gene mutant plasmid library.

[0146] The primer sequences used here are shown in Table 1.

[0147] [Table 1]

[0148] As described above, the prepared enzyme expression vector was used to transform E. coli K12 strain by electroporation (Non-Patent Literature 20). Homologous recombination on the chromosome was induced (Non-Patent Literature 20). Strains in which the vector was inserted into the chromosome by homologous sequence recombination were selected from LB medium containing 25 mg / L of kanamycin. DNA was obtained from the three selected E. coli transformant strains using a DNA-spin plasmid DNA purification kit (Intron) according to the manufacturer's protocol, and the nucleotide sequence was analyzed by sequencing. The nucleotide sequence analysis confirmed that the CysM mutants derived from the three E. coli transformant strains consisted of the amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, respectively. In the CysM mutant consisting of the amino acid sequence of SEQ ID NO: 5, it was confirmed that the 77th amino acid residue, proline, of the parent sequence, Mycobacterium smegmatis-derived CysM mutant was replaced with alanine, and the 218th amino acid residue, alanine, was replaced with glycine. In the CysM mutant consisting of the amino acid sequence of Sequence ID No. 7, it was confirmed that the 55th amino acid residue, alanine, of the parent sequence derived from Mycobacterium smegmatis is replaced with valine, the 77th amino acid residue, proline, is replaced with alanine, and the 218th amino acid residue, alanine, is replaced with glycine. In the CysM mutant consisting of the amino acid sequence of Sequence ID No. 9, it was confirmed that the 7th amino acid residue, leucine, of the parent sequence derived from Mycobacterium smegmatis is replaced with proline, the 77th amino acid residue, proline, is replaced with alanine, and the 218th amino acid residue, alanine, is replaced with glycine. [Examples]

[0149] Creation of CysM mutant expression vectors and expressing bacterial strains 2-1. Construction of CysM mutant (P77A or P77S) expression vectors and expressing bacterial strains To create a strain expressing a CysM mutant containing the mutation identified in Example 1, a CysM mutant expression vector was first prepared.

[0150] Specifically, to construct a CysM(P77A) expression vector, the nucleotide sequence (SEQ ID NO: 14) encoding Msm-T (Patent Document 8) (SEQ ID NO: 13), a mutant of CysM(Msm-OPSS) derived from wild-type Mycobacterium smegmatics with five amino acid residues deleted from the C-terminus, was used as a template. The upstream fragment of the cysM(P77A) gene was obtained by PCR using primer pairs of SEQ ID NOs: 16 and 20, and the downstream fragment of the cysM(P77A) gene was obtained by PCR using the same template and primer pairs of SEQ ID NOs: 17 and 21. As a control group, to construct a CysM(P77S)(Msm-T-HA2) expression vector, Msm-T-HA2 was used as a template, and the cysM(P77S) gene fragment was obtained by PCR using primer pairs of SEQ ID NOs: 16 and 17. The PCR conditions involved denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 1 minute, repeated 30 times, and then polymerization at 72°C for 5 minutes.

[0151] As described above, the secured gene fragments were introduced into pUC vectors containing the cysK promoter (pUC_PcysK). The vector prepared in Example 1 was used as the pUC_PcysK vector. First, the secured cysM(P77A) and cysM(P77S) gene fragments were cloned into pUC_PcysK vectors that had been cleaved with the restriction enzyme EcoRV using an infusion cloning kit. Cloning was carried out by reacting at 50°C for 60 minutes.

[0152] The primer sequences used here are shown in Table 2.

[0153] [Table 2]

[0154] By doing so, we obtained the pUC_PcysK-cysM(P77A) and pUC_PcysK-cysM(P77S)(Msm-T-HA2) vectors, respectively. These vectors were then used to transform the E. coli K12 strain in the same manner as in Example 1, and CysM mutant (P77A or P77S) expressing strains were secured.

[0155] 2-2. Construction of CysM mutant (P77A / A218G) expression vector and expression strain To create a strain expressing a CysM mutant containing the two combinations of mutations identified in Example 1, a CysM mutant expression vector was first prepared.

[0156] Specifically, to construct a cysM(P77A / A218G) expression vector, the upstream fragment of the cysM(P77A / A218G) gene was secured by PCR using cysM-4 (SEQ ID NO: 3) as a template and primer pairs of SEQ ID NO: 16 and 22. The downstream fragment of the cysM(P77A / A218G) gene was then secured by PCR using the same template and primer pairs of SEQ ID NO: 17 and 23. The PCR conditions were denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, polymerization at 72°C for 1 minute, repeated for 30 cycles, and then polymerization at 72°C for 5 minutes.

