# Patent

## Title
O-phosphoserine sulfhydrylase mutants and method for production of cysteine using the same

## Abstract

Disclosed is an O-phosphoserine sulfhydrylase (OPSS) mutant which has a Mycobacterium smegmatis-derived amino acid sequence corresponding to that of SEQ ID NO: 1 which is devoid of three to seven C-terminal amino acid residues. Also, a nucleic acid molecule encoding the OPSS mutant, an expression vector carrying the nucleic acid molecule, and a transformant transformed with the expression vector are disclosed. In addition, a method is provided for producing cysteine in which O-phospho-L-serine (OPS) is reacted with a sulfide in the presence of the OPSS mutant. The OPSS mutant has improved enzymatic activity and can be applied to the environmentally friendly production of L-cysteine through a simple enzymatic conversion reaction.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the priority benefit of Korean Patent Application No. 10-2010-0102665, filed Oct. 20, 2010. The contents of this patent application is incorporated herein by reference in its entirety.
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is HANO—003—00US_ST25.txt. The text file is 27 KB, was created on Oct. 20, 2011, and is being submitted electronically via EFS-Web.
Technical Field
The present invention relates to an O-phosphoserine sulfhydrylase (also referred to as “OPSS”) mutant which has a Mycobacterium smegmatis-derived amino acid sequence corresponding to that of SEQ ID NO: 1 which is deleted three to seven C-terminal amino acid residues. The present invention also relates to a nucleic acid molecule encoding the OPSS mutant, an expression vector carrying the nucleic acid molecule, and a transformant transformed with the expression vector. In addition, the present invention is concerned with a method for the production of cysteine by reacting O-phospho-L-serine (OPS) with a sulfide in the presence of the OPSS mutant.
BACKGROUND ART
L-cysteine is an amino acid that plays an important role in sulfur metabolism in all living organisms. It is used in the biosynthesis of proteins, such as hair keratin, glutathione, biotin, methionine, and other sulfur-containing metabolites as well as serving as a precursor of coenzyme A. In addition, the biosynthesis of cysteine is known to be closely associated with the biosynthesis of other amino acids including L-serine, L-glycine, and L-methionine. Industrially, L-cysteine and its derivatives find applications in a variety of fields including the pharmaceutical industry (for treatment of bronchial diseases), the cosmetics industry (in hair shampoo, compositions for permanent waves, etc.), and the food industry (antioxidants, flavorant enhancers, dough aids, etc.).
Traditionally, L-cysteine was obtained industrially by acid hydrolysis of human hair or animal feathers (Biotechnology of the Amino Acids Production edited by Ko Aida, p 217-223, 1986). However, not only does the production of cysteine from hair or feathers ensure a yield of as low as 7-8%, but also the use of hydrochloric acid or sulfuric acid produces a significant amount of environment polluting waste. Further, consumers may have a strong aversion to extraction from hair or feathers. These problems have caused a push for the development of environmentally friendly production processes of L-cysteine, leading to the fermentation of L-cysteine utilizing microorganisms.
Representative among the microbial production of L-cysteine is the biological conversion of D, L-ATC using a microorganism (Ryu O H, Ju J Y, and Shin C S, Process Biochem., 32:201-209, 1997). This conversion process is, however, difficult to apply industrially due to the low solubility of the precursor D, L-ATC. Another method of L-cysteine production is direct fermentation using E. coli (Patent No. EP 0885962B; Wada M and Takagi H, Appl. Microbiol. Biochem., 73:48-54, 2006). Excessive accumulation of L-cysteine within microorganisms incur intracellular toxicity, resulting in the limitation for production of L-cysteine at a high concentration. To overcome this drawback, L-cysteine-exporting proteins were employed, however there have been no significant improvements in productivity.
Referring to the biosynthesis pathway of L-cysteine in bacteria and plants, O-acetyl-serine (OAS) acts as an intermediate precursor providing the carbon backbone of L-cysteine (Kredich N M and Tomkins G M, J. Biol. Chem., 241: 4955-4965, 1966). The enzyme O-acetylserine sulfhydrylase (OASS), using hydrogen sulfide as a sulfur donor, catalyses the conversion of O-acetylserine to S-sulfocysteine and finally to cysteine, releasing acetate. Alternatively, SO4 may be reduced to thiosulfate for use as a sulfur donor in cysteine production (Nakamura T, Kon Y, Iwahashi H, and Eguchi Y, J. Bacteriol., 156: 656-662, 1983). The cysteine biosynthesis pathway via OAS use the two enzymes of serine acetyltransferase (CysE), which catalyzes the conversion of serine to OAS, and cysteine synthase (CysK), which catalyzes the conversion of OAS to cysteine. Notably among them serine acetyltransferase (CysE) is highly sensitive to feedback inhibition by the final product cysteine (Wada M and Takagi H, Appl. Microbiol. Biochem., 73:48-54, 2006). Therefore, an altered enzyme that is insensitive to feedback inhibition is needed, however is difficult to develop.
DISCLOSURE
Technical Problem
Leading to the present invention, intensive and thorough research into the production of L-cysteine at a high yield conducted by the present inventors aimed at overcoming the problems encountered in the prior art, resulted in the finding that there exists O-phosphoserine sulfhydrylase (OPSS) in Aeropyrum pernix, Mycobacterium tuberculosis, and Trichomonas vaginalis that takes an O-phospho-L-serine (OPS)-specific pathway, rather than the OAS-specific pathway, to synthesize L-cysteine (Mino K and Ishikawa K, FEBS letters, 551: 133-138, 2003; Burns K E, Baumgart S, Dorrestein P C, Zhai H, McLafferty F W, and Begley T P, J. Am. Chem. Soc., 127: 11602-11603, 2005; Westrop G D, Goodall G, Mottram J C, and Coombs G H, J. Biol. Chem., 281: 25062-25075, 2006) and that the OPSS of M. tuberculosis, which catalyzes the conversion of OPS to cysteine with the additional enzymes mec+ and cysO, can use Na2S as a sulfur donor in converting OPS to cysteine even in the absence of the additional enzymes when five C-terminal amino acid residues are removed therefrom (Agren D, Schnell R and Schneider G, FEBS letters, 583: 330-336, 2009).
Technical Solution
It is therefore an object of the present invention to provide an O-phosphoserine sulfhydrylase (also referred to as “OPSS”) mutant which has a Mycobacterium smegmatis-derived amino acid sequence corresponding to that of SEQ ID NO: 1 which is deleted three to seven C-terminal amino acid residues.
It is another object of the present invention to provide a nucleic acid molecule encoding the OPSS mutant
It is a further object of the present invention to provide an expression vector carrying the nucleic acid molecule.
It is still a further object of the present invention to provide a transformant transformed with the expression vector.
It is still another object of the present invention to provide a method for the conversion of O-phospho-L-serine into cysteine with a sulfide in the presence of the OPSS mutant.
Advantageous Effects
As described above, the OPSS mutant with improved enzymatic activity which is essential for the enzymatic conversion of O-phosphoserine into L-cysteine can be used to produce L-cysteine from OPS on mass scale at a high yield in a simple manner.



DESCRIPTION OF DRAWINGS
FIG. 1 is a graph showing the activity of OPSS according to temperature.
FIG. 2 is a set of graphs showing the pH sensitivity of OPSS.
FIG. 3 is a photograph showing the expression level of the enzyme in a pET system and a pCL-Pcj1 system as analyzed by SDS PAGE.
FIG. 4 is a graph showing the enzymatic activity of OPSS to convert purified OPS fermentation broth into cysteine.
FIG. 5 is a graph showing the enzymatic activity of OPSS to convert OPS fermentation broth into cysteine.
FIG. 6 is a graph showing the production of OPS and cysteine which is converted by OPSS using OPS as a substrate on a 1 L jar scale.
FIG. 7 is a schematic view showing the map of a pCL-Pcj1 expression vector carrying a gene coding for an OPSS mutant.



BEST MODE
In accordance with an aspect thereof, the present invention provides a Mycobacterium smegmatics-derived OPSS mutant with the same amino acid sequence as that of SEQ ID NO: 1, with the exception of lacking 3 to 7 C-terminal amino acid residues of the wild-type O-phosphoserine sulfhydrylase amino acid sequence.
In one embodiment of the present invention, the OPSS mutant of the present invention may have one of the amino acid sequences of SEQ ID NOS: 2, 3, and 4. The OPSS mutant having an amino acid sequence of SEQ ID NO: 2 may be further modified to have a substitution of the proline residue (Pro) at position 77 with a serine residue (Ser). In addition, the OPSS mutant having an amino acid sequence of SEQ ID NO: 2 may be further modified to have a substitution of the threonine residue (Thr) at position 131 with an alanine residue (Ala), the lysine residue (Lys) at position 137 with an asparagine residue (Asp) and the threonine residue (Thr) at position 238 with a serine residue (Ser). Further, amino acid sequences that share homology of at least 50% preferably 60%, 70%, 75%, and 80%, more preferably 85%, 90% and 95%, and most preferably 97% to 99% with the above-mentioned mutants, fall within the scope of the present invention.
The term “Mycobacterium smegmatics” refers to a bacillus-shape strain in the phylum Actinobacteria that is fine, straight, or slightly curved, has irregular branches, and can be stained with the basic dye aniline. In the present invention, it was found that when its amino acid sequence was modified, the O-phosphoserine sulfhydrylase derived from the strain could catalyze the biosynthesis of L-cysteine with an increased yield.
As used herein, the term “O-phosphoserine sulfhydrylase (OPSS)” refers to an enzyme that catalyzes the conversion of OPS into cysteine. The enzyme was first found in Aeropyrum pernix and named (Mino K and Ishikawa K, FEBS letters, 551: 133-138, 2003, SEQ ID NO: 6).
Various well-known methods may be used to obtain OPSS. Illustrative examples of the methods include, but are not limited to, gene synthesis techniques based on codon optimization by which enzymes of interest can be obtained at a high yield, and bioinformatic screening methods of useful enzyme resources based on massive stores of genetic information of microorganisms. In one embodiment of the present invention, OPSS enzymes that utilize OPS as a substrate to synthesize cysteine were selected from various microbes. In this regard, cell pellets obtained using suitable medium and culture conditions known in the art were (lyzed), followed by the purification of the supernatant containing the enzyme to afford the OPSS enzyme.
As used herein, the term “mutant” refers to a culture or an individual that shows a heritable or non-heritable stable alteration in phenotype. When used in conjunction with OPSS(O-phosphoserine sulfhydrylase), the term “mutant” is intended to mean an OPSS enzyme that is genetically altered such that its activity can be effectively improved, compared to the wild-type.
On the basis of the report that Mycobacterium tuberculosis H37Rv-derived OPSS mutants deleted five C-terminal amino acid residues show increased affinity for an S2− group-containing sulfur source even in the absence of additional enzymes, an OPSS mutant was prepared by deleting five C-terminal amino acid residues from the OPSS of Mycobacterium smegmatics. The OPSS mutant according to the present invention may have a Mycobacterium smegmatis-derived amino acid sequence corresponding to that of SEQ ID NO: 1 which is deleted three to seven, preferably five, C-terminal amino acid residues.
In one embodiment of the present invention, the mutant having the amino acid sequence of SEQ ID NO: 2 was observed to exhibit a conversion rate of 100% within one hour after it was applied to the conversion of OPS into cysteine (Table 5).
Amino acid substitution may further increase the enzymatic activity of the OPSS mutant of the present invention. As long as it is well known in the art, any method may be used to improve the enzyme. In the present invention, preferably, random mutagenesis was employed to bring about an improvement in the enzymatic activity of the OPSS mutant. In detail, after OPSS was allowed to undergo random mutagenesis, mutants with improved enzyme activity were selected using the screening system developed on the basis of HTS (high-throughput screening) by the present inventors. As a result, the OPSS mutants, Msm-T-HA2 and Msm-T-EP3, which have improved enzymatic activity, were obtained by conducting HTS screening on an Msm-T gene. Msm-T-HA2 is an OPSS mutant that has the same amino acid sequence as SEQ ID NO: 2, with the exception that the proline residue (Pro) at position 77 is substituted with a serine residue (Ser). Msm-T-EP3 is an OPSS mutant that has the same amino acid residue as SEQ ID NO: 2, with the exception that substitution mutation occurs with an alanine residue (Ala) for the threonine residue (Thr) at position 131, with an asparagine residue (Asp) for the lysine residue (Lys) at position 137, and with a serine residue (Ser) for the threonine residue (Thr) at position 238. Preferably, the OPSS mutants Msm-T-HA2 and Msm-T-EP3 have amino acid sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
In one embodiment of the present invention, both Msm-T-HA2 and Msm-T-EP3 were found to exhibit higher enzymatic activity than that of Msm-T having an amino acid residue of SEQ ID NO: 2 (Tables 5 and 6). In detail, the OPSS mutants Msm-T-HA2 and Msm-T-EP3 were measured to exhibit 5-fold and 1.2-fold increased conversion rates in an early stage of the conversion reaction, compared to the control Msm-T. In addition, even when the Msm-T-HA2 mutant of which cysteine synthesis activity is 4-fold higher than that of the Msm-T enzyme and was used in an amount corresponding to 40% of Msm-T, the final cysteine conversion rate was similar.
The term “homology”, as used herein, is intended to refer to the percent of identity between two polypeptides. The correspondence between one sequence to another can be determined by techniques known in the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single strand-specific nuclease, and size determination of the digested fragments.
As used herein, the term “homologous” in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a “common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.). Such proteins (and their encoding genes) have sequence homology, as reflected by their sequence similarity. However, in common usage and in the instant application, the term “homologous,” when modified with an adverb such as “highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin.
As used herein, the term “sequence similarity” in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin. In a specific embodiment, two amino acid sequences are “substantially homologous” or “substantially similar” when at least 21% (preferably at least about 50%, and most preferably about 75%, 90%, 95%, 96%, 97%, or 99%) of the amino acids match over the defined length of the amino acid sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. The hybridization conditions defined are within the scope of the art (e.g. Sambrook et al., 1989, infra).
The OPSS mutants of the present invention can catalyze the transfer of a thiol group (SH group) to OPS to produce cysteine. Preferably, the conditions that allow the OPSS mutants of the present invention to exert their optimal activity include i) the presence of 0-2 mM PLP (pyridoxal-5′-phosphate) or 0-100 mM DTT (dithiothreitol) as a cofactor, ii) a reaction temperature of from 25 to 60° C., and iii) pH of from 6.0 to 10.0, but are not limited thereto.
An assay for the enzymatic activity of OPSS that catalyzes the synthesis of cysteine by transferring a thiol group to the OPS substrate was disclosed together with the nomenclature of Ape-OPSS (Ape-O-phosphoserine sulfhydrylase). Particularly, because Ape-OPSS has the activity of converting OAS into cysteine by the transfer of a thiol group, the assay is based on the measurement of the activity of E. coli cysteine synthesis enzyme (OASS, O-acetylserine sulphydrylase, EC 4.2.99.8)) (Mino K and Ishikawa K, FEBS letters, 551: 133-138, 2003).
In the assay according to one embodiment of the present invention, PLP, which provides a thiol group for OAS (O-acetylserine) or OPS, serving as a cofactor of OASS or OPSS in the cysteine conversion, is added at a concentration of 0.2 mM. Also, DTT is added not only to prevent the oxidation of air-exposed cysteine into cysteine, but also to quantitate the amount of cysteine already oxidized, thanks to its reducing power. Preferably, when 25 mM DTT or 0.2 mM PLP was added, the cysteine conversion rate was increased by 2.3 times. That is, PLP and DTT have positive influences on the conversion of OPS into cysteine.
The enzymes Ape-OPSS, Mtb-OPSS, and Mtb-T have an optimal reaction temperature of 60° C. or 37° C., with an optimal pH of 7.4, as reported previously (Mino K and Ishikawa K, FEBS letters, 551: 133-138, 2003; Agren D, Schnell R and Schneider G, FEBS letters, 583: 330-336, 2009). Based on the report, the conversion reaction conditions for the OPSS mutants with improved enzymatic activity can be optimized.
In one embodiment of the present invention, the OPSS mutants can catalyze the conversion at a temperature of from 37° C. to 80° C. In detail, the Ape-OPSS enzyme from Archea spp., which can grow even at high temperatures, shows a higher enzymatic activity at 60° C. than at 37° C. Also, the high-heat stability of the enzyme itself leads to an optimal temperature of 60° C. On the other hand, Msm-T shows optimal enzymatic activity at 37° C. and is vulnerable to heat treatment at 60° C. The OPSS enzymes were found to retain conversion activity over a pH range of from 6.0 to 10.0. Optimal enzymatic activity was detected at pH7.4 in Ape-OPSS and a pH of 8.0 to 9.0 in Msm-T. Hence, Msm-T is stable over a wider range of pH than is Ape-OPSS.
In accordance with another aspect thereof, the present invention provides a nucleic acid molecule encoding the OPSS mutant.
As used herein, the term “nucleic acid molecule” is intended to encompass DNA and RNA molecules. Nucleotides, which make up the structural units of nucleic acid molecules, include not only naturally occurring nucleotides, but sugar moiety- or base moiety-modified analogues (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).
In accordance with a further aspect thereof, the present invention provides an expression vector carrying the nucleic acid molecule.
A “vector” refers to any vehicle for the cloning of and/or transfer of a nucleic acid into a host cell. A vector may be a replicon to which another DNA segment may be attached so as to bring about the replication of the attached segment. A “replicon” refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control. The term “vector” includes both viral and non-viral vehicles for introducing the nucleic acid into a host cell in vitro, ex vivo, or in vivo. The term “vector” may also include minicircle DNAs. For example, the vector may be a plasmid without bacterial DNA sequences. The removal of bacterial DNA sequences that are rich in CpG regions has been shown to decrease transgene expression silencing and resulting in more persistent expression from plasmid DNA vectors (see e.g., Ehrhardt, A. et al. (2003) Hum Gene Ther 10: 215-25; Yet, N. S. (2002) MoI Ther 5: 731-38; Chen, Z. Y. et al. (2004) Gene Ther 11: 856-64). The term “vector” may also include transposons such as Sleeping Beauty (Izsvak et al. J. MoI. Biol. 302:93-102 (2000)), or artificial chromosomes.
As a vector, the pET system (Novagen) using a T7 promoter is well known in the art. In the present invention, various expression systems known in the art may be used without limitation.
In one embodiment of the present invention, an expression system using a CJ1 promoter, developed by the present inventors, for expressing an exogenous gene (see, Korean Patent Laid-Open Publication No. 10-2006-0068505, FIG. 7) was employed.
In one embodiment of the present invention, the expression levels of OPSS between the pET system comprising a T7 promoter and the CJ1 system comprising a CJ1 promoter were compared given the same conditions. As a result, the CJ1 system showed a higher expression level of OPSS than did the pET system (FIG. 3). In addition, overexpression of OPSS required a low temperature (18° C.) and a long period of time in the pET system, but a high temperature (37° C.) and a short period of time in the CJ1 system. Therefore, it is preferred that the CJ1 promoter is used to effectively obtain OPSS, but the present invention is not limited thereto.
In accordance with a still further aspect thereof, the present invention provides a transformant transformed with the expression vector.
As used herein, the term “transformation” in all its grammatical forms and spelling variations refers to the artificial genetic alteration of a cell resulting from the introduction of a foreign gene to the host cell so that the introduced gene can replicate itself or as a factor incorporated into the chromosome.
The vector of the present invention can be introduced into host cells by suitable standard techniques known in the art, examples of which include but are not limited to electroporation, calcium phosphate co-precipitation, retroviral infection, microinjection, DEAE-dextran, and cationic liposome calcium.
The term “host cell transformed with a recombinant vector,” as used herein, refers to a host cell that anchors a recombinant vector therein carrying a gene of interest. The host cell suitable for use in the present invention may be prokaryotic or eukaryotic. Examples include enterobacteria and coryneform bacteria, with preference for Escherichia spp. and Serratia spp., with the highest preference being E. coli. 
In accordance with still another aspect thereof, the present invention provides a method for production of cysteine, comprising of converting OPS with a sulfide in the presence of the OPSS mutant of the present invention.
The mutant of the present invention may be applied to the mass production of cysteine. When the transformant expressing the mutant is used, the mass production of cysteine may be accomplished under optimal culture conditions that are well known in the art. Therefore, the method for mass production of cysteine comprises culturing the transformant in an optimal condition that is well known in the art.
For use as a substrate, OPS may be in pure form or may be in the form of a fermentation culture containing OPS. Pure OPS may be commercially available, as identified in Catalog No. P0878 from Sigma-Aldrich or CAS407-41-0 from Wako. However, the OPS-containing culture obtained by microbial fermentation has economic advantages over commercially available pure OPS in that the OPS-containing culture can be used without additional purification and the cofactor PLP necessary for the conversion can be obtained in the fermented culture.
Any sulfur compound may be used in the present invention, as long as it may be converted to a thiol group (SH). Preferably, Na2S, H2S, or S2O3, eitherin the form of liquid or gas, may be used.
In one embodiment of the present invention, Na2S was used as a sulfur source. Na2S may be added at a molar concentration 0.1 to 3 times as high as that of OPS used in the enzymatic conversion. In detail, Msm-T-HA2, which has a cysteine conversion rate of 80%, which is equivalent to that of Msm-T, is used at a concentration of 50 μg/mL in a reaction condition comprising 50 mM OPS fermentation broth or 60 mM purified OPS fermentation broth, 100 mM or 120 mM Na2S, and 0.2 mM PLP.
It will be apparent to and readily understood by those skilled in the art that an enzyme conversion process can be optimized and scaled up with highly active enzymes. In one embodiment, when 19.317 g/L OPS fermentation broth was incubated in the presence of 50 mg of Msm-T-HA2, cysteine was produced at a concentration of up to 9.075 g/L. In a 1 L jar, OPS was converted into cysteine at a rate of 71.83% in the presence of the mutant (FIG. 6).
Abbreviation and Terminology
To better explain the present invention and to instruct those technic in the art to perform the present invention, a description is given of the following words and methods. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise,” “comprising,” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of “including, but not limited to.” The singular terms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. For example, the term “comprising a cell” means “including one cell or a plurality of such cells, but not limited thereto.”
When used in reference to a list of two or more items, the word “or” covers all of the following interpretations of the word: any of the items in the list, all of the items in the list, and any combination of the items in the list. Unless stated otherwise, all of the technical and scientific terms used herein have the same meanings as are understood to those skilled in the art to which the present invention belongs. Pertinent methods and materials are disclosed below, but similar or equivalent methods and materials may be utilized in the practices or experiments of the present invention. The materials, methods, and examples given below are set forth to illustrate, but are not to be construed as limiting the present invention. Other features and advantages of the present invention will be apparent from the following description and accompanying claims.
Deletion: A mutation in which one or more nucleotides or amino acid residues has been removed from a nucleic acid molecule or a protein, respectively.
MODE FOR INVENTION
A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed to limit the present invention.
Example 1
Development of OPSS
Aeropyrum pernix, Mycobacterium tuberculosis, and Trichomonas vaginalis are reported to have OPSS, an enzyme that use OPS, instead of OAS in E. coli, as a substrate for the synthesis of cysteine (Mino K and Ishikawa K, FEBS letters, 551: 133-138, 2003; Burns K E, Baumgart S, Dorrestein P C, Zhai H, McLafferty F W, and Begley T P, J. Am. Chem. Soc., 127: 11602-11603, 2005; Westrop G D, Goodall G, Mottram J C, and Coombs G H, J. Biol. Chem., 281: 25062-25075, 2006). Based on the report, the present inventors found two types of OPSS that convert OPS into cysteine, from Aeropyrum pernix and Mycobacterium tuberculosis H37Rv. Among them, the Mycobacterium tuberculosis H37Rv-derived OPSS enzyme was used for screening amino acid homology. As a result, three OPSS mutants Msm-OPSS, Rjo-OPSS, and Nfa-OPSS were secured from Mycobacterium smegmatis str. MC2 155, Rhodococcus jostii RHA1, and Nocardia farcinica IFM 10152, respectively.
To obtain OPSS from each strain, a pET28a vector system (Novagen), which is typically used for enzyme expression, was constructed. Each templates and primers for use in cloning the five different OPS sulfhydrylase enzymes and the resulting recombinant plasmids are summarized in Table 1, below. Suitable combinations of template and the primers, as given in Table 1, were used for PCR for amplifying each OPSS genes. The PCR products and the pET28a vector were digested with NdeI and HindIII (37° C. for 3 hours). Each of the gene fragments was ligated to the digested pET28a vector (Novagen). Base sequencing confirmed the construction of the expression vectors carrying the each OPSS genes. The enzyme expression vectors were introduced into E. coli (DE3) to produce strains capable of expressing five OPSS enzymes. Enzyme names are given in Table 1, below.










