MICROORGANISM PRODUCING O-PHOSPHOSERINE AND METHOD OF PRODUCING L-CYSTEINE OR DERIVATIVES THEREOF FROM O-PHOSPHOSERINE USING THE SAME

Abstract

The present invention provides methods for the production of cysteine or derivates thereof by culturing a microorganism having reduced activity of endogenous phosphoserine phosphatase. The O-phosphoserine produced by such an organism can then be reacted with a sulfide in the presence of a sulfydrylase or a microorganism expressing a sulfhydrylase to produce cysteine or a derivative thereof. Microorganisms having the properties noted above are also provided herein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 13/278,102, which was filed on Oct. 20, 2011, now pending, which claims the priority benefit of Korean Patent Application Nos. 10-2011-0086081, filed Aug. 26, 2011 and 10-2010-0102664, filed Oct. 20, 2010. The contents of these patent applications are incorporated herein by reference in their entireties.

STATEMENT REGARDING SEQUENCE LISTING

[0002] 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_004_00US_ST25.txt. The text file is 28 KB, was created on Oct. 20, 2011, and is being submitted electronically via EFS-Web.

TECHNICAL FIELD

[0003] The present invention relates to a method for production of cysteine or its derivatives using O-phosphoserine as an intermediate and recombinant microorganism for use in production of O-phosphoserine.

BACKGROUND ART

[0004] L-cysteine is an amino acid that plays an important role in sulfur metabolism of 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), and the food industry (antioxidants, flavorant enhancers, dough aids, etc.).

[0005] L-cysteine was once obtained industrially by acid hydrolysis of human hairs 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 hairs or feathers ensure a yield of as low as 7-8%, but also the use of hydrochloric acid or sulfuric acid produces a lot of waste resulting in environmental pollution. Further, extraction from hairs or feathers may induce the user to have a strong aversion thereto. These problems have caused a push for the development of environmentally friendly production processes of L-cysteine. The main contemporary route involves fermentation utilizing microorganisms.

[0006] Representative among the microbial production of L-cysteine is 1) 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. 2) Another method of L-cysteine production is direct fermentation using E. coli (Patent No. EP0885962B; Wada M and Takagi H, Appl. Microbiol. Biochem., 73:48-54, 2006). Excessive accumulation of L-cysteine within microorganisms incurs intracellular toxicity, exhibiting a limitation in the production of L-cysteine at a high concentration. To overcome this drawback, L-cysteine-exporting proteins are employed, but there have been no significant improvements in productivity.

[0007] Referring to the biosynthesis pathway of L-cysteine in microorganisms 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 cysteine. Alternatively, SO.sub.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. Bacteriol., 156: 656-662, 1983). Therefore, cystein may be produced using microorganisms accumulating OAS and QASS using various sulfur donors (U.S. Pat. No. 6,579,705). The cysteine biosynthesis pathway via OAS uses the two enzymes of serine acetyltransferase (CysE), which catalyzes the conversion of OAS from serine, and cysteine synthase (CysK), which catalyzes the conversion of OAS to cysteine. 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).

DISCLOSURE

Technical Problem

[0008] Leading to the present invention, the present inventors found out the existence of 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 through intensive research (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, can use Na.sub.2S 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 (Argen D, Schnell R and Schneider G, FEBS letters, 583: 330-336, 2009). In the present invention, a microorganism is mutated to accumulate OPS therein, following incubation to convert OPS into cysteine in the presence of the OPSS enzyme. Nowhere has this method been previously described.

Technical Solution

[0009] It is an object of the present invention to provide a method for producing cysteine or a derivative thereof. It is another object of the present invention to provide a recombinant microorganism for the production of O-phosphoserine.

Advantageous Effects

[0010] The method of the present invention in which O-phosphoserine is produced at high yield by a recombinant microorganism and is used for conversion into cysteine, as it is, is more friendly to the environment and ensures higher efficiency in the production of cysteine than do chemical synthesis methods. The cysteine and its derivatives produced by the fermentation and bioconversion of the present invention can be widely used in the production of animal and human foods and food additives.

DESCRIPTION OF DRAWINGS

[0011] FIG. 1 is a schematic diagram showing the accumulation of O-phosphoserine by microbial fermentation and the enzymatic conversion of the accumulated O-phosphoserine into L-cysteine.

[0012] FIG. 2 is a graph showing the activity of OPS sulfhydrylase according to temperatures.

[0013] FIG. 3 is a set of graphs showing pH sensitivity of OPS sulfhydrylase.

[0014] FIG. 4 is a photograph showing the expression level of Msm-T in a pET system and a pCL-Pcj1 system as analyzed by SDS PAGE.

[0015] FIG. 5 is a graph showing the enzymatic activity of OPS sulfhydrylase to convert purified OPS fermentation broth into cysteine.

[0016] FIG. 6 is a graph showing the enzymatic activity of OPS sulfhydrylase to convert OPS fermentation broth into cysteine.

BEST MODE

[0017] As used herein, the term "cysteine conversion" is intended to refer to the catalytic reaction of O-phosphoserine sulfhydrylase (OPSS) which results in the conversion of the substrate O-phosphoserine (OPS) into the product cysteine, that is, it refers to the catalytic reaction of converting OPS into cysteine.

[0018] As used herein, the term "cysteine conversion rate" refers to the percentage of the amount of the product cysteine to the amount of the starting material OPS. Under optimal reaction conditions, 1 mole of OPS is converted into 1 mole of cysteine. For example, if 100 moles of OPS is converted into 100 moles of cysteine, the cysteine conversion rate is 100%.

[0019] In accordance with an aspect thereof, the present invention provides a method for producing cysteine or a derivative thereof, comprising:

[0020] 1) culturing a recombinant microorganism in which the activity of endogeneous phosphoserine phosphatase (SerB) is reduced, to produce O-phosphoserine (OPS); and 2) reacting the OPS of step 1) with a sulfide in the presence of O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing OPSS, to produce cysteine or a derivative thereof.

[0021] The SerB is a protein that has the activity of hydrolyzing OPS into L-serine. Thus, a microorganism which has reduced endogeneous SerB activity is characterized by the accumulation of OPS therein. The SerB is not limited to, may comprise any amino acid sequences, which exhibits SerB activity, and may have preferably the amino acid sequence of SEQ ID NO: 1 or 2. However, as long as it exhibits SerB activity, any amino acid sequence is used, which preferably has a homology of 90% or higher, more preferably 96% or higher, far more preferably 98% or higher, and most preferably 99% or higher with that of SEQ ID NO: 1 or 2. The reduced SerB activity means a decrease in SerB activity, compared to that of a prior-modified strain, and encompasses the disrupting of SerB. Various techniques for reduction of SerB activity are well known in the art. Illustrative examples include the deletion of a chromosomal serB, the introduction of mutation into the chromosomal serB to reduce endogenous SerB activity, the introduction of mutation into a regulatory region for the serB to reduce endogenous SerB activity, the substitution of the chromosomal serB with a gene mutated to reduce the endogenous SerB activity, the introduction of mutation into a regulatory region for the serB to reduce endogenous SerB activity, and the introduction of an antisense oligonucleotide complementary to a transcript of the serB to inhibit the translation of the mRNA, but methods for reducing the SerB activity are not limited to these. These techniques may be applied to the reducing the activity of other enzymes in the present invention.

[0022] The disruption of endogenous SerB results in the introduction of serