O-acylhomoserine sulfhydrylase mutants and uses thereof

Abstract

The present application relates to O-acylhomoserine sulfhydrylase mutants and uses thereof.

Description

[Technical Field]

[0001] The present application relates to O-acylhomoserine sulfhydrylase mutants and uses thereof. [Background technology]

[0002] Methionine is an amino acid essential for protein synthesis and is widely used in feed and food additives, as well as in food ingredients, plant nutrition, and pharmaceutical applications. Methionine plays an important role in the transmethylation reaction in vivo, converting it to cysteine ​​via homocysteine ​​and cystathionine. It also plays an important role in the enzyme conversion process related to transsulfuration, as it provides sulfur.

[0003] Homocysteine ​​is an intermediate metabolic product produced in the metabolism of methionine in the body. It is a non-proteinogenic amino acid and is generally converted to cysteine ​​or methionine in the body and exists only in small amounts. Homocysteine ​​functions as a strong oxidizing agent, and excessive accumulation in the body can cause problems such as cytotoxicity. However, it is widely used in feed, food additives, food ingredients, plant nutrients, biopolymers, and pharmaceuticals.

[0004] A technique for producing methionine and homocysteine ​​by a biological method is disclosed in Patent Document 1. This method is called a two-stage method, and includes a precursor production method by fermentation and a subsequent enzymatic conversion production method.

[0005] By developing this two-step process, we were able to solve the problems of substrate toxicity specific to sulfide, feedback regulation of the bacterial strain by methionine and SAM, and intermediate product decomposition activity specific to cystathionine gamma synthase, O-succinylhomoserine sulfhydrylase, and O-acetylhomoserine sulfhydrylase. Furthermore, because it can selectively produce only L-methionine, it is an excellent process compared to conventional chemical synthesis processes that simultaneously produce DL-methionine. Furthermore, it is an excellent process in that organic acids, more specifically succinic acid and acetic acid, are simultaneously produced as by-products in the same reaction.

[0006] In the two-step enzymatic conversion process, an enzyme having the activity of cystathionine gamma synthase, O-succinylhomoserine sulfhydrylase, or O-acetylhomoserine sulfhydrylase is used. In such two-step enzymatic conversion processes, an enzyme that can efficiently produce a product is required. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2008 / 013432 [Patent Document 2] U.S. Patent No. 7,662,943 [Patent Document 3] U.S. Patent No. 10,584,338 [Patent Document 4] U.S. Patent No. 10,273,491 [Patent Document 5] International Publication No. 2010 / 098629

Patent document 6

Patent document 7

Non-licensed literature

[0008]

Non-licensed literature 1

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

[0009] The present application aims to provide an O-acylhomoserine sulfhydrylase mutant in which at least one of the amino acids corresponding to positions 4, 90, 106, 242, 261, 290, and 395 from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid.

[0010] Another object of the present application is to provide a polynucleotide encoding the mutant.

[0011] Furthermore, the present application aims to provide a microorganism comprising an O-acylhomoserine sulfhydrylase mutant in which at least one of the amino acids corresponding to positions 4, 90, 106, 242, 261, 290, and 395 from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid, or a polynucleotide encoding the same.

[0012] Furthermore, the present application aims to provide a method for producing L-methionine or a precursor thereof, which comprises a step of contacting an O-acylhomoserine sulfhydrylase mutant in which at least one of the amino acids corresponding to positions 4, 90, 106, 242, 261, 290, and 395 from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid, or a microorganism containing the mutant or a polynucleotide encoding the mutant, with O-acylhomoserine. [Means for solving the problem]

[0013] These will be described in detail below. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. Furthermore, this application is not limited to the specific descriptions below. Furthermore, many papers and patent documents are referenced throughout this specification, and citations thereof are provided. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety, thereby more clearly explaining the state of the art to which this application pertains and the contents of this application.

[0014] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present application described herein which equivalents are intended to be encompassed by this application.

[0015] One aspect of the present application provides an O-acylhomoserine sulfhydrylase mutant in which at least one of the amino acids corresponding to positions 4, 90, 106, 242, 261, 290, and 395 from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid.

[0016] In the present application, "acylhomoserine" refers to a compound in which an acyl group is bound to homoserine, and includes both succinylhomoserine and acetylhomoserine. For example, the acylhomoserine may be O-succinylhomoserine or O-acetylhomoserine.

[0017] In the present application, O-acylhomoserine sulfhydrylase may be a protein having at least one activity of O-acetylhomoserine sulfhydrylase, O-acetylhomoserine transsulfurase, O-succinylhomoserine sulfhydrylase, and O-succinylhomoserine transsulfurase.

[0018] In this application, "sulfhydrylation" is used interchangeably with "sulfhydration" and refers to a reaction that provides a sulfhydryl (-SH) functional group to a specific molecule. For the purposes of this application, the term refers to, but is not limited to, a reaction in the process of methionine synthesis. The enzyme involved in "sulfhydration" is also called "sulfhydrylase."

[0019] In the present application, "O-acetylhomoserine" refers to the first specific intermediate in methionine biosynthesis found in microorganisms, and is produced from L-homoserine and acetyl-CoA catalyzed by homoserine acetyltransferase at the branch point of threonine biosynthesis.

[0020] In this application, "O-acetylhomoserine sulfhydrylase" refers to an enzyme that has the activity of synthesizing L-methionine using O-acetylhomoserine, which is a precursor of L-methionine, and methyl mercaptan. O-acetylhomoserine sulfhydrylase has the following three activities: L-cysteine ​​+ O-acetylhomoserine => acetic acid + cystathionine Sulfide (HS-) + O-acetylhomoserine => Acetate + Homocysteine Methyl mercaptan + O-acetylhomoserine => acetic acid + L-methionine

[0021] In the present application, "O-succinylhomoserine sulfhydrylase" refers to an enzyme that has the activity of synthesizing L-methionine using O-succinylhomoserine and methyl mercaptan. O-succinylhomoserine sulfhydrylase has the following three activities: L-cysteine ​​+ O-succinylhomoserine => succinic acid + cystathionine Sulfide (HS-) + O-succinylhomoserine => succinic acid + homocysteine Methyl mercaptan + O-succinylhomoserine => succinic acid + L-methionine

[0022] Some enzymes designated as O-acetylhomoserine sulfhydrylases may use O-succinylhomoserine as a substrate and may also have O-succinylhomoserine sulfhydrylase activity. In one embodiment, the O-acylhomoserine sulfhydrylase of the present application has both O-acetylhomoserine sulfhydrylase and O-succinylhomoserine sulfhydrylase activity, but is not necessarily limited thereto.

[0023] The O-acylhomoserine sulfhydrylase of the present application can be an O-acylhomoserine sulfhydrylase or a polypeptide having the activity of an O-acylhomoserine sulfhydrylase that is modified to produce the O-acylhomoserine sulfhydrylase provided by the present application.

[0024] Specifically, the polypeptide may be a naturally occurring or wild-type polypeptide, a mature polypeptide thereof, or a mutant or functional fragment thereof, and may be any polypeptide that serves as a parent for the O-acylhomoserine sulfhydrylase mutant of the present application.

[0025] In one embodiment, the O-acylhomoserine sulfhydrylase of the present application may be derived from a microorganism of the Rhodobacteraceae family. In one embodiment, the O-acylhomoserine sulfhydrylase may be derived from a microorganism of the genus Rhodobacter. In another embodiment, the O-acylhomoserine sulfhydrylase may be derived from a microorganism of the genus Cereibacter. In any of the above embodiments, the O-acylhomoserine sulfhydrylase of the present application may be derived from Rhodobacter sphaeroides. On the other hand, since the name of Rhodobacter sphaeroides has been changed to Luteovulum sphaeroides and Cereibacter sphaeroides, the microorganism is not limited to the above names, and any microorganism classified in the same taxon as Rhodobacter sphaeroides is acceptable.

[0026] As one example, in an O-acylhomoserine sulfhydrylase to be mutated in the present application, the amino acid corresponding to position 4 of SEQ ID NO: 1 may be alanine (A), the amino acid corresponding to position 90 may be aspartic acid (D), the amino acid corresponding to position 106 may be threonine (T), the amino acid corresponding to position 242 may be valine (V), the amino acid corresponding to position 261 may be leucine (L), the amino acid corresponding to position 290 may be histidine (H), and / or the amino acid corresponding to position 395 may be alanine (A).

[0027] As one example, the O-acylhomoserine sulfhydrylase of the present application may be the polypeptide of SEQ ID NO: 1. In any of the above-mentioned examples, the O-acylhomoserine sulfhydrylase of the present application is a polypeptide having about 60%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to the polypeptide of SEQ ID NO: 1, but is not limited thereto. Any O-acylhomoserine sulfhydrylase having the same or equivalent activity as a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is included in the O-acylhomoserine sulfhydrylase. Furthermore, even if a polypeptide / protein has an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, or added, the polypeptide / protein is still included in the polypeptide / protein to be mutated in the present application, as long as it has such homology or identity and exhibits O-acylhomoserine sulfhydrylase activity.

[0028] As one example, the polypeptide of SEQ ID NO: 6 (WP_002722742.1) is a polypeptide that has an amino acid sequence in which a portion of the sequence is deleted compared to the polypeptide of SEQ ID NO: 1 and exhibits O-acylhomoserine sulfhydrylase activity, and therefore is included in the polypeptides to be mutated in the present application. As other examples, the polypeptides of SEQ ID NOs: 2, 3, 4, 5, and 7 are polypeptides that have an amino acid sequence in which a portion of the sequence is substituted compared to the polypeptide of SEQ ID NO: 1 and exhibit O-acylhomoserine sulfhydrylase activity, and therefore are included in the polypeptides to be mutated in the present application.

[0029] As one example, in an O-acylhomoserine sulfhydrylase to be mutated in the present application, at least one of the amino acids corresponding to positions 3, 65, 104, and 196 from the N-terminus of SEQ ID NO: 1 may be an amino acid selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0030] In any of the above-described Examples, in the O-acylhomoserine sulfhydrylase to be mutated in the present application, the amino acid corresponding to the third position from the N-terminus of SEQ ID NO: 1 may be isoleucine or an amino acid other than isoleucine. For example, the amino acid corresponding to the third position may be asparagine (N).

[0031] In any of the above-described Examples, in the O-acylhomoserine sulfhydrylase to be mutated in the present application, the amino acid corresponding to the 65th position from the N-terminus of SEQ ID NO: 1 may be phenylalanine or an amino acid other than phenylalanine. For example, the amino acid corresponding to the 65th position may be tyrosine (Y).

[0032] In any of the above-described Examples, in the O-acylhomoserine sulfhydrylase to be mutated in the present application, the amino acid corresponding to the 104th position from the N-terminus of SEQ ID NO: 1 may be valine (V) or an amino acid other than valine. For example, the amino acid corresponding to the 104th position may be alanine (A).

[0033] In any of the above-described Examples, in the O-acylhomoserine sulfhydrylase to be mutated in the present application, the amino acid corresponding to the 196th position from the N-terminus of SEQ ID NO: 1 may be valine or an amino acid other than valine. For example, the amino acid corresponding to the 196th position may be threonine (T).

[0034] In any of the above-described Examples, in the O-acylhomoserine sulfhydrylase to be mutated in the present application, the amino acid corresponding to the third position from the N-terminus of SEQ ID NO: 1 may be asparagine (N), the amino acid corresponding to the 65th position may be tyrosine (Y), and the amino acid corresponding to the 104th position may be alanine (A).

[0035] In any of the above-described Examples, the O-acylhomoserine sulfhydrylase to be mutated in the present application may have, contain, consist of, or essentially consist of the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7. In any of the above-described Examples, the O-acylhomoserine sulfhydrylase comprises an amino acid sequence having about 60% or more sequence homology or identity to the polypeptide of SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, for example, about 60%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 98.5%, or 99% or more, but is not limited thereto.

[0036] In this application, "homology" or "identity" refers to the degree of similarity between two given amino acid or nucleotide sequences, expressed as a percentage. Homology and identity are often used interchangeably.

[0037] Sequence homology or identity of conserved polynucleotides or polypeptides can be determined by standard sequence algorithms, optionally with default gap penalties established by the program used. Substantially homologous or identical sequences will generally hybridize to all or part of the sequence under moderately or highly stringent conditions. Hybridization, of course, also includes hybridization to polynucleotides containing common codons or codons that take into account codon degeneracy in the polynucleotide.

[0038] Whether any two polynucleotide or polypeptide sequences have homology, similarity, or identity can be determined using known computer algorithms, such as the "FASTA" program, with default parameters, as described in, for example, Non-Patent Document 1. Alternatively, the Needleman-Wunsch algorithm (Non-Patent Document 3) can be used, as implemented in the Needle program (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) (version 5.0.0 or later) of the EMBOSS package (which includes the GCG program package (Non-Patent Document 4), BLASTP, BLASTN, and FASTA (Non-Patent Documents 5, 6, and 7)). For example, BLAST or Clustal W from the National Center for Biotechnology Information can be used to determine homology, similarity, or identity.

[0039] Homology, similarity, or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program such as that disclosed in Non-Patent Document 3, as disclosed in, for example, Non-Patent Document 8. Briefly, the GAP program defines the number of similar sequence symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program include: (1) a binary comparison matrix (identity takes a value of 1, non-identity a value of 0) and a weighted comparison matrix (or EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix) as disclosed in Non-Patent Document 9; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap open penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0040] In this application, the term "variant" refers to a polypeptide that differs from the amino acid sequence of the variant by conservative substitution and / or modification of at least one amino acid, but maintains its functions or properties. 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 performance of the variant may be improved, unchanged, or decreased compared to the polypeptide of the variant. Some variants also include variants in which at least one portion, such as an N-terminal leader sequence or a transmembrane domain, has been deleted. Other variants include variants in which a portion has been deleted from the N- and / or C-termini of a mature protein. The term "variant" may refer to any term that refers to a mutation, such as a mutant type, modification, mutant polypeptide, mutated protein, or mutation (in English, terms such as modification, modified polypeptide, modified protein, mutant, mutein, and divergent are used).

[0041] The variant may also include deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the N-terminus of the variant may be linked to a signal (or leader) sequence involved in co- or post-translational protein translocation. The variant may also be linked to other sequences or linkers to allow identification, purification, or synthesis.

[0042] The O-acylhomoserine sulfhydrylase mutant provided in the present application means a polypeptide having an activity of O-acylhomoserine sulfhydrylase having any sequence, or a mutant O-acylhomoserine sulfhydrylase comprising a substitution of at least one other amino acid for the amino acids corresponding to the 4th, 90th, 106th, 242nd, 261st, 290th, and 395th amino acids from the N-terminus of SEQ ID NO: 1.

[0043] The O-acylhomoserine sulfhydrylase mutant provided by the present application may be a polypeptide comprising an amino acid sequence in which at least one of the amino acids corresponding to the 4th, 90th, 106th, 242nd, 261st, 290th, and 395th positions from the N-terminus of SEQ ID NO: 1 has been substituted with another amino acid.

[0044] The "other amino acid" may be any amino acid different from the amino acid before substitution. It goes without saying that "a specific amino acid has been substituted" in the present application means that the amino acid has been substituted with an amino acid different from the amino acid before substitution, even if it is not specifically stated that the amino acid has been substituted with another amino acid.

[0045] Amino acids are commonly classified based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues.

[0046] As an example of such classification, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; amino acids with nonpolar side chains (nonpolar amino acids) include glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline; and amino acids with polar or hydrophilic side chains (polar amino acids) include serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Other examples include electrically charged amino acids (arginine, lysine, histidine, glutamic acid, and aspartic acid) and uncharged amino acids (neutral amino acids) (glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine). Phenylalanine, tryptophan, and tyrosine are classified as aromatic amino acids. Valine, leucine, and isoleucine are classified as branched-chain amino acids. As another example, the 20 amino acids are classified by size and divided into five groups in order of decreasing relative volume: glycine, alanine, serine; cysteine, proline, threonine, aspartic acid, asparagine; valine, histidine, glutamic acid, glutamine; isoleucine, leucine, methionine, lysine, arginine; and phenylalanine, tryptophan, tyrosine. However, the groups are not necessarily limited to these.

[0047] "Corresponding to," as used herein, means the amino acid residue at the recited position in the polypeptide, or an amino acid residue that is similar, identical, or equivalent to the recited residue in the polypeptide. Identifying the amino acid at the corresponding position will determine the specific amino acid of the sequence to which the particular sequence refers. "Corresponding region," as used herein, generally refers to a similar or corresponding position in a related or reference protein.

[0048] For example, when any amino acid sequence is aligned with SEQ ID NO: 1, each amino acid residue in the amino acid sequence can be numbered based on the number and position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 1. For example, the sequence alignment algorithm in the present application can identify the amino acid positions or positions where modifications such as substitutions, insertions, deletions, etc. occur when compared with a query sequence (referred to as a "reference sequence").

[0049] For such alignment, for example, the Needleman-Wunsch algorithm (Non-Patent Document 3) or the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Non-Patent Document 2) can be used, but the present invention is not limited to these. Sequence alignment programs, pairwise sequence comparison algorithms, and the like known in the art can also be used as appropriate.

[0050] As one example, the O-acylhomoserine sulfhydrylase mutant provided by the present application may include an amino acid sequence in which one, two, three, four, five, six, or seven amino acids at positions corresponding to the 4th, 90th, 106th, 242nd, 261st, 290th, and 395th amino acids from the N-terminus of SEQ ID NO: 1 are substituted with other amino acids.

[0051] In any of the above-mentioned examples, the O-acylhomoserine sulfhydrylase mutant provided by the present application may include a substitution of the amino acid corresponding to the 90th position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0052] In any of the above-described embodiments, the mutant may be one in which the amino acid corresponding to the 90th position from the N-terminus of SEQ ID NO: 1 is substituted with a nonpolar amino acid. For example, the amino acid may be an amino acid selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In any of the above-described embodiments, the mutant may be one in which the amino acid corresponding to the 90th position from the N-terminus of SEQ ID NO: 1 is substituted with alanine (A).

[0053] In any of the above-mentioned examples, the O-acylhomoserine sulfhydrylase mutant provided by the present application may include a substitution of the amino acid corresponding to the fourth position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0054] In any of the above-described embodiments, the O-acylhomoserine sulfhydrylase mutant provided by the present application may have the amino acid corresponding to the fourth position from the N-terminus of SEQ ID NO: 1 substituted with an amino acid having a charged side chain selected from arginine, lysine, histidine, glutamic acid, and aspartic acid. For example, the amino acid may be an amino acid selected from basic amino acids arginine, lysine, and histidine. In any of the above-described embodiments, the mutant may have the amino acid corresponding to the fourth position from the N-terminus of SEQ ID NO: 1 substituted with lysine (K).

[0055] In any of the above-mentioned examples, the O-acylhomoserine sulfhydrylase mutant provided by the present application may include a substitution of the amino acid corresponding to the 106th position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0056] In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to position 106 from the N-terminus of SEQ ID NO: 1 is substituted with an amino acid selected from polar amino acids serine, cysteine, tyrosine, asparagine, and glutamine. In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to position 106 from the N-terminus of SEQ ID NO: 1 is substituted with cysteine ​​(C).

[0057] In any of the above-mentioned examples, the O-acylhomoserine sulfhydrylase mutant provided by the present application may include a substitution of the amino acid corresponding to the 290th position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0058] In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to position 290 from the N-terminus of SEQ ID NO: 1 is substituted with an amino acid selected from polar amino acids serine, threonine, cysteine, tyrosine, asparagine, and glutamine. In any of the above-mentioned embodiments, the mutant may be one in which the amino acid corresponding to position 290 from the N-terminus of SEQ ID NO: 1 is substituted with cysteine ​​(C).

[0059] In any of the above-mentioned examples, the O-acylhomoserine sulfhydrylase mutant provided by the present application may include a substitution of the amino acid corresponding to position 242 from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0060] In any of the above-described embodiments, the mutant may be one in which the amino acid corresponding to position 242 from the N-terminus of SEQ ID NO: 1 is substituted with a nonpolar amino acid. For example, the amino acid may be an amino acid selected from glycine, alanine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In any of the above-described embodiments, the mutant may be one in which the amino acid corresponding to position 242 from the N-terminus of SEQ ID NO: 1 is substituted with leucine (L).

[0061] In any of the above-mentioned examples, the O-acylhomoserine sulfhydrylase mutant provided by the present application may include a substitution of the amino acid corresponding to the 261st position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0062] In any of the above-described embodiments, the mutant may be one in which the amino acid corresponding to position 261 from the N-terminus of SEQ ID NO: 1 is substituted with a nonpolar amino acid. For example, the amino acid may be an amino acid selected from glycine, alanine, valine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In any of the above-described embodiments, the mutant may be one in which the amino acid corresponding to position 261 from the N-terminus of SEQ ID NO: 1 is substituted with valine (V).

[0063] In any of the above-mentioned examples, the O-acylhomoserine sulfhydrylase mutant provided by the present application may include a substitution of the amino acid corresponding to the 395th position from the N-terminus of SEQ ID NO: 1 with another amino acid.

[0064] In any of the above-described embodiments, the mutant may be one in which the amino acid corresponding to position 395 from the N-terminus of SEQ ID NO: 1 is substituted with a nonpolar amino acid. For example, the amino acid may be an amino acid selected from glycine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, and proline. In any of the above-described embodiments, the mutant may be one in which the amino acid corresponding to position 395 from the N-terminus of SEQ ID NO: 1 is substituted with valine (V).

[0065] In any of the above-mentioned Examples, the O-acylhomoserine sulfhydrylase mutant provided by the present application may include at least one of a basic amino acid substitution for the amino acid corresponding to the 4th position from the N-terminus of SEQ ID NO: 1, a nonpolar amino acid substitution for the amino acid corresponding to the 90th position, a polar amino acid substitution for the amino acid corresponding to the 106th position, a nonpolar amino acid substitution for the amino acid corresponding to the 242nd position, a nonpolar amino acid substitution for the amino acid corresponding to the 261st position, a polar amino acid substitution for the amino acid corresponding to the 290th position, and a nonpolar amino acid substitution for the amino acid corresponding to the 395th position.

[0066] In any of the above-mentioned Examples, the O-acylhomoserine sulfhydrylase mutant provided by the present application may include at least one of a substitution of the amino acid corresponding to the 4th position from the N-terminus of SEQ ID NO: 1 with lysine (K), a substitution of the amino acid corresponding to the 90th position with alanine (A), a substitution of the amino acid corresponding to the 106th position with cysteine ​​(C), a substitution of the amino acid corresponding to the 242nd position with leucine (L), a substitution of the amino acid corresponding to the 261st position with valine (V), a substitution of the amino acid corresponding to the 290th position with cysteine ​​(C), and a substitution of the amino acid corresponding to the 395th position with valine (V).

[0067] In any of the above-described embodiments, the O-acylhomoserine sulfhydrylase mutant provided by the present application may include at least one of a substitution of lysine (K) for alanine (A) corresponding to the fourth position from the N-terminus of SEQ ID NO: 1 (A4K), a substitution of alanine (A) for aspartic acid (D) corresponding to the 90th position (D90A), a substitution of cysteine ​​(C) for threonine (T) corresponding to the 106th position (T106C), a substitution of leucine (L) for valine (V) corresponding to the 242nd position, a substitution of leucine (L) for valine (V) corresponding to the 261st position (L261V), a substitution of cysteine ​​(C) for histidine (H) corresponding to the 290th position (H290C), and a substitution of valine (V) for alanine (A) corresponding to the 395th position (V261V).

[0068] In any of the above-mentioned embodiments, the O-acylhomoserine sulfhydrylase mutant provided by the present application may include substitutions of amino acids corresponding to positions 4, 90, 106, and 290 from the N-terminus of SEQ ID NO: 1 with other amino acids.

[0069] In any of the above-mentioned embodiments, the O-acylhomoserine sulfhydrylase mutant provided by the present application may include substitutions of amino acids corresponding to positions 4, 90, 106, 242, 261, 290, and 395 from the N-terminus of SEQ ID NO: 1 with other amino acids.

[0070] In any of the above-mentioned embodiments, the O-acylhomoserine sulfhydrylase mutant provided by the present application may have one, two, three, four, five, six, or seven amino acids substituted with other amino acids at positions corresponding to the 4th, 90th, 106th, 242nd, 261st, 290th, and 395th amino acids from the N-terminus of SEQ ID NO: 1, and may include an amino acid sequence having at least 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% or more homology or identity to the amino acid sequence represented by SEQ ID NO: 1.

[0071] In any of the above-described embodiments, the O-acylhomoserine sulfhydrylase variant provided by the present application may consist of the amino acid sequence of SEQ ID NO: 8, or may comprise an amino acid sequence having at least 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% or more homology or identity thereto.

[0072] In any of the above-described embodiments, the O-acylhomoserine sulfhydrylase variant provided by the present application may consist of, have, include, or consist essentially of an amino acid sequence selected from SEQ ID NOs: 12, 13, and 14. For example, the O-acylhomoserine sulfhydrylase variant may have at least 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, or 100%, homology or identity to SEQ ID NOs: 12, 13, and 14. In the O-acylhomoserine sulfhydrylase mutant, the amino acids corresponding to the 4th, 90th, 106th, 242nd, 261st, 290th, and 395th positions from the N-terminus of SEQ ID NO: 12, 13, or 14 may be identical to those of SEQ ID NO: 12, 13, or 14.