TABLE 1





Enzyme
Vector
Template
Primer







Ape-OPSS
pET28a-Ape-
Synthetic DNA
SEQ ID NOS: 7 and 8



OPSS


Mtb-OPSS
pET28a-Mtb-
Mtb genomic
SEQ ID NOS: 9 and 10



OPSS
DNA


Msm-OPSS
pET28a-Msm-
Msm genomic
SEQ ID NOS: 11 and 12



OPSS
DNA


Rjo-OPSS
pET28a-Rjo-
Rjo genomic
SEQ ID NOS: 13 and 14



OPSS
DNA


Nfa-OPSS
pET28a-Nfa-
Nfa genomic
SEQ ID NOS: 15 and 16



OPSS
DNA









Expression of the enzymes was conducted according to the instructions of the pET system manufacturer (Novagen). Single colonies of each strain from the LB plates were inoculated into 5 mL of LB broth and incubated at 37° C. for 16 hours while shaking at 200 rpm. The cultures were transferred to 25 mL of fresh LB broth (in 250 mL flasks) and incubated to an OD600 of 0.5 ̃0.6 (for 2 ̃3 hours) in the same condition, immediately after which 1 mM IPTG was added to the media to induce the enzymes to be expressed during incubation at 18° C. for 18 hours while shaking at 120 rpm. The enzymes were purified using Ni-NTA columns for His-tag, with the aid of His SpinTrap (GE Healthcare). Of the five OPSS enzymes isolated, four were found to be in soluble forms, with one (Rjo-OPSS) being an inclusion body, as analyzed by 14% SDS-PAGE electrophoresis.
Example 2
Assay of OPSS for Cysteine Synthesis Activity
The four OPSS enzymes obtained from various microorganism strains were assayed for the ability to catalyze the conversion of OPS to cysteine. With regard to assay conditions and methods (cysM enzyme assay), reference was made to previous reports (Mino K and Ishikawa K, FEBS letters, 551: 133-138, 2003; Burns K E, Baumgart S, Dorrestein P C, Zhai H, McLafferty F W, and Begley T P, J. Am. Chem. Soc., 127: 11602-11603, 2005; Westrop G D, Goodall G, Mottram J C and Coombs G H, J. Biol. Chem., 281: 25062-25075, 2006). The amount of the substrate used is represented by a unit of mL. Assay conditions for enzyme activity are summarized in Table 2, below.