[0157] The primer sequences used here are shown in Table 3.

[0158] [Table 3]

[0159] As described above, the secured gene fragment was cloned into the pUC_PcysK vector of Example 1, which had been cleaved with the restriction enzyme EcoRV, using an infusion cloning kit. Cloning was carried out at 50°C for 60 minutes. In this way, the pUC_PcysK-cysM(P77A / A218G) vector was obtained, and this vector was transformed into the E. coli K12 strain in the same manner as in Example 1 to secure a CysM mutant (P77A / A218G) expressing strain. [Examples]

[0160] Evaluation of cysteine ​​conversion ability of CysM mutant-expressing bacterial strains. 3-1. Evaluation of cysteine ​​conversion ability of CysM mutant (P77A or P77S) expressing strains. The pET system manual (novagen) was used as the method for expressing the enzyme. Specifically, single colonies of each strain from Example 2-1 were selected from LB agar plates containing kanamycin, inoculated into 5 ml of LB liquid medium, and cultured at 37°C and 200 rpm for 16 hours. Next, these were inoculated into LB liquid medium containing kanamycin and cultured at 33°C and 200 rpm for 18 hours. Xylene equivalent to 2% of the culture medium was added to the culture medium to obtain the enzyme. The enzymes obtained in this process were identified by 14% SDS-PAGE (Figure 1), and soluble CysM (Msm-OPSS) mutants were obtained.

[0161] As described above, the soluble CysM mutants K12 / pUC_Ppro-cysM(P77S)(Msm-T-HA2) and K12 / pUC_Ppro-cysM(P77A) were used to comparatively analyze the enzyme activity that converts OPS to cysteine. The conditions for evaluating cysteine ​​synthesis activity (CysM enzyme assay) are shown in Table 4.

[0162] [Table 4]

[0163] In Table 1, the remaining solution after removing the enzyme was mixed and incubated at 37°C for 5 minutes. Then, 25 μL of xylene-treated soluble CysM mutant was added and reacted at 37°C for 10 minutes. 100 μL of the completed reaction solution was taken and reacted with 900 μL of a mixture of 100 mM Tris-HCl (pH 8.5) and 10 mM DTT (dithiothreitol) for 30 minutes, after which the reaction was stopped with 0.1 M HCl. The cysteine ​​concentration in the reaction solution was quantified by HPLC, and the cysteine ​​conversion rate and specific activity (cysteine ​​concentration / time / enzyme amount) after 10 minutes of reaction were compared to evaluate the cysteine ​​synthesis titer. The results are shown in Table 5.

[0164] [Table 5]

[0165] As can be seen from the results in Table 5, the cysM(P77A)-expressing strain showed a 46% increase in specific activity and improved cysteine ​​synthesis activity compared to the control strain, cysM(P77S)(Msm-T-HA2)-expressing strain.

[0166] 3-2. Evaluation of cysteine ​​conversion ability of CysM mutant (P77A / A218G) expressing strains Using the soluble CysM mutant K12 / pUC_PcysK-cysM(P77A / A218G) obtained by the method of Example 2-2, the enzyme activity of converting OPS to cysteine ​​was comparatively analyzed. The experiment was carried out in the same manner as in Example 3-1. The cysteine ​​concentration in the reaction solution was quantified by HPLC, and the cysteine ​​conversion rate and specific activity (cysteine ​​concentration / time / enzyme amount) after 10 minutes of reaction were compared to evaluate the cysteine ​​synthesis titer. The results are shown in Table 6.

[0167] [Table 6]

[0168] As can be seen from the results in Table 6, the cysM(P77A / A218G) expressing strain showed a 13% increase in specific activity and improved cysteine ​​synthesis activity compared to the control strain, the cysM(P77A) expressing strain.

[0169] 3-3. Evaluation of cysteine ​​conversion ability of strains expressing CysM mutants (L7P / P77A / A218G or A55V / P77A / A218G) In the library prepared as a result of Example 1, the enzyme activity of the CysM mutants K12 / pUC_PcysK-cysM(P77A / A218G), K12 / pUC_PcysK-cysM(L7P / P77A / A218G), and K12 / pUC_PcysK-cysM(A55V / P77A / A218G) obtained by screening was comparatively analyzed to convert OPS to cysteine. The experiment was performed in the same manner as in Example 3-1. The cysteine ​​concentration in the reaction solution was quantified by HPLC, and the cysteine ​​conversion rate and specific activity (cysteine ​​concentration / time / enzyme amount) after 10 minutes of reaction were compared to evaluate the cysteine ​​synthesis titer. The results are shown in Table 7.

