Description
Title of Invention: O-PHOSPHOSERINE SULFHYDRYLASE
MUTANTS AND METHOD FOR PRODUCTION OF CYSTEINE
USING THE SAME
Technical Field
[1] 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: 1which 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.
[2]
Background Art
[3] 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, glu
tathione, 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.).
[4] 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.
[5] Representative among the microbial production of L-cysteine is the biological
conversion of D, L-ATC using a microorganism (Ryu OH, Ju JY, and Shin CS,
Process Biochem., 32:201-209, 1997). This conversion process is, however, difficult toapply 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.
[6] 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 NM and Tomkins GM, 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, S0 4 may be reduced to thiosulfate for use as
a sulfur donor in cysteine production (Nakamura T, Kon Y, Iwahashi H, and Eguchi Y,
J . Bacterid., 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.
[7]
Disclosure of Invention
Technical Problem
[8] 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 tu
berculosis, 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 KE,
Baumgart S, Dorrestein PC, Zhai H, McLafferty FW, and Begley TP, J. Am. Chem.
Soc, 127: 11602-11603, 2005; Westrop GD, Goodall G, Mottram JC, and Coombs
GH, 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 areremoved therefrom (Agren D, Schnell R and Schneider G, FEBS letters, 583: 330-336,
2009).
[9]
Solution to Problem
[10] It is therefore an object of the present invention to provide an O-phosphoserine
sulfhydrylase (also referred to as "OPSS") mutant which has aMycobacterium
smegmatis-derived amino acid sequence corresponding to that of SEQ ID NO: 1 which
is deleted three to seven C-terminal amino acid residues.
[11] It is another object of the present invention to provide a nucleic acid molecule
encoding the OPSS mutant
[12] It is a further object of the present invention to provide an expression vector carrying
the nucleic acid molecule.
[13] It is still a further object of the present invention to provide a transformant
transformed with the expression vector.
[14] 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 of Invention
[15] 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.
[16]
Brief Description of Drawings
[17] FIG. 1 is a graph showing the activity of OPSS according to temperature.
[18] FIG. 2 is a set of graphs showing the pH sensitivity of OPSS.
[19] FIG. 3 is a photograph showing the expression level of the enzyme in a pET system
and a pCL-Pcj 1 system as analyzed by SDS PAGE.
[20] FIG. 4 is a graph showing the enzymatic activity of OPSS to convert purified OPS
fermentation broth into cysteine.
[21] FIG. 5 is a graph showing the enzymatic activity of OPSS to convert OPS fer
mentation broth into cysteine.
[22] 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.
[23] FIG. 7 is a schematic view showing the map of a pCL-Pcjl expression vector
carrying a gene coding for an OPSS mutant.
[24]
Best Mode for Carrying out the Invention[25] In accordance with an aspect thereof, the present invention provides a My
cobacterium smegmatics-derrved 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.
[26] 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.
[27] 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.
[28] 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).
[29] 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 bioin-
formatic 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 su
pernatant containing the enzyme to afford the OPSS enzyme.
[30] 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 anOPSS enzyme that is genetically altered such that its activity can be effectively
improved, compared to the wild-type.
[31] 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 aMycobacterium smegmatis-derrved amino acid sequence corresponding to
that of SEQ ID NO: 1which is deleted three to seven, preferably five, C-terminal
amino acid residues.
[32] 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).
[33] 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 mu
tagenesis 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.
[34] 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 amountcorresponding to 40% of Msm-T, the final cysteine conversion rate was similar.
[35] 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 de
termined 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 polynu
cleotides under conditions that form stable duplexes between homologous regions,
followed by digestion with single strand- specific nuclease, and size determination of
the digested fragments.
[36] As used herein, the term "homologous" in all its grammatical forms and spelling
variations refers to the relationship between proteins that possess a "common evo
lutionary origin," including proteins from superfamilies (e.g., the immunoglobulin su-
perfamily) 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.
[37] 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 em
bodiment, 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).
[38] 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.
[39] 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).
[40] 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 25mM DTT or 0.2mM PLP was added, the cysteine
conversion rate was increased by 2.3 times. That is, PLP and DTT have positive in
fluences on the conversion of OPS into cysteine.
[41] The enzymes Ape-OPSS, Mtb-OPSS, and Mtb-T have an optimal reaction tem
perature 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.
[42] 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.
[43] In accordance with another aspect thereof, the present invention provides a nucleic
acid molecule encoding the OPSS mutant.
[44] 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)).