TABLE 2










OPS



Stock sol'n
Final Conc.
Blank
sulfhydrase









6xhis-enzyme

—
40 (50 mg)



1M HEPES
100 mM HEPES
100
100



(pH 7.4)



0.5M Na2S
 10 mM Na2S
20
20



10 mM PLP
 0.2 mM PLP
20
20



100 mM OPS
 5 mM OPS
0
50



DW

790
750



Total

1000
1000










Reaction solutions excepting the enzymes were incubated at 37° C. for 5 min, after which 50 mg of purified OPSS was added to the reaction solution. At predetermined times during incubation at 37° C., 100 mL of the enzyme reactions were taken and mixed with 100 mL of 33.2% TCA to stop the enzymatic reaction. The cysteine concentrations of the enzyme reactions were quantitatively analyzed by measuring absorbance at OD560 according to the Gaitonde method. Cysteine synthesis activities of the four different OPS sulfhydrylase enzymes are summarized in Table 3, below. The cysteine synthesis titers of the OPSS enzymes are expressed as cysteine conversion rates with reaction time.









TABLE 3









Cysteine Conversion Rate (%)











10 min
30 min
60 min
















Ape-OPSS
63.4
89.7
97.4



Mtb-OPSS
1.7
4.8
10.1



Msm-OPSS
12.8
25
43.7



Nfa-OPSS
0.1
0.1
0.2










As can be seen in Table 3, the OPSS enzymes derived from Aeropyrum pernix and Mycobacterium tuberculosis H37Rv were confirmed to have the activity of using OPS as a substrate to synthesize cysteine. The cysteine synthesis activity of the novel Mycobacterium smegmatis str. MC2 155-derived OPSS, which was obtained by screening amino acid homology with the Mtb-OPSS enzyme, was first found. On the other hand, the novel Nocardia farcinica IFM 10152-derived OPSS, obtained by the homology screening, exhibited insufficient activity of converting O-phosphoserine into cysteine.
As seen in the data of Table 3, the conversion rate from OPS into cysteine of Ape-OPSS reached near 100% in one hour.
The final conversion rate of the Msm-OPSS enzyme, which was newly selected through enzyme screening on the basis of previously reported Mycobacterium tuberculosis H37Rv-derived OPSS, was 43.7% which is 4.3 times as high as that of Mtb-OPSS.
Example 3
Preparation of Mtb-T and Msm-T, Mutants Deleted 5 C-Terminal Amino Acid Residues of Mtb-OPSS and Msm-OPSS
Mycobacterium tuberculosis H37Rv-derived OPSS (Mtb-OPSS), which catalyzes the conversion of OPS to cysteine with the aid of the additional enzymes mec+ and cysO, is able to use an S2− containing sulfur source in converting OPS to cysteine even in the absence of the additional enzymes when five C-terminal amino acid residues are removed therefrom. On the basis of this fact, Mtb-T (SEQ ID NO: 24), which can rapidly convert OPS in the presence of S2− as a sulfur source, was obtained. From Msm-OPSS that shares a high amino acid homology with Mtb-OPSS, Msm-T was also obtained by deleting 5 C-terminal amino acid residues. Expression vectors carrying the two enzyme mutants were constructed. In this regard, pfu PCR was performed on the genomic DNA of Mycobacterium tuberculosis H37Rv and Mycobacterium smegmatis str. MC2 155 in the presence of each pairs of primers of SEQ ID NOS: 17 and 18 and SEQ ID NOS: 19 and 20. The OPSS gene fragments thus obtained were treated with NdeI and HindIII and were cloned into the pET28a vector digested with the same restriction enzymes to construct recombinant expression vectors named pET28a-Mtb-T and pET28a-Msm-T, respectively. The recombinant expression vectors were introduced into E. coli (DE3). The expression of the two OPSS mutants obtained under the same condition as in Example 1 was confirmed by 14% SDS PAGE. As a result, Mtb-T (SEQ ID NO: 5) and Msm-T (SEQ ID NO: 2) were obtained.
Example 4
Assay of Mtb-T and Msm-T for Cysteine Synthesis Activity
Mtb-T and Msm-T, obtained above, were evaluated for enzymatic activity by measuring final cysteine conversion rates. Enzymatic activity was assayed in the same manner as in Example 2. The converted cysteine was quantitatively analyzed using the Gaitonde method and the results are summarized in Table 4, below.









TABLE 4









Cysteine Conversion Rate (%)











10 min
30 min
60 min
















Mtb-T
9.5
18.6
37.1



Msm-T
20.3
54.6
100










As seen in Table 4, the Msm-T mutant allowed the conversion of cysteine from the substrate at a rate of 100% in one hour.
Example 5
Assay for Cysteine Synthesis Activity of Msm-T-Derived Mutants Msm-T-HA2 and Msm-T-EP3
On the basis of Msm-T, which can convert OPS into cysteine at a rate of 100%, two enzyme mutants with improved activity were obtained using an enzyme evolutionary method, and named Msm-T-HA2 (SEQ ID NO: 25) and Msm-T-EP3 (SEQ ID NO: 26) Random mutagenesis using hydroxylamine treatment and an error-prone PCR kit (Clontech. Diversity PCR random mutagenesis kit) were introduced into Msm-T to construct a library of Msm-T mutants which were then screened to select OPSS mutants with improved enzymatic activity.
The OPSS mutants Msm-T-HA2 and Msm-T-EP3, obtained by HTS screening on an Msm-T gene, were found to have amino acid sequences of SEQ ID NOS: 3 and 4, respectively, as analyzed by base sequencing.
The mutants Msm-T-HA2 and Msm-T-EP3 and the control Msm-T were assayed for enzymatic activity, and the results are summarized in Table 5 and 6. The enzyme activity assay was conducted in the same manner as in Example 2.









TABLE 5









Cysteine Conversion Rate (%)











10 min
30 min
60 min
















Msm-T
20.3
54.6
100



Msm-T-HA2
101.6
101.2
97.5



Msm-T-EP3
24
65.7
100.7





















TABLE 6







Msm-T
Msm-T-HA2
Msm-T-EP3



















Specific activity
14.96
44.82
16.72


(Cysteine (μmole)/min/mg)









As can be seen in Tables 5 and 6, the OPSS mutants Msm-T-HA2 and Msm-T-EP3, obtained by an enzyme evolutionary method, were measured to exhibit 5-fold and 1.2-fold increased conversion rates, respectively, within 10 min after initiation of the reaction, compared to the control Msm-T. Also, they were compared in terms of specific activity (product concentration/time/enzyme amount), which is widely used in the determination of enzyme activity. The specific activity of Msm-HA2 was three-fold increased compared to that of the control Msm-T, indicating that the OPSS mutant is improved in enzyme activity, with the capacity of synthesizing cysteine in higher amounts per time per unit enzyme.
Example 6
Requirement of Cofactor for OPSS Activity
To examine the effect of cofactors on the cysteine conversion of OPSS, the cysteine conversion rate of Msm-T was measured in the absence or presence of PLP and DTT. In this regard, the substrates of 50 mM OPS broth and 100 mM Na2S were reacted at 37° C. for 30 min in the presence of 25 mM DTT or 0.2 mM PLP. The cysteine thus produced was quantitatively analyzed using the Gaitonde method. The results are summarized in Table 7, below.