[0170] [Table 7]

[0171] As can be seen from the results in Table 7, the cysM(L7P / P77A / A218G) expressing strain and the cysM(A55V / P77A / A218G) expressing strain showed improved cysteine ​​synthesis activity, with specific activity increasing by 41% and 72.5%, respectively, compared to the control strain, the cysM(P77A / A218G) expressing strain.

[0172] From the above explanation, a person skilled in the art to which this application pertains will understand that this application can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the above embodiments are merely illustrative and not limiting. This application should be interpreted as including all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

Claims

1. In the amino acid sequence of Sequence ID No. 1, 0 to 7 amino acid residues are deleted from the C-terminus, and the amino acid corresponding to position 77 is replaced with alanine. O-phosphoserine sulfhydrase mutant.

2. The O-phosphoserine sulfhydrase mutant has five amino acid residues deleted from the C-terminus in the amino acid sequence of SEQ ID NO:

1. A variant according to claim 1.

3. The O-phosphoserine sulfhydrase variant has 80% or more sequence identity or homology to SEQ ID NO:

1. A variant according to claim 1.

4. The O-phosphoserine sulfhydrase variant consists of the amino acid sequence of SEQ ID NO:

3. A variant according to claim 1.

5. The O-phosphoserine sulfhydrase mutant is further characterized in which the amino acid corresponding to the position selected from the group consisting of the 7th, 55th, 218th, and combinations thereof in the amino acid sequence of Sequence ID No. 1 is substituted with another amino acid. A variant according to claim 1.

6. In the amino acid sequence of Sequence ID No. 1, the amino acid corresponding to the 7th position is substituted with proline. The variant according to claim 5.

7. In the amino acid sequence of Sequence ID No. 1, the amino acid corresponding to the 55th position is substituted with valine. The variant according to claim 5.

8. In the amino acid sequence of Sequence ID No. 1, the amino acid corresponding to the 218th position is substituted with glycine. The variant according to claim 5.

9. The O-phosphoserine sulfhydrase variant consists of at least one amino acid sequence selected from the group consisting of SEQ ID NOs. 5, SEQ ID NOs. 7, and SEQ ID NOs.

9. The variant according to claim 5.

10. Encoding the O-phosphoserine sulfhydrase variant according to any one of claims 1 to 9, Polynucleotide.

11. A polynucleotide comprising the O-phosphoserine sulfhydrase variant described in any one of claims 1 to 9 or the polynucleotide encoding the same, Microorganisms.

12. The aforementioned microorganism exhibits improved cysteine ​​production capacity compared to a microorganism containing wild-type O-phosphoserine sulfhydrylase having the amino acid sequence of Sequence ID No. 1 or a polynucleotide encoding it. The microorganism according to claim 11.

13. The aforementioned microorganism is a microorganism of the genus Escherichia. The microorganism according to claim 12.

14. The aforementioned microorganism of the genus Escherichia is Escherichia coli. The microorganism according to claim 13.

15. The step of reacting a mixture of O-phosphoserine and a sulfide with the O-phosphoserine sulfhydrase variant described in any one of claims 1 to 9, A method for producing cysteine ​​or its derivatives.

16. The O-phosphoserine is purified O-phosphoserine, or a microbial fermentation liquid containing O-phosphoserine. The method according to claim 15.

17. The sulfide is Na ２ S, NaSH, (NH ４ ) ２ S, H ２ S, S ２ O ３ and Na ２ S ２ O ３ and is at least one selected from the group consisting of The method according to claim 15.

18. In the amino acid sequence of Sequence ID No. 1, 0 to 7 amino acid residues are deleted from the C-terminus, and the amino acid corresponding to position 77 is replaced with alanine. Use of O-phosphoserine sulfhydrase variants in the production of cysteine ​​or its derivatives.

19. A vector comprising an O-phosphoserine sulfhydrase variant according to any one of claims 1 to 9, a polynucleotide encoding the same, or the polynucleotide said, Use in the production of cysteine ​​or its derivatives by microorganisms.

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