[45] In accordance with a further aspect thereof, the present invention provides an ex
pression vector carrying the nucleic acid molecule.
[46] 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) Mol 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. Mol. Biol. 302:93-102 (2000)), or
artificial chromosomes.
[47] 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.
[48] 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.
[49] 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.
[50] In accordance with a still further aspect thereof, the present invention provides a
transformant transformed with the expression vector.
[51] 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 in
troduction of a foreign gene to the host cell so that the introduced gene can replicate
itself or as a factor incorporated into the chromosome.
[52] 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, mi
croinjection, DEAE-dextran, and cationic liposome calcium.
[53] 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. Thehost cell suitable for use in the present invention may be prokaryotic or eukaryotic.
Examples include enterobacteria and coryneform bacteria, with preference for E s
cherichia spp. and Serratia spp., with the highest preference being E. coli.
[54] 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.
[55] 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.
[56] For use as a substrate, OPS may be in pure form or may be in the form of a fer
mentation 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.
[57] 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 S20 3, eitherin the form of
liquid or gas, may be used.
[58] 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.
[59] 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).
[60]
[61] Abbreviation and Terminology
[62] 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 andthe 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".
[63] 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.
[64] 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 de
scription and accompanying claims.
[65] 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.
[66]
Mode for the Invention
[67] 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.
[68]
[69] EXAMPLE 1: Development of OPSS
[70]
[71] 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 KE, Baumgart S, Dorrestein PC, Zhai H, McLafferty FW, and
Begley TP, J. Am. Chem. Soc, 127: 11602-11603, 2005; Westrop GD, Goodall G,
Mottram JC, and Coombs GH, 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, andNfa-OPSS were secured from Mycobacterium smegmatis str. MC2 155, Rhodococcus
jostii RHA1, and Nocardiafarcinica IFM 10152, respectively.
[72] 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 re
combinant 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 Ndel and
Hindlll (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 ex
pression 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.
[73]
[74] Table 1
[Table 1]
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 1mM 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 ofHis 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.
[77]
[78] EXAMPLE 2 : Assay of OPSS for Cysteine Synthesis Activity
[79]
[80] 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 KE, Baumgart S,
Dorrestein PC, Zhai H, McLafferty FW, and Begley TP, J. Am. Chem. Soc, 127:
11602-1 1603, 2005; Westrop GD, Goodall G, Mottram JC and Coombs GH, 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.
[81]
[82] Table 2
[Table 2]
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 con
centrations of the enzyme reactions were quantitatively analyzed by measuring ab-
sorbance 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 conversionrates with reaction time.
[85]
[86] Table 3
[Table 3]
[88] 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 My
cobacterium 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 Nocardiafarcinica IFM 10152-derived OPSS, obtained by the homology
screening, exhibited insufficient activity of converting O-phosphoserine into cysteine.
[89] As seen in the data of Table 3, the conversion rate from OPS into cysteine of Ape-
OPSS reached near 100% in one hour.
[90] The final conversion rate of the Msm-OPSS enzyme, which was newly selected
through enzyme screening on the basis of previously reported Mycobacterium tu
berculosis H37Rv-derived OPSS, was 43.7% which is 4.3 times as high as that of Mtb-
OPSS.
[91]
[92] EXAMPLE 3 : Preparation of Mtb-T and Msm-T, Mutants deleted 5 C-
Terminal Amino Acid Residues of Mtb-OPSS and Msm-OPSS
[93]
[94] Mycobacterium tuberculosis H37Rv-derived OPSS (Mtb-OPSS), which catalyzes the
conversion of OPS to cysteine with the aid of the additional enzymes mec-i- 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 thetwo 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
Ndel and Hindlll and were cloned into the pET28a vector digested with the same r e
striction enzymes to construct recombinant expression vectors named pET28a-Mtb-T
and pET28a-Msm-T, respectively. The recombinant expression vectors were in
troduced into E. coli (DE3). The expression of the two OPSS mutants obtained under
the same condition as in Example 1was confirmed by 14% SDS PAGE. As a result,
Mtb-T (SEQ ID NO: 5) and Msm-T (SEQ ID NO: 2) were obtained.
[95]
[96] EXAMPLE 4 : Assay of Mtb-T and Msm-T for Cysteine Synthesis Activity
[97]
[98] 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.
[99]
[100] Table 4
[Table 4]
[101]
[102] 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.
[103]
[104] EXAMPLE 5:Assay for Cysteine Synthesis Activity of Msm-T-Derived Mutants
Msm-T-HA2 and Msm-T-EP3
[105]
[106] 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 toconstruct a library of Msm-T mutants which were then screened to select OPSS
mutants with improved enzymatic activity.