TABLE 7







Msm-T
Cysteine Conversion Rate (%)









(−) PLP, (−) DTT
23.62



(+) PLP, (−) DTT
33.21



(−) PLP, (+) DTT
40.08



(+) PLP, (+) DTT
54.65










As seen in Table 7, the cysteine conversion rate in the presence of both PLP and DTT was 2.3 times as large as that in the absence of both PLP and DTT. Thus, both PLP and DTT were observed to have a positive influence on the conversion.
Example 7
Activity of OPSS by Temperature
The cysteine conversion rates of Ape-OPSS and Msm-T according to temperatures were examined. The enzymatic activity at 37° C. and 60° C. was measured at 2, 5, 10, 30, and 60 min after reaction. The reaction was conducted under the condition of 100 mM HEPES (pH 7.4), 5 mM OPS, 10 mM Na2S, 0.2 mM PLP, and CysM 50 μg/mL. The amount of produced cysteine was determined using the Gaitonde method. The results are shown in FIG. 1.
In the condition of a buffer, as shown in FIG. 1, Ape-OPSS showed a faster initial reaction rate at 37° C. as well as higher reactivity at 60° C. than did Msm-T.
Example 8
Heat Stability of OPSS
Ape-OPSS and Msm-T were analyzed for heat stability. Each of the enzymes was diluted to a concentration of 2 mg/mL in an OPS broth and thermally treated at 37° C. and 60° C. for 10, 30, 60, 120, and 240 min, followed by reaction at 37° C. for 30 min under the condition of 5 mM OPS, 10 mM Na2S, 0.2 mM PLP, and 100 mM HEPES (pH 7.4). For this reaction, 10 μg/mL Ape-OPSS and 50 μg/mL Msm-T were employed. The amounts of the produced cysteine were measured using the Gaitonde method and are summarized in Table 8, below.









TABLE 8









Relative activity (%)



Heating time (min)














(—)
10 min
30 min
60 min
120 min
240 min

















Ape-OPSS
100
102
107
100
107
101


Msm-T
100
82
50
32
19
8









In spite of heat treatment at 60° C. for 4 hours, as seen in Table 8, Ape-OPSS was observed to retain its activity intact while the activity of Msm-T was maintained at 37° C., but decreased by 50% upon heat treatment at 60° C. for 30 min.
An examination was made on the retention of enzymatic activity at 37° C. when Msm-T was used in an amount of 50 μg/mL, which is a practical concentration in OPS broth. In the absence of Na2S, 50 μg/mL Msm-T was treated, together with 50 mM OPS broth and 0.2 mM PLP, at 37° C. for 0.5, 1, 2, 4, and 6 hours, after which Na2S was added to induce the enzymatic reaction. After the reaction for 30 min, the activity of Msm-T was measured. The amounts of the produced cysteine were determined using the Gaitonde method and are given in Table 9, below.









TABLE 9









Time














(—)
30 min
60 min
120 min
240 min
360 min

















Cysteine
100
88
73
47
11
3


Conversion Rate


(%)









As can be seen in Table 9, the activity of Msm-T was decreased below 50% 2 hours after reaction at 37° C. in OPS broth.
Example 9
pH Sensitivity of OPSS
The cysteine conversion rates of Ape-OPSS and Msm-T according to pH were measured. In 100 mM of buffer, Ape-OPSS and Msm-T, each having a concentration of 50 μg/mL, were subjected to reaction at 37° C. for 10 min. In this regard, a K-phosphate buffer with a pH of 6.4/7.0/7.4/8.0, a Tris-HCl buffer with a pH of 7.0/7.4/8.0/8.5/8.8, and a Na-carbonate buffer with a pH of 8.0/8.5/9.0/10.0 were used. The quantitative analysis of the produced cysteine was conducted using the Gaitonde method. The results are summarized in FIG. 2.
As seen in FIG. 2, Msm-T exhibited the highest activity at a pH of from 8.0 to 9.0 irrespective of buffer. As for Ape-OPSS, its highest activity was detected in K-phosphate (pH 7.4), with an optimal pH differing from one buffer to another.
Example 10
Effect of Ions on the Activity of OPSS
Effects of ions on the activity of the OPSS enzymes were examined as follows. In a reaction mixture containing 5 mM OPS, 10 mM Na2S, 0.2 mM PLP, and 100 mM HEPES (pH 7.4), the enzymes were subjected to reaction at 37° C. for 30 min in the presence of (NH4)2SO4 [1, 3, 5, 10, 20 g/L], KH2PO4 [0.5, 1, 2, 4, 8 g/L], or NH4Cl [0.2, 0.5, 1, 2 g/L]. Ape-OPSS and Msm-T were used at a concentration of 10 μg/mL and 50 μg/mL, respectively. The amounts of the produced cysteine were determined using the Gaitonde method and are summarized in Table 10, below.










TABLE 10









Relative activity (%)










NH4Cl
Ape-OPSS
Msm-T












0
100.00
100.00


0.2
86.26
91.49


0.5
73.35
91.30


1
49.11
67.11


2
27.72
47.12









No changes were detected in the cysteine conversion rate when (NH4)2SO4 or KH2PO4 was added to the reaction mixture. On the other hand, as can be seen in Table 10, the cysteine conversion rate was decreased with an increase in NH4Cl concentration. Particularly, the maximal enzyme activity was decreased by more than 70% when 2 g/L NH4 was added. Therefore, NH4Cl and NH4 were observed to have a negative effect on the conversion activity of OPSS.
Example 11
Effect of Sulfur Source on the Cysteine Synthesis Activity of OPSS
An experiment was conducted to examine the effect of sulfur sources on the cysteine synthesis activity of each enzyme. In a reaction mixture containing 5 mM OPS, 0.2 mM PLP, and 100 mM HEPES, each enzyme (50 μg/mL Ape-OPSS, 50 μg/mL Msm-T) was subjected to reaction at 37° C. for 1 hour in the presence of 10 mM Na2S, NaSH, or Na2S2O3. The amounts of the produced cysteine were measured using the Gaitonde method. Ape-OPSS was observed to prefer Na2S2O3 as a sulfur source, whereas Msm-T prefers Na2S. The results are summarized in Table 11, below.









TABLE 11









Relative activity (%)