[0073] Furthermore, it goes without saying that variants having an amino acid sequence in which a portion of the sequence has been deleted, modified, substituted, conservatively substituted, or added are also included in the present application, as long as the variant has such homology or identity and exhibits efficacy equivalent to that of the variant of the present application.

[0074] For example, the variants may have additions or deletions of sequences at the N-terminus, C-terminus and / or internally of the amino acid sequence that do not alter the function of the variants of the present application, naturally occurring mutations, silent mutations or conservative substitutions.

[0075] The term "conservative substitution" refers to the substitution of an amino acid with another amino acid having similar structural and / or chemical properties. Such amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. Typically, conservative substitutions have little or no effect on the activity of a protein or polypeptide.

[0076] In any of the above-mentioned embodiments, the O-acylhomoserine sulfhydrylase mutant provided by the present application may further include modifications that improve the activity of the O-acylhomoserine sulfhydrylase, or modifications to the extent that the activity is maintained.

[0077] In any of the above-mentioned embodiments, in the O-acylhomoserine sulfhydrylase mutant provided by the present application, at least one of the amino acids corresponding to positions 3, 65, 104, and 196 from the N-terminus of SEQ ID NO: 1 may be an amino acid selected from glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0078] In any of the above-mentioned Examples, in the O-acylhomoserine sulfhydrylase mutant provided by the present application, the amino acid corresponding to the third position from the N-terminus of SEQ ID NO: 1 may be isoleucine or an amino acid other than isoleucine. For example, the amino acid corresponding to the third position may be asparagine (N).

[0079] In any of the above-mentioned Examples, in the O-acylhomoserine sulfhydrylase mutant provided by the present application, the amino acid corresponding to the 65th position from the N-terminus of SEQ ID NO: 1 may be phenylalanine or an amino acid other than phenylalanine. For example, the amino acid corresponding to the 65th position may be tyrosine (Y).

[0080] In any of the above-described Examples, in the O-acylhomoserine sulfhydrylase mutant provided by the present application, the amino acid corresponding to the 104th position from the N-terminus of SEQ ID NO: 1 may be valine (V) or an amino acid other than valine. For example, the amino acid corresponding to the 104th position may be alanine (A).

[0081] In any of the above-mentioned Examples, in the O-acylhomoserine sulfhydrylase mutant provided by the present application, the amino acid corresponding to the 196th position from the N-terminus of SEQ ID NO: 1 may be valine or an amino acid other than valine. For example, the amino acid corresponding to the 196th position may be threonine (T).

[0082] In any of the above-mentioned examples, in the O-acylhomoserine sulfhydrylase mutant provided by the present application, the amino acid corresponding to the third position from the N-terminus of SEQ ID NO: 1 may be asparagine (N), the amino acid corresponding to the 65th position may be tyrosine (Y), and the amino acid corresponding to the 104th position may be alanine (A).

[0083] Another aspect of the present application provides polynucleotides encoding the variants of the present application.

[0084] In this application, the term "polynucleotide" refers to a nucleotide polymer in which nucleotide monomers are linked in a long chain by covalent bonds, and refers to a DNA or RNA chain longer than a certain length.

[0085] For example, a polynucleotide encoding an O-acylhomoserine sulfhydrylase of the present application may comprise a sequence encoding the polypeptide of SEQ ID NO: 1 or a sequence having 70% or more identity thereto. For example, the polynucleotide encoding the O-acylhomoserine sulfhydrylase may consist of the sequence of SEQ ID NO: 9, or may consist essentially of said sequence.

[0086] As an example, a polynucleotide encoding an O-acylhomoserine sulfhydrylase mutant of the present application has, contains, consists of, or essentially consists of a nucleotide sequence that is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, or 97% or more, but less than 100%, homologous or identical to the sequence of SEQ ID NO: 9 or 10, but is not limited to these. Here, in the sequence having homology or identity, a codon encoding an amino acid corresponding to at least one position selected from the 4th, 90th, 106th, 242nd, 261st, 290th, and 395th positions from the N-terminus of SEQ ID NO: 1 may be a codon encoding an amino acid different from the amino acid at the aforementioned position in SEQ ID NO: 1.

[0087] Furthermore, the polynucleotide of the present 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 the present application. The term "stringent conditions" refers to conditions that allow specific hybridization between polynucleotides. Such conditions are specifically described in the literature (see Non-Patent Documents 11 and 12). For example, conditions include those under which polynucleotides with high homology or identity, such as polynucleotides with a homology or identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, hybridize with each other, but polynucleotides with lower homology or identity do not hybridize with each other; or conditions including washing once, specifically two to three times, at a salt concentration and temperature equivalent to those used in conventional Southern hybridization, namely, 60°C, 1×SSC, 0.1% SDS, specifically, 60°C, 0.1×SSC, 0.1% SDS, more specifically, 68°C, 0.1×SSC, 0.1% SDS.

[0088] Hybridization requires that two nucleic acids have complementary sequences, even if mismatches between bases are possible depending on the stringency of 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. Thus, the polynucleotides of the present application may include not only substantially similar nucleic acid base sequences, but also isolated nucleic acid fragments that are complementary to the entire sequence.

[0089] Specifically, polynucleotides having homology or identity to the polynucleotides of the present application can be detected using the hybridization conditions described above, in which the hybridization step is performed at a Tm value of 55° C. The Tm value may be, but is not limited to, 60° C., 63° C., or 65° C., and can be appropriately adjusted by those skilled in the art depending on the purpose.

[0090] The appropriate stringency for hybridizing the polynucleotides depends on the length of the polynucleotides and the degree of complementation, variables known in the art (eg, Non-Patent Document 11).

[0091] Yet another aspect of the present application provides a vector comprising a polynucleotide encoding the variant of the present application, the vector being, but not limited to, an expression vector for expressing the polynucleotide in a host cell.

[0092] In this application, the term "vector" may include a DNA product comprising a base sequence of a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so as to enable expression of the target polypeptide in a suitable host. The expression control region includes a promoter that initiates transcription, an optional operator sequence for regulating the transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence that regulates the termination of transcription and translation. When transformed into a suitable microorganism, the vector can replicate and function independently of the host genome and is integrated into the genome itself.

[0093] The vector used in the present application is not particularly limited, and any vector known in the art may be used. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage or cosmid vectors. Examples of plasmid vectors that may be used include pDZ, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET. Specifically, pDZ, pDC, pDCM2, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, and pUCtk vectors may be used.

[0094] For example, a polynucleotide encoding a target polypeptide can be inserted into a chromosome using a vector for intracellular chromosomal introduction. The insertion of the polynucleotide into a chromosome can be achieved by any method known in the art, including, but not limited to, homologous recombination. A selection marker for determining whether or not the polynucleotide has been inserted into the chromosome may be further included. The selection marker is used to select cells transformed with the vector, i.e., to determine whether or not the target nucleic acid molecule has been inserted. Markers that confer selectable phenotypes, such as drug resistance, auxotrophy, 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 survive or exhibit a different phenotype, allowing the selection of transformed cells.

[0095] In the present application, "transformation" refers to the introduction of a vector containing a polynucleotide encoding a target polypeptide into a host cell or microorganism, thereby expressing the polypeptide encoded by the polynucleotide in the host cell. The transformed polynucleotide may be any polynucleotide that can be expressed in the host cell, regardless of whether it is located intrachromosomally or extrachromosomally. The polynucleotide may also include DNA and / or RNA encoding the target polypeptide. The polynucleotide may be introduced in any form that can be expressed in the host cell. For example, the polynucleotide may be introduced into the host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. Typically, the expression cassette contains a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the polynucleotide. The expression cassette may be in the form of a self-replicating expression vector. The polynucleotide may also be introduced into the host cell in its own form and operably linked to sequences necessary for expression in the host cell, but is not limited thereto.

[0096] Furthermore, the term "operably linked" means that the polynucleotide sequence is functionally linked to a promoter sequence that initiates and mediates transcription of the polynucleotide encoding the variant of the present application.

[0097] Yet another aspect of the present application provides a microorganism comprising a variant of the present application or a polynucleotide encoding said variant.

[0098] The microorganism of the present application may comprise a variant of the present application, a polynucleotide encoding it, or a vector comprising said polynucleotide.