[107] 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, r e
spectively, as analyzed by base sequencing.
[108] 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.
[109]
[110] Table 5
[Table 5]
[111]
[112]
[113]
[114] 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.
[115]
[116] EXAMPLE 6 : Requirement of Cofactor for OPSS Activity[117]
[118] 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.
[119]
[120] Table 7
[Table 7]
[121]
[122] 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.
[123]
[124] EXAMPLE 7 : Activity of OPSS by Temperature
[125]
[126] 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.
[127] 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.
[128]
[129] EXAMPLE 8 : Heat Stability of OPSS
[130]
[131] 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 minunder the condition of 5 mM OPS, 10 mM Na2S, 0.2 mM PLP, and 100 mM HEPES
(pH 7.4). For this reaction, 10 µ /mL Ape-OPSS and 50 µ /mL Msm-T were
employed. The amounts of the produced cysteine were measured using the Gaitonde
method and are summarized in Table 8, below.
[132]
[133] Table 8
[Table 8]
[134]
[135] 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.
[136] 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.
[137]
[138] Table 9
[Table 9]
[139]
[140] 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.
[141]
[142] EXAMPLE 9 : pH Sensitivity of OPSS[143]
[144] 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.
[145] As seen in FIG. 2, Msm-T exhibited the highest activity at a pH of from 8.0 to 9.0 ir
respective 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.
[146]
[147] EXAMPLE 10: Effect of Ions on the Activity of OPSS
[148]
[149] 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)
2S0 4 [1, 3, 5, 10, 20 g/L], KH2P0 4 [0.5, 1, 2, 4, 8 g/L], or NH4C 1
[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.
[150]
[151] Table 10
[Table 10]
No changes were detected in the cysteine conversion rate when (NH4)
2S0 or KH2PO
4was 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 NFLCl concentration. Par-ticularly, the maximal enzyme activity was decreased by more than 70% when 2 g/L
NH4 was added. Therefore, NH4C 1 and NH4 were observed to have a negative effect
on the conversion activity of OPSS.
[154]
[155] EXAMPLE 11: Effect of Sulfur Source on the Cysteine Synthesis Activity of
OPSS
[156]
[157] 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 Na2S20 3. The amounts of the produced cysteine were measured using the Gaitonde
method. Ape-OPSS was observed to prefer Na2S20 3 as a sulfur source, whereas Msm-
T prefers Na2S. The results are summarized in Table 11, below.
[158]
[159] Table 11
[Table 11]
[160]
[161] EXAMPLE 12: Construction of Expression Vector Carrying OPSS (pCL-Pcj1
System) and Expression of the Enzyme
[162]
[163] PCR was performed using primers of SEQ ID NOS: 2 1 and 22, with the
pET28a-Msm-T vector serving as a template. The PCR product thus obtained was
treated with EcoRV and Hindlll, and cloned to construct a recombinant vector named
pCL-P(CJl)-Msm-T (pCJl-MsmT CysM, FIG. 7). To examine a difference in the ex
pression level of Msm-tc between the pET system and the pCL-Pcj 1 system, strains for
expressing the enzyme were prepared. The pET system was introduced into Rosetta
(DE3) while the pCL-Pcjl 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 1mM IPTG wasadded 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.
[164] In the same condition, as can be seen in FIG. 3, the pCL-Pcjl 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 0.1mM allowed the system to express the enzyme.
Therefore, the pCL-Pcj 1 system can sufficiently function in substitution for the pET.
[165]
[166] EXAMPLE 13: Cysteine Synthesis of OPSS with the Use of Purified OPS Fer¬
mentation Broth as Substrate
[167]
[168] 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.
[169] 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.
[170]
[171] EXAMPLE 14: Cysteine Synthesis of OPSS with the Use of OPS Fermentation
Broth as Substrate
[172]
[173] 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.
[174] 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.
[175]
[176] EXAMPLE 15: Cysteine Conversion Rate According to OPSS Concentration[177]
[178] 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 prede
termined 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.
[179]
[180] Table 12
[Table 12]
Cysteine Conversion Rate (%)
[181]
[182] As can be seen in Table 12, even when the Msm-T-HA2 mutant of which cysteine
synthesis activity was improved and wasused 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.
[183]
[184] EXAMPLE 16: Cysteine Conversion Rate by OPS Concentrations
[185]
[186] 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 fer-mentation broth to induce the conversion reaction. The enzyme was used in an amount
of 50 µ for Msm-T and 20 µ 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.