Enzyme
Na2S
NaSH
Na2S2O3
















Ape-OPSS
100.0
95.2
142.3



Msm-T
106.7
98.3
66.2










Example 12
Construction of Expression Vector Carrying OPSS (pCL-Pcj1 System) and Expression of the Enzyme
PCR was performed using primers of SEQ ID NOS: 21 and 22, with the pET28a-Msm-T vector serving as a template. The PCR product thus obtained was treated with EcoRV and HindIII, and cloned to construct a recombinant vector named pCL-P(CJ1)-Msm-T (pCJ1-MsmT CysM, FIG. 7). To examine a difference in the expression level of Msm-tc between the pET system and the pCL-Pcj1 system, strains for expressing the enzyme were prepared. The pET system was introduced into Rosetta (DE3) while the pCL-Pcj1 system employed the K12G strain. Single colonies taken from LB plates were inoculated into 5 mL of LB broth and cultured at 37° C. for 16 hours while shaking at 200 rpm. These cultures were transferred to 25 mL of fresh LB broth containing kanamycine or spectinomycine and 0.2% glucose (in 250 mL flasks) and incubated to an OD600 of 0.5-0.6, immediately after which 1 mM IPTG was added to the media to induce the enzymes to be expressed. During incubation at 37° C. while shaking at 200 rpm, the expression levels of the enzyme were measured at various culture times (8, 16, 24 hours). The enzyme expression levels of the two systems were analyzed on 14% SDS PAGE and are shown in FIG. 3.
In the same condition, as can be seen in FIG. 3, the pCL-Pcj1 system ensures a higher expression level of the enzyme than does the pET system. In addition, there is an improvement in culture condition because a temperature of as high as 37° C. and an IPTG concentration of as low as 0.1 mM allowed the system to express the enzyme. Therefore, the pCL-Pcj1 system can sufficiently function in substitution for the pET.
Example 13
Cysteine Synthesis of OPSS with the Use of Purified OPS Fermentation Broth as Substrate
The conversion rates from purified OPS to cysteine of Msm-T and Ape-OPSS were determined. In the presence of 75 μg/mL of each of the enzymes and 0.2 mM PLP, 60 mM OPS purified from OPS fermentation broth was reacted with 120 mM Na2S at 37° C. or 70° C. for 30, 60, 90, and 120 min. The reaction was conducted only at 37° C. for Msm-T, but at both 37° C. and 70° C. for Ape-OPSS. The amounts of the produced cysteine were measured using the Gaitonde method. The results are shown in FIG. 4.
As seen in FIG. 4, a purified OPS fermentation broth served well as a substrate for the enzymatic conversion into cysteine. Particularly, the cysteine conversion rate of Ape-OPSS was increased at 70° C. even upon the use of the purified OPS fermentation broth.
Example 14
Cysteine Synthesis of OPSS with the Use of OPS Fermentation Broth as Substrate
When an OPS fermentation broth was used as a substrate, the cysteine conversion rates of Msm-T and Ape-OPSS were measured according to the concentrations of the enzymes. In the presence of 5 μg/mL or 50 μg/mL of each of Msm-T and Ape-OPSS and 0.2 mM PLP, 50 mM of OPS fermentation broth was reacted with 100 mM Na2S at 37° C. The amounts of the produced cysteine were measured using the Gaitonde method. The results are shown FIG. 5.
As seen in FIG. 5, the highest conversion rate was detected in 50 μg/mL Msm-T. In addition, upon the use of OPS fermentation broth as a substrate, the activity of Msm-T was higher than that of Ape-OPSS.
Example 15
Cysteine Conversion Rate According to OPSS Concentration
To an OPS broth containing OPS at a concentration of 9.76 g/L, Na2S was added in an amount twice as large as the mole number of OPS, followed by incubation at 37° C. for 5 min. Thereafter, purified Msm-T was added in an amount of 50 mg while purified Msm-T-HA2 was used in an amount of 5 μg, 10 μg, 20 μg, and 50 μg. At predetermined times during incubation at 37° C., 100 mL of the enzyme reaction mixture was taken and mixed with 100 mL of 33.2% TCA to stop the enzymatic reaction. The cysteine concentrations of the enzyme reactions were quantitatively analyzed by measuring absorbance at OD560 according to the Gaitonde method. Cysteine synthesis activities of the OPSS enzymes according to enzyme concentrations are summarized in Table 12, below.







TABLE 12







Cysteine Conversion Rate (%)












Time
0 min
10 min
20 min
30 min
60 min
















Msm-T
50 μg
0.00
20.69
48.45
62.68
59.82


Msm-T-HA2
 5 μg
0.00
0.00
4.77
10.66
27.23


Msm-T-HA2
10 μg
0.00
10.46
24.78
37.43
57.32


Msm-T-HA2
20 μg
0.00
14.90
31.31
48.09
59.94


Msm-T-HA2
50 μg
2.33
44.02
56.72
62.17
63.52









As can be seen in Table 12, even when the Msm-T-HA2 mutant of which cysteine synthesis activity was improved and was used in an amount corresponding to 40% of that of Msm-T, the final cysteine conversion rate (%) was similar. In addition, when used in the same amount, the Msm-T-HA2 mutant showed faster initial reaction activity compared to Msm-T.
Example 16
Cysteine Conversion Rate by OPS Concentrations
To examine the effect of OPS concentration on the conversion rate of Msm-T and Msm-T-HA2, predetermined amounts of purified OPS were added to OPS fermentation broth to induce the conversion reaction. The enzyme was used in an amount of 50 μg for Msm-T and 20 μg for Msm-T-HA2. The amounts of cysteine in the reaction solution were measured using the Gaitonde method. The results are summarized in Tables 13 to 15.







TABLE 13







Cysteine Conversion Rate (OPS measured 10.65 g/l)









Time














0 min
10 min
30 min
60 min
120 min
180 min

















Msm-T 50 ug
0
23.03
65.38
65.70
61.95
55.35


Msm-T-HA2
0
13.70
43.82
61.32
63.77
53.74


20 ug
















TABLE 14







Cysteine Conversion Rate (OPS measured 36.09 g/l)









Time














0 min
10 min
30 min
60 min
120 min
180 min

















Msm-T 50 ug
0
1.15
10.23
28.07
97.84
100.34


Msm-T-HA2
0
2.33
12.04
27.57
77.65
80.01


20 ug
















TABLE 15







Cysteine Conversion Rate (OPS measured 55.6 g/l)









Time














0 min
10 min
30 min
60 min
120 min
180 min

















Msm-T 50 ug
0
0
2.36
7.41
42.69
66.67


Msm-T-HA2
0
0.32
4.76
12.15
50.16
62.46


20 ug









When the concentration of OPS was about 30 g/L, as seen in Tables 13 to 15, the highest conversion rates were detected as being 100% for Msm-T and 80% for Msm-T-HA2. When the concentration of OPS exceeded 50 g/L, both the conversion rate and the conversion percentage were found to decrease.
The data of Tables 13 to 15 was used to select an optimal concentration ratio between OPS and Msm-T-HA2 for a conversion process using a high concentration of OPS, and to reduce the reaction time of Msm-T-HA2 compared to that of Msm-T.
Example 17
Cysteine Conversion Rate by Na2S Concentrations
To examine the effect of the amount of Na2S used as a sulfur source on the cysteine conversion rate, OPS conversion reactions were conducted in the presence of 20 μg of Msm-T-HA2 when Na2S was used at a concentration of 160 mM, 320 mM, and 480 mM, which corresponded to the amounts equivalent to and twice and three times as large as the mole number of OPS, respectively. The resulting cysteine conversion rates are summarized in Table 16, below.







TABLE 16







Cysteine Conversion Rate (OPS 29.76 g/l)









Time














0 min
10 min
30 min
60 min
120 min
180 min

















160 mM Na2S
0
0
0
0
0
0


320 mM Na2S
0
13.43
34.58
78.78
67.76
74.95


480 mM Na2S
0
14.90
25.99
54.42
51.24
58.25









As can be seen in Table 16, the cysteine conversion rate peaked when the mole number of Na2S was twice as large as that of the OPS used. From the results, it is understood that an optimal condition is set when the molar ratio of Na2S to OPS is 2.
Example 18
Cysteine Conversion Rate by pH
To examine the effect of pH on the enzymatic conversion of OPS into cysteine, a conversion reaction was conducted in the presence of 20 μg of Msm-T-HA2 at various pH values. The results are summarized in Table 17, below.