[0099] The term "microorganism (or strain)" as used herein includes all wild-type microorganisms and naturally or artificially genetically modified microorganisms, and refers to a microorganism in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by strengthening or inactivating the activity of an endogenous gene, and which has been genetically modified for the production of a desired polypeptide, protein, or product. The genetic modification includes strengthening and / or weakening the activity of a polypeptide involved in the specific mechanism.

[0100] The microorganism of the present application is, but is not limited to, a microorganism comprising at least one of a variant of the present application, a polynucleotide encoding a variant of the present application, and a vector comprising a polynucleotide encoding a variant of the present application; a microorganism that has been modified to express a variant of the present application or a polynucleotide encoding a variant of the present application; a microorganism (e.g., a recombinant strain) that expresses a variant of the present application or a polynucleotide encoding a variant of the present application; or a microorganism (e.g., a recombinant strain) that has the activity of a variant of the present application.

[0101] The microorganism of the present application may be either a prokaryotic or eukaryotic microorganism, as long as it is a microorganism that produces the O-acylhomoserine sulfhydrylase variant provided by the present application, or contains the O-acylhomoserine sulfhydrylase variant and produces the target reactant. Examples include microbial strains belonging to the genera Escherichia, Erwinia, Serratia, Providencia, Corynebacteria, Pseudomonas, Leptospira, Salmonella, Brevibacteria, Hypomonas, Chromobacterium, and Norcardia, or fungi or yeast. In one example, the microorganism may be a microorganism of the genus Escherichia or a microorganism of the genus Corynebacterium. In any of the above embodiments, the microorganism may be, but is not limited to, Escherichia coli or Corynebacterium glutamicum.

[0102] In the present application, "enhancing" a polypeptide activity means improving the activity of the polypeptide compared to its endogenous activity. The term "enhancing" is used interchangeably with "activation," "up-regulation," "overexpression," and "increase." Here, activation, enhancement, up-regulation, overexpression, and enhancement all encompass the development of an activity not originally present, as well as an improvement in activity compared to endogenous activity or activity prior to modification. The term "endogenous activity" refers to the activity of a specific polypeptide inherently possessed by a parent strain or unmodified microorganism prior to phenotypic change, when the trait is altered through genetic mutation due to natural or artificial factors. This term is also used interchangeably with "activity prior to modification." "Enhancing," "up-regulating," "overexpressing," or "improving" a polypeptide activity compared to its endogenous activity means an improvement in the activity and / or concentration (expression level) of a specific polypeptide inherently possessed by a parent strain or unmodified microorganism prior to phenotypic change.

[0103] The enhancement may be achieved by introducing a foreign polypeptide, or by enhancing the activity and / or increasing the concentration (expression level) of an endogenous polypeptide. Whether the activity of the polypeptide has been enhanced can be confirmed by an increase in the level of activity of the polypeptide, the expression level, or the amount of a product produced from the polypeptide.

[0104] Various methods well known in the art can be applied to enhance the activity of the polypeptide, and any method can be used as long as it can enhance the activity of the target polypeptide compared to the unmodified microorganism. Specifically, methods such as, but not limited to, conventional methods in molecular biology using genetic engineering and / or protein engineering well known to those skilled in the art (e.g., Non-Patent Documents 13 and 14).

[0105] Specifically, the enhancement of a polypeptide of the present application can be achieved by: 1) increasing the intracellular copy number of a polynucleotide encoding the polypeptide; 2) replacing the expression regulatory region of a gene on a chromosome encoding the polypeptide with a sequence with stronger activity; 3) modifying the nucleotide sequence encoding the start codon or 5'UTR region of a gene transcript encoding the polypeptide; 4) modifying the amino acid sequence of the polypeptide so that the activity of the polypeptide is enhanced; 5) modifying the polynucleotide sequence encoding the polypeptide so that the activity of the polypeptide is enhanced (for example, modifying the polynucleotide sequence of the polypeptide gene so that the polypeptide is encoded as a polypeptide modified so that the activity of the polypeptide is enhanced); 6) introducing a foreign polypeptide that exhibits the activity of the polypeptide or a foreign polynucleotide encoding it; 7) optimizing the codons of the polynucleotide encoding the polypeptide; 8) analyzing the tertiary structure of the polypeptide and selectively modifying or chemically modifying exposed portions; or 9) a combination of two or more selected from 1) to 8) above, but is not limited to these.

[0106] More specifically, the 1) intracellular copy number of a polynucleotide encoding a polypeptide may be increased by introducing into a host cell a vector to which a polynucleotide encoding the polypeptide is operably linked, the vector replicating and functioning independently of the host. Alternatively, the polynucleotide encoding the polypeptide may be introduced into a chromosome in the host cell at one or more copies. The introduction into a chromosome may be achieved by, but not limited to, introducing into the host cell a vector capable of inserting the polynucleotide into a chromosome in the host cell. The vector is as described above.

[0107] The expression regulatory region (or expression regulatory sequence) of a gene on a chromosome encoding a polypeptide (2) can be replaced with a sequence with stronger activity by, for example, generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by replacing the expression regulatory region with a sequence with higher activity, so as to further enhance the activity of the expression regulatory region. Examples of the expression regulatory region include, but are not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences regulating the termination of transcription and translation. For example, this can be achieved by replacing the original promoter with a strong promoter, but is not limited to this.

[0108] Examples of known strong promoters include, but are not limited to, the CJ1 to CJ7 promoters (Patent Document 2), 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 3), the O2 promoter (Patent Document 4), the tkt promoter, and the yccA promoter.

[0109] The nucleotide sequence encoding the start codon or 5'UTR region of the gene transcript encoding the polypeptide (3) can be modified, for example, by substituting it with a nucleotide sequence encoding another start codon that has a higher polypeptide expression rate than the endogenous start codon, but is not limited to this.

[0110] The modification of the amino acid sequence or polynucleotide sequence in 4) and 5) can be carried out by, but is not limited to, generating a sequence mutation through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide to enhance the activity of the polypeptide, or by substituting an amino acid sequence or polynucleotide sequence improved to have higher activity or improved activity. Specifically, the substitution can be carried out by, but is not limited to, inserting the polynucleotide into a chromosome by homologous recombination. The vector used here may further contain a selection marker for confirming whether or not it has been inserted into the chromosome. The selection marker is as described above.

[0111] 6) Introduction of a foreign polynucleotide that exhibits the activity of a polypeptide may be carried out by introducing into a host cell a foreign polynucleotide that encodes a polypeptide that exhibits the same or similar activity as the polypeptide. The foreign polynucleotide may be of any origin or sequence, as long as it exhibits the same or similar activity as the polypeptide. The introduction can be carried out by a known transformation method appropriately selected by those skilled in the art, and the introduced polynucleotide is expressed in the host cell as described above, thereby producing the polypeptide and improving its activity.

[0112] 7) Optimizing the codons of a polynucleotide encoding a polypeptide may be carried out by optimizing the codons of an endogenous polynucleotide so as to increase transcription or translation within a host cell, or by optimizing the codons of an exogenous polynucleotide so as to achieve optimized transcription and translation within a host cell.

[0113] 8) Analyzing the tertiary structure of a polypeptide and selecting and altering or chemically modifying exposed portions may be carried out, for example, by comparing the sequence information of the polypeptide to be analyzed with a database storing sequence information of known proteins, determining candidate template proteins based on the degree of sequence similarity, confirming the structure based on the candidate template proteins, and selecting and altering or modifying exposed portions to be altered or chemically modified.

[0114] Such enhancement of polypeptide activity can be achieved by, but is not limited to, improving the activity, concentration, or expression level of the corresponding polypeptide compared to the activity or concentration of the polypeptide expressed in a wild-type or unmodified microbial strain, or by increasing the amount of product produced from the polypeptide.

[0115] In the microorganisms of the present application, partial or complete modification of a polynucleotide can be induced by, but is not limited to, (a) homologous recombination using a vector for chromosomal introduction into the microorganism, or genome editing using engineered nucleases (e.g., CRISPR-Cas9), and / or (b) light and / or chemical treatment, such as ultraviolet light or radiation. Methods for partially or completely modifying the gene include methods using DNA recombination techniques. For example, partial or complete deletion of a gene can be achieved by introducing a nucleotide sequence or vector containing a nucleotide sequence homologous to the target gene into the microorganism and causing homologous recombination. The introduced nucleotide sequence or vector may contain, but is not limited to, a dominant selection marker.