[187]
[188] Table 13
[Table 13]
Cysteine Conversion Rate (OPS measured 10.65 g/1)
[189]
[190]
[191]
[192]
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.
[195] 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.
[196]
[197] EXAMPLE 17: Cysteine Conversion Rate by Na2S Concentrations
[198]
[199] 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.
[200]
[201] Table 16
[Table 16]
Cysteine Conversion Rate (OPS 29.76 g/1)
[202]
[203] 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 u n
derstood that an optimal condition is set when the molar ratio of Na2S to OPS is 2.
[204]
[205] EXAMPLE 18: Cysteine Conversion Rate by pH
[206]
[207] 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.
[208][209] Table 17
[Table 17]
Cysteine Conversion Rate ( )
[210]
[211] As can be seen, an optimal pH for the conversion reaction was measured to be
between 8.5 and 9.5.
[212]
[213] EXAMPLE 19: Conversion Process on 1 L Jar Scale
[214]
[215] 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 1mL
tube condition, a conversion reaction process was established for Msm-T-HA2.
[216] 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 µιη) 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 µιη). 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 of the enzyme reactions
was taken and mixed with 100 of 33.2% TCA to stop the enzymatic reaction. The
samples taken were diluted in 0.1N HC1 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.
[217]Table 18
[Table 18]
[219]
[220] 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.
[221] 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.
[222]
[223] Table 19
[Table 19]
Cysteine/cystine Conversion Rate ( )
[224]
[225] Table 20
[Table 20]
Cysteine/cystine (
[226]
Industrial ApplicabilityAs 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
A Mycobacterium smegmatis-derrved O-phosphoserine sulfhydrylase
(OPSS) mutant, having the same amino acid sequence as that of SEQ
ID NO: 1, wherein said Mycobacterium smegmatis-derrved O-
phosphoserine sulfhydrylase (OPSS) mutant lacks three to seven C-
terminal amino acid residues of a wild-type OPSS amino acid
sequence.
The Mycobacterium smegmatis-d d OPSS mutant of claim 1,
lacking five C-terminal amino acid residues.
The Mycobacterium smegmatis-d d OPSS mutant of claim 2,
having an amino acid sequence of SEQ ID NO: 2.
The Mycobacterium smegmatis-d d OPSS mutant of claim 1,
wherein a proline residue (Pro) at position 77 is substituted with a
serine residue (Ser).
The Mycobacterium smegmatis-d d OPSS mutant of claim 4,
having an amino acid sequence of SEQ ID NO: 3.
The Mycobacterium smegmatis-d d OPSS mutant of claim 1,
wherein a threonine (Thr) at position 131, a lysine residue (Lys) at
position 137, and a threonine residue (Thr) at position 238 are sub
stituted with an alanine residue (Ala), an asparagine residue (Asp), and
a serine residue (Ser), respectively.
The Mycobacterium smegmatis-d d OPSS mutant of claim 6,
having an amino acid sequence of SEQ ID NO: 4.
The Mycobacterium smegmatis-d d OPSS mutant of claim 1,
showing optimal activity under conditions comprising:
i) the addition of about 0.001 to about 2 mM PLP
(pyridoxal-5' -phosphate) or about 0.001 to about 100 mM DTT
(dithiothreitol) as a cofactor;
ii) a reaction temperature range of 25 ~ 60°C ; and
iii) a pH range of 6.0 ~ 10.0.
A nucleic acid molecule encoding the OPSS mutant as in one of claims
1 to 8.
An expression vector carrying a nucleic acid molecule encoding the
OPSS mutant as in one of claims 1 to 8.
A transformant transformed with the expression vector of claim 10.
A method for producing cysteine comprising reacting O-
phospho-L-serine (OPS) with sulfides in the presence of the OPSSmutant as in one of claims 1 to 8.
[Claim 13] The method for producing cysteine of claim 12, wherein the OPS is in a
purified form or in a form of a fermentation culture containing OPS.
[Claim 14] The method for producing cysteine of claim 12, comprising a sulfide
selected from the group consisting of Na2S, NaSH, (NH4)
2SH, H2S, and
S20 3, all being in a gas or liquid state.
[Claim 15] The method for producing cysteine of claim 12, wherein the sulfide is
used in an amount of 0.1 ~ 3 times to a mole concentration of OPS.
[Claim 16] Use of the OPSS mutant as in one of claims 1 to 8 in the biosynthesis
of cysteine from OPS.