TABLE 17







Cysteine Conversion Rate (%)









Time













0 min
10 min
30 min
60 min
120 min
















pH 8.5 (OPS 35.02 g/l)
0.24
4.74
20.74
40.19
69.13


pH 9 (OPS 34.59 g/l)
0.19
6.00
24.03
43.86
63.88


pH 9.5 (OPS 35.34 g/l)
0.26
9.85
28.48
44.82
64.68


pH 10 (OPS 34.59 g/l)
0.25
10.97
28.54
40.19
58.14









As can be seen, an optimal pH for the conversion reaction was measured to be between 8.5 and 9.5.
Example 19
Conversion Process on 1 L Jar Scale
The reaction process for converting OPS broth into cysteine was scaled up to a 1 L jar. Based on the OPSS characteristics and conversion reaction data set under the 1 mL tube condition, a conversion reaction process was established for Msm-T-HA2.
Preceding a conversion reaction on a 1 L jar scale, OPS fermentation broth obtained in a 1 L jar was centrifuged (10,000 rpm, 10 min, 4° C.) and the supernatant was passed through a membrane (0.45 μm) to remove cells. 72 g of Na2S was added to the filtered OPS broth (19.317 g/L), followed by filtering out the precipitate through Whatman filter paper (6 μm). After the addition of 10 mM PLP, the reaction mixture was pre-incubated at 37° C. for 5 min, while shaking at 200 rpm. Finally, a conversion reaction was performed in the presence of 50 mg of Msm-T-HA2 on a 1 L jar scale. At 0, 10, 30, 60, 120, and 180 min during incubation at 37° C., 100 μl, of the enzyme reactions was taken and mixed with 100 μL of 33.2% TCA to stop the enzymatic reaction. The samples taken were diluted in 0.1 N HCl and analyzed for cysteine and cystine contents using LC. Also, the Gaitonde method was used to determine the amount of cysteine. Concentrations of the substrates and the enzyme used in the conversion reaction process are summarized in Table 18, below. The amount of the substrates used is represented by a unit of mL.









TABLE 18









pH 9.01 after addition of Na2S,



pH 9.16 after filtration












OPS Fermentation Broth

Final
volume
















Final OPS concentration
101.47
mM




Msm-T-HA2 (2.59 μg/μL)
50
μg
19



Na2S (72 g)
300
mM
0



10 mM PLP
0.1
mM
10



OPS Broth (19.317 g/l)
104.42
mM
1000



D. W


0



Total


1029










The concentrations of cysteine in the reaction mixture obtained after the conversion reaction performed under the condition of Table 18 were quantitatively analyzed by the Gaitonde method and LC, and the results are shown in FIG. 6. When 19.317 g/L OPS broth was incubated for 2 hours in the presence of 50 mg of Msm-T-HA2, as seen in FIG. 6, the amount of produced cysteine or cystine peaked to up to 9.075 g/L, indicating that cysteine and cystine can be produced from OPS at a rate of 71.83% by an enzymatic conversion process on 1 L jar scale.








TABLE 19







Cysteine/cystine Conversion Rate (%)









Time














0 min
10 min
30 min
60 min
120 min
180 min

















OPS
10.81
28.08
51.26
67.21
71.83
69.45


broth
















TABLE 20







Cysteine/cystine (g/L)









Time














0 min
10 min
30 min
60 min
120 min
180 min

















OPS
1.366
3.548
6.476
8.492
9.075
8.775


broth









INDUSTRIAL APPLICABILITY
As described hitherto, the OPSS mutants of the present invention can be useful for the mass production of L-cysteine, which and whose derivatives can find applications in a variety of fields including the pharmaceutical industry (for treatment of bronchial diseases), the cosmetics industry (in hair shampoo, compositions for permanent waves, etc.), and the food industry (antioxidants, flavorant enhancers, dough aids, etc.).

## Claims

1. An isolated Mycobacterium smegmatis-derived O-phosphoserine sulfhydrylase (OPSS) mutant, having O-phosphoserine sulfhydrylase activity and the same amino acid sequence as that of SEQ ID NO: 1 except for deletion of the last three to seven C-terminal amino acids of SEQ ID NO: 1, and (i) substitution of the proline residue (Pro) at position 77 of SEQ ID NO: 1 with a serine residue (Ser), or (ii) substitution of the threonine residue (Thr) at position 131, the lysine residue (Lys) at position 137, and the threonine residue at position 238 of SEQ ID NO: 1 with an alanine residue (Ala), an asparagine residue (Asp), and a serine residue (Ser), respectively.

2. The isolated Mycobacterium smegmatis-derived OPSS mutant of claim 1, wherein the last five C-terminal amino acid residues of SEQ ID NO: 1 are deleted.

3. The isolated Mycobacterium smegmatis-derived OPSS mutant of claim 1, having the amino acid sequence of SEQ ID NO: 3.

4. The isolated Mycobacterium smegmatis-derived OPSS mutant of claim 1, having the amino acid sequence of SEQ ID NO: 4.

5. The isolated Mycobacterium smegmatis-derived OPSS mutant of claim 1, showing optimal activity under conditions comprising:
i) presence of PLP (pyridoxal-5′-phosphate) at a concentration of about 0.001 to about 2 mM, or presence of DTT (dithiothreitol) at a concentration of about 0.001 to about 100 mM;
ii) a reaction temperature range of 25 to 60° C.; and
iii) a pH range of 6.0 to 10.0.

6. A method for producing cysteine comprising reacting O-phospho-L-serine (OPS) with a sulfide in the presence of the isolated Mycobacterium smegmatis-derived OPSS mutant of claim 1.

7. The method of claim 6, wherein the isolated Mycobacterium smegmatis-derived OPSS mutant has the amino acid sequence of SEQ ID NO: 3 or 4.

8. The method of claim 6, wherein the isolated Mycobacterium smegmatis-derived OPSS mutant shows optimal activity under conditions comprising:
(i) presence of PLP (pyridoxal-5′-phosphate) at a concentration of about 0.001 to about 2 mM, or presence of DTT (dithiothreitol) at a concentration of about 0.001 to about 100 mM;
(ii) a reaction temperature range of 25 to 60° C.; and
(iii) a pH range of 6.0 to 10.0.

9. The method for producing cysteine of claim 6, wherein the OPS is in a purified form or in a fermentation culture.

10. The method for producing cysteine of claim 6, wherein the sulfide is selected from the group consisting of Na2S, NaSH, (NH4)2SH, H2S, and S2O3, all being in a gas or liquid state.

11. The method for producing cysteine of claim 6, wherein the sulfide is used at a mole concentration which is 0.1 to 3 times higher than the mole concentration of OPS.