[0116] In the present application, "attenuation" of a polypeptide refers to a decrease in activity compared to the endogenous activity or the absence of activity. The term "attenuation" is interchangeable with other terms such as inactivation, deficiency, down-regulation, decrease, reduce, and attenuation.

[0117] The attenuation includes at least one of the following: a reduction or elimination of the activity of the polypeptide itself compared to the activity of the polypeptide originally possessed by the microorganism due to, for example, a mutation in the polynucleotide encoding the polypeptide; a reduction in the overall level and / or concentration (expression level) of polypeptide activity in the cell compared to that of a native strain due to, for example, inhibition of gene expression of the encoding polynucleotide or inhibition of translation into the polypeptide; complete absence of expression of the polynucleotide; and absence of polypeptide activity even if the polynucleotide is expressed. The term "endogenous activity" refers to the activity of a specific polypeptide originally possessed by a parent strain, wild-type, or unmodified microorganism before the trait change, when genetic mutation is caused by natural or artificial factors. This term is used interchangeably with "activity before modification." "Inactivation," "deficiency," "reduction," "down-regulation," "reduction," or "attenuation" of a polypeptide activity compared to the endogenous activity refers to a reduction in the activity of a specific polypeptide originally possessed by a parent strain or unmodified microorganism before the trait change.

[0118] The activity of such polypeptides can be attenuated by applying various methods well known in the art, including, but not limited to, those described in Non-Patent Documents 14 and 15.

[0119] Specifically, the activity of a polypeptide of the present application can be attenuated by 1) deleting all or part of a gene encoding the polypeptide, 2) modifying an expression regulatory region (or expression regulatory sequence) so as to reduce the expression of a gene encoding the polypeptide, 3) modifying the amino acid sequence constituting the polypeptide so as to delete or attenuate the activity of the polypeptide (for example, by deleting / substituting / adding one or more amino acids in the amino acid sequence), or 4) modifying the gene sequence encoding the polypeptide so as to delete or attenuate the activity of the polypeptide (for example, by converting the polypeptide to encode a polypeptide modified so as to delete or attenuate the activity of the polypeptide). The modification can be carried out by, but is not limited to, any of the following methods: (1) deleting / substituting / adding one or more nucleic acid bases in the nucleic acid base sequence of a polypeptide gene; (2) modifying the nucleic acid sequence encoding the start codon or 5'UTR region of a gene transcript encoding a polypeptide; (3) introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcript encoding a polypeptide; (4) adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide so that a secondary structure that prevents ribosome attachment is formed; (5) adding a promoter to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide so that reverse transcription occurs (reverse transcription engineering, RTE); or (6) combining two or more selected from 1) to 8).

[0120] For example, 1) deleting a part or all of the gene encoding the polypeptide may be carried out by deleting the entire polynucleotide encoding the endogenous target polypeptide in the chromosome, or by substituting a polynucleotide with a partial deletion of nucleotides or a marker gene.

[0121] Furthermore, the modification of the expression regulatory region (or expression regulatory sequence) described above in 2) may be carried out by generating a mutation in the expression regulatory region (or expression regulatory sequence) by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or by substituting a sequence having a lower activity. The expression regulatory region includes, but is not limited to, a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating the termination of transcription and translation.

[0122] Furthermore, the base sequence encoding the start codon or 5'UTR region of the gene transcription product encoding the polypeptide (5) can be modified, for example, by substituting it with a base sequence encoding another start codon that has a lower polypeptide expression rate than the endogenous start codon, but this is not limited to this.

[0123] Furthermore, modifying the amino acid sequence or polynucleotide sequence of 3) and 4) above can be carried out by, but is not limited to, generating a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, in the amino acid sequence of the polypeptide or the polynucleotide sequence encoding the polypeptide so as to attenuate the activity of the polypeptide, or by substituting an amino acid sequence or polynucleotide sequence that has been improved to have lower activity or to eliminate activity. For example, gene expression can be inhibited or reduced by, but is not limited to, introducing a mutation into a polynucleotide sequence to form a stop codon.

[0124] 6) Introducing an antisense oligonucleotide (e.g., antisense RNA) that binds complementarily to the gene transcription product encoding the polypeptide may be performed, for example, as described in Non-Patent Document 16.

[0125] 7) Adding a sequence complementary to the Shine-Dalgarno sequence before the Shine-Dalgarno sequence of a gene encoding a polypeptide so that a secondary structure that prevents ribosome attachment is formed may be achieved by disabling or slowing down mRNA translation.

[0126] 8) Adding a promoter to the 3' end of the ORF (open reading frame) of a gene sequence encoding a polypeptide so as to induce reverse transcription (reverse transcription engineering, RTE) may be carried out by creating an antisense nucleotide complementary to the gene transcript encoding the polypeptide, thereby reducing the activity.

[0127] Yet another aspect of the present application provides a method for producing an O-acylhomoserine sulfhydrylase reactant, comprising the step of contacting the O-acylhomoserine sulfhydrylase mutant, or a microorganism containing the mutant or a polynucleotide encoding it, with O-acylhomoserine.

[0128] In the present application, the term "O-acylhomoserine sulfhydrylase reactant" refers to a substance produced by a reaction mediated by O-acylhomoserine sulfhydrylase using O-acylhomoserine as a substrate.

[0129] O-acylhomoserine sulfhydrylase reactants of the present application include cystathionine, homocysteine, L-methionine, acetate, and succinate.

[0130] In one embodiment, the method for producing an O-acylhomoserine sulfhydrylase reactant may be a method for producing cystathionine, which includes contacting an O-acylhomoserine sulfhydrylase mutant, or a microorganism containing the mutant or a polynucleotide encoding the mutant, with O-acylhomoserine. The method for producing cystathionine may include contacting the mutant or the microorganism with O-acylhomoserine and L-cysteine.

[0131] In one embodiment, the method for producing an O-acylhomoserine sulfhydrylase reactant may be a method for producing homocysteine, comprising contacting an O-acylhomoserine sulfhydrylase mutant, or a microorganism containing the mutant or a polynucleotide encoding the mutant, with O-acylhomoserine. The method for producing homocysteine ​​may comprise contacting the mutant or the microorganism with O-acylhomoserine and sulfide.

[0132] In one embodiment, the method for producing an O-acylhomoserine sulfhydrylase reactant may be a method for producing L-methionine, comprising the step of contacting an O-acylhomoserine sulfhydrylase mutant, or a microorganism containing the mutant or a polynucleotide encoding the mutant, with O-acylhomoserine. For example, the method for producing L-methionine may comprise the step of contacting the mutant or the microorganism with O-acylhomoserine and methyl mercaptan. For another example, the method for producing L-methionine may comprise the step of contacting the mutant or the microorganism with O-acylhomoserine and thiosulfate.

[0133] In any of the above-described embodiments, the method for producing an O-acylhomoserine sulfhydrylase reactant may be a method for producing acetic acid, comprising the step of contacting an O-acylhomoserine sulfhydrylase mutant, or a microorganism containing the mutant or a polynucleotide encoding the mutant, with O-acetylhomoserine.

[0134] In any of the above-described embodiments, the method for producing an O-acylhomoserine sulfhydrylase reactant may be a method for producing succinic acid, comprising the step of contacting an O-acylhomoserine sulfhydrylase mutant, or a microorganism containing the mutant or a polynucleotide encoding the mutant, with O-succinylhomoserine.

[0135] In any of the above-mentioned examples, the O-acylhomoserine of the present application may be obtained from an L-methionine precursor-producing microorganism. The term "L-methionine precursor-producing microorganism" in the present application refers to prokaryotic and eukaryotic microorganisms that produce L-methionine precursors. L-methionine precursor-producing microorganisms are disclosed in, for example, Patent Document 1. In any of the above-mentioned examples, the O-acylhomoserine may be in a purified form or may be a microbial fermentation broth containing O-acylhomoserine.

[0136] In any of the foregoing embodiments, the contacting step may be carried out in the presence of a sulfur source. In any of the foregoing embodiments, the sulfur source may have a thiol functional group, such as a CHS group or an SH group. In any of the foregoing embodiments, the sulfur source may be selected from methyl mercaptan (CHSH), sodium sulfide (NaSH), thiosulfate (SO), alkanesulfonates including methanesulfonate and ethanesulfonate, sulfates, sulfites, hydrogen sulfides such as HS, sulfides, sulfide derivatives, organic and inorganic sulfur-containing compounds such as thioglycolates, thiocyanates, thioureas, or mixtures thereof.

[0137] In any of the above-described embodiments, the sulfur source may be a liquid or gaseous solution or gas. For example, when the sulfur source is methyl mercaptan, the gaseous methyl mercaptan may be liquefied or dissolved in a sodium hydroxide (NaOH) solution. In any of the above-described embodiments, the methyl mercaptan may be liquid sodium methyl mercaptan (CH3S-Na), gaseous or liquefied methyl mercaptan (CH3SH), or methyl mercaptan mixed with dimethylsulfide (DMS). The mixture of DMS and methyl mercaptan is disclosed in Patent Document 5.

[0138] In any of the above-described embodiments, the contacting step involves replacing the CH3S- residue of methyl mercaptan with the succinate residue of O-succinylhomoserine or the acetate residue of O-acetylhomoserine to produce L-methionine.

[0139] In any of the above embodiments, the contacting step involves substituting the SH residue of sodium sulfide with the succinate residue of O-succinylhomoserine or the acetate residue of O-acetylhomoserine to produce homocysteine.

[0140] Yet another aspect of the present application provides a method for producing an O-acylhomoserine sulfhydrylase reactant, comprising the step of culturing a microorganism containing an O-acylhomoserine sulfhydrylase mutant or a polynucleotide encoding the same in a medium containing O-acylhomoserine.

[0141] The O-acylhomoserine sulfhydrylase mutant and the microorganism are as described above.

[0142] The medium and other culture conditions used to culture the microorganism of the present application may be any medium used to culture known microorganisms. The microorganism of the present application can be cultured in a conventional medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, etc., under aerobic or anaerobic conditions, with temperature, pH, etc., adjusted.

[0143] As an example, the method for producing an O-acylhomoserine sulfhydrylase reactant of the present application may include a step of recovering the O-acylhomoserine sulfhydrylase reactant from the culture medium or the microorganism. Depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture, the target substance can be recovered from the culture medium using a suitable method known in the art.

[0144] The method may further comprise a purification step, which may be carried out by any suitable method known in the art. [Effects of the Invention]

[0145] The novel mutant of the present application can be used to produce L-methionine and / or its precursors in high yields. [Brief explanation of the drawings]

[0146] [Figure 1] 1 is a graph showing the activity of the enzyme mutants using sodium methyl mercaptan as a substrate. [Figure 2] 1 shows the activity of 40 enzyme mutants with excellent titers identified by screening, compared with that of a control group. The negative control is a negative control group containing no acylhomoserine sulfhydrylase enzyme, and the standard control is the acylhomoserine sulfhydrylase of SEQ ID NO: 2. [Figure 3] FIG. 1 shows the amount of methionine produced over time at each activity level of mutant enzymes (D90A, QM). [Figure 4]FIG. 1 shows the results of the methionine production reaction using mutant enzymes (D90A, QM) under different pH conditions and pretreatment times. [Figure 5] FIG. 1 shows the results of the methionine production reaction using a mutant enzyme (Variant 3) under various pH conditions and pretreatment times. [Figure 6] FIG. 1 shows the expression of the control enzyme and mutant enzymes (QM and Variant 3) by SDS-PAGE. [Figure 7] 1 is a graph showing the methionine conversion activity of a control enzyme and Variant 3. DETAILED DESCRIPTION OF THE INVENTION

[0147] The present application will be described in more detail below with reference to examples and experimental examples. However, these examples and experimental examples are merely illustrative of the present application, and the present application is not limited to these examples and experimental examples.

[0148] Comparative Example 1. Preparation of O-acylhomoserine sulfhydrylase derived from the Rhodobacteraceae family To generate O-acylhomoserine sulfhydrylase mutants that improve methionine production, we engineered O-acylhomoserine sulfhydrylase.

[0149] The nucleotide sequence (SEQ ID NO: 10) was obtained from the O-acylhomoserine sulfhydrylase (SEQ ID NO: 2) developed in Patent Document 6, and a plasmid containing the nucleotide sequence (based on pUCtk (SEQ ID NO: 11)) was constructed. The plasmid was transformed into Escherichia coli K12, which was then cultured overnight on an LB plate containing 50 μg / L kanamycin. Colonies were then selected. The selected colonies were inoculated into a deep 96-well plate containing 1 ml of LB medium containing 50 μg / L kanamycin and cultured overnight at 33°C and 1000 rpm. A portion of the culture was thoroughly mixed with 50% glycerol to prepare a glycerol stock plate. The enzyme strain isolated from the glycerol stock plate was inoculated into a deep 96-well plate containing 1 ml of 2X YT medium containing 50 μg / L kanamycin and cultured for 16 hours at 33°C and 1000 rpm. The enzyme fungus culture plate was centrifuged at 4000 G for 5 minutes to remove the supernatant. The cells were then disrupted using Bugsbuster Protein Extraction Reagent (Merck) according to the provided method. The cell lysate was collected and the total protein amount was quantified using Bio-Rad protein assay solution (BIO-Rad, USA). Protein expression was also confirmed using SDS-PAGE. The collected cell lysate was then used in the enzyme conversion reaction. The protein of SEQ ID NO: 2 prepared was used as a control in the following examples. [Example]

[0150] Construction of O-acylhomoserine sulfhydrylase mutants - 1 To generate highly active enzymes, we performed an amino acid mutation method using a saturation mutation library based on the HTP method (Patent Document 7). Cell lysates were extracted from the isolated mutants using the same method as in Comparative Example 1, and the activity of the mutants was evaluated using methyl mercaptan (Tokyo Chemical Industry Co., Ltd., Japan) as a substrate. The methyl mercaptan was prepared as a liquid by adding methyl mercaptan to a caustic soda solution (sodium methyl mercaptan, CH3S-Na, 4.7 M, 33%). The reaction mixture for the methionine conversion reaction using sodium methyl mercaptan as the substrate is shown in Table 1, and the reaction was carried out in a medium-depth well plate. Just before the reaction, a 300 mM stock solution of sodium methyl mercaptan was prepared, and 20 μl of the solution was added to 180 μl of the reaction mixture and mixed well. Then, 6 μl of the enzyme extract was added to each well, and the mixture was stored in a 40°C incubator for 1 hour. The final reaction was then terminated by transferring the solution to a TCA plate. The methionine, acetic acid, and O-acetylhomoserine (OAH) concentrations of the completed reaction mixture were measured by HPLC to evaluate the enzyme's conversion activity. Figure 1 shows the activity compared with a standard control containing the enzyme prepared in Comparative Example 1 and a negative control containing no O-acylhomoserine sulfhydrylase.

[0151] [Table 1] Component: Volume (mL): Volume (mL) Sodium phosphate dibasic: disodium hydrogen phosphate Citric acid:

[0152] The strain containing the mutant with the highest enzyme activity was selected, and the plasmid was isolated and analyzed by sequencing. The analysis of the base sequence confirmed that the mutant was a mutant (SEQ ID NO: 12) in which the 90th amino acid residue, aspartic acid (D), of O-acylhomoserine sulfhydrylase (SEQ ID NO: 2) was replaced with alanine (A), and this mutant was named D90A mutant. [Example]

[0153] Construction of O-acylhomoserine sulfhydrylase mutants - 2 Based on the saturation mutation library constructed in Example 1, combinatorial consolidation mutation was used to construct combinatorial mutants (Patent Document 7) that combined effective mutations from mutants exhibiting excellent activity. The constructed mutants were evaluated for their conversion activity using the same method as in Example 1. The activity of the mutants is shown in Figure 2. Among these, the mutant with the highest conversion activity was selected and designated the QM mutant. The QM mutant was subjected to a methionine conversion reaction using methyl mercaptan as a substrate, as in Example 1. The results confirmed that the QM mutant exhibited improved methionine conversion activity compared to the control or the D90A mutant in Example 1. As shown in Figure 3, the QM mutant produced the highest methionine over the course of the reaction. Analysis of the amino acid sequence of the QM mutant confirmed that it contained the A4K, D90A, T106C, and H290C mutations compared to the control (SEQ ID NO: 13). [Example]

[0154] Evaluation of mutant stability under different pH conditions To confirm the enzymatic stability of the selected mutants (D90A, QM) under each pH condition, the cell lysate obtained in Comparative Example 1 was exposed to reaction solutions at pH 5.8, 6.5, and 7.5 for 0, 1, 2, and 5 hours, respectively. Then, a methionine conversion experiment using methyl mercaptan as a substrate was performed in the same manner as in Example 1. As shown in Figure 4, the enzymatic reaction at pH 5.8, 6.5, and 7.5 confirmed that the QM mutant had the highest activity. Furthermore, the enzymatic activity was maintained regardless of the exposure time to the cell lysate. Therefore, it was confirmed that the activity of the mutants discovered in this application was stably maintained at a high level at various pH levels. [Example]

[0155] Construction of O-acylhomoserine sulfhydrylase mutants - 3 Based on the QM mutants selected in Example 2, secondary combination mutants were obtained by the method of combining and integrating effective mutations of mutants that showed excellent activity in Example 1, as in Example 2.

[0156] The mutants obtained as described above were subjected to an experiment to determine their methionine conversion ability using acetylhomoserine and sodium methyl mercaptan as substrates under various pH conditions. The most excellent mutant among these was named Variant 3.

[0157] As shown in Figure 5, Variant 3 had the highest conversion activity and enzyme activity stability compared to the control group under all pH conditions. Analysis of the amino acid sequence of Variant 3 confirmed that it contained the A4K, D90A, T106C, V242L, L261V, H290C, and A395V mutations compared to the control group in Comparative Example 1 (SEQ ID NO: 14). [Example]

[0158] Evaluation of the scale-up activity of selected mutants in the methionine conversion reaction To evaluate the activity of O-acylhomoserine sulfhydrylase variant 3 selected in Example 4 in a larger scale system, evaluation was carried out in a flask and a 30-liter reactor system.

[0159] 5-1. Production of enzyme extract using flask evaluation method To produce enzyme extracts of the control, QM mutant, and mutant 3, E. coli K12 cells containing the gene vectors encoding the enzymes were plated onto LB plates containing 50 μg / L kanamycin and cultured overnight in an incubator at 30°C. The entire culture was collected and inoculated into 50 ml of 2X YT medium containing 1% glucose and 50 μg / L kanamycin, followed by culture at 33°C and 200 rpm for 16 hours. Five ml of the culture was measured for OD600 and set aside for protein analysis by SDS-PAGE. The remaining culture was centrifuged at 4000 xg for 5 minutes and the supernatant was removed. The remaining pellet was dissolved in 5 ml of 100 mM PBS (pH 7.5) and dispensed in 500 μl aliquots into a 96-well deep-well plate. The plates were then treated with 10 μl of xylene and placed in a shaking incubator at 1150 rpm for 60 minutes. A 60 μl aliquot of the xylene-treated sample was mixed with 540 μl of 100 mM PBS (pH 7.5) to prepare the enzyme disruption dilution solution for the enzymatic conversion reaction. Total protein was quantified using Bio-Rad protein assay kit (BIO-Rad, USA). Protein expression was also confirmed using SDS-PAGE. As shown in Figure 6, the protein expression results for each enzyme mutant clone demonstrated superior protein expression levels for the QM and mutant 3 mutants compared to the control group at the same OD600.

[0160] 5-2. Evaluation of enzyme conversion activity in flask culture The titer of the conversion reaction was measured using the cell lysate and methyl mercaptan sodium solution obtained by the method of Example 5-1. Citrate-phosphate buffer solutions consisting of 0.1 M citric acid and 0.2 M sodium phosphate dibasic (NaHPO) were prepared to pH 7.5, pH 6.5, and pH 5.7, and then conversion reaction solutions were prepared with the compositions shown in Table 2. The methyl mercaptan sodium solution was preferentially removed, and the remaining conversion reaction solutions were mixed first, followed by incubation at 40°C for 5 minutes, followed by the addition of methyl mercaptan sodium to allow the reaction.

[0161] [Table 2] Volume: Sodium methylmercaptan: Sodium methylmercaptan

[0162] The reaction was terminated 1 hour after the start of the reaction, and then the OAH and methionine concentrations were measured using HPLC to determine the enzymatic conversion reaction activity as shown in Table 3. The number of enzyme units (units) was calculated in mg / mL / min based on the conversion rate of methionine per minute.

[0163] [Table 3] Variants Specific Activity:Specific activity Variant

[0164] As a result, it was confirmed that Mutant 3 had the highest enzyme activity under all pH conditions.

[0165] 5-3. Production and conversion of enzyme extract in a 30-liter fermenter This example was carried out using a 30 L batch reactor (CNS Co., Ltd., 30 L liquid fermenter) to scale up and evaluate the methionine enzymatic conversion reactor system using the conversion activity of the O-acylhomoserine sulfhydrylase mutant confirmed in the flask. The enzyme extract production and conversion reaction method used were the same as those for producing L-methionine from an O-acetylhomoserine culture solution according to a previous patent (Patent Document 6).

[0166] A graph of methionine conversion by the reaction is shown in Figure 7. This confirmed that even under scaled-up conditions, the conversion activity of Mutant 3 was higher than that of conventional O-acylhomoserine sulfhydrylase.

[0167] As described above, conversion was evaluated at pH 5.7. The results showed that the control group showed a conversion activity of 87%, while mutant 3 showed a 100% conversion rate, and the reaction time was also shortened.

[0168] From the above description, those skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing the technical idea or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present application should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalent concepts.

Claims

1. At least one of the amino acids corresponding to the 4th, 90th, 106th, 242nd, 261st, 290th and 395th positions from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid; O-acylhomoserine sulfhydrylase mutants.

2. The O-acylhomoserine sulfhydrylase is an acylhomoserine sulfhydrylase derived from a microorganism of the Rhodobacteraceae family. The O-acylhomoserine sulfhydrylase mutant according to claim 1.

3. The O-acylhomoserine sulfhydrylase mutant has amino acids corresponding to positions 3, 65, and 104 substituted with other amino acids. The O-acylhomoserine sulfhydrylase mutant according to claim 1.

4. The mutant has amino acids corresponding to the 4th, 90th, 106th, and 290th positions from the N-terminus of SEQ ID NO: 1 substituted with other amino acids. The O-acylhomoserine sulfhydrylase mutant according to claim 1.

5. The mutant has the amino acids at positions 242, 261, and 395 from the N-terminus of SEQ ID NO: 1 substituted with other amino acids. The O-acylhomoserine sulfhydrylase mutant according to claim 4.

6. The mutant is from the N-terminus of SEQ ID NO: 1 Substitution of the amino acid corresponding to the fourth position with a basic amino acid, Substitution of the amino acid corresponding to position 90 with a nonpolar amino acid; Substitution of the amino acid corresponding to position 106 with a polar amino acid; Substitution of the amino acid corresponding to position 242 with a nonpolar amino acid; Substitution of the amino acid corresponding to position 261 with a nonpolar amino acid; Substitution of the amino acid corresponding to position 290 with a polar amino acid, and at least one substitution of the amino acid corresponding to position 395 with a non-polar amino acid; The O-acylhomoserine sulfhydrylase mutant according to claim 1.

7. The mutant is from the N-terminus of SEQ ID NO: 1 Substitution of the amino acid corresponding to the fourth position with lysine (K); Substitution of the amino acid corresponding to position 90 with alanine (A); Substitution of the amino acid corresponding to position 106 with cysteine ​​(C); Substitution of the amino acid corresponding to position 242 with leucine (L); Substitution of the amino acid corresponding to position 261 with valine (V); a substitution of the amino acid corresponding to position 290 with cysteine ​​(C); and at least one substitution of the amino acid corresponding to position 395 with valine (V); The O-acylhomoserine sulfhydrylase mutant according to claim 1.

8. A polynucleotide encoding the variant of any one of claims 1 to 7.

9. The present invention includes an O-acylhomoserine sulfhydrylase mutant in which at least one of the amino acids corresponding to the 4th, 90th, 106th, 242nd, 261st, 290th, and 395th positions from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid, or a polynucleotide encoding the mutant. Microorganisms.

10. The method comprises a step of contacting an O-acetylhomoserine sulfhydrylase mutant in which at least one of the amino acids corresponding to the 4th, 90th, 106th, 242nd, 261st, 290th, and 395th positions from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid, or a microorganism containing the mutant or a polynucleotide encoding the same, with O-acylhomoserine; A method for producing an O-acylhomoserine sulfhydrylase reactant.

11. the O-acylhomoserine sulfhydrylase reactant is selected from L-methionine, acetate, succinate, homocysteine, and cystathionine; The method for producing L-methionine or a precursor thereof according to claim 10.

12. The method comprises the step of culturing a microorganism containing an O-acylhomoserine sulfhydrylase mutant in which at least one of the amino acids corresponding to the 4th, 90th, 106th, 242nd, 261st, 290th and 395th positions from the N-terminus of SEQ ID NO: 1 is substituted with another amino acid, or a polynucleotide encoding the same, in a medium containing O-acylhomoserine; A method for producing an O-acylhomoserine sulfhydrylase reactant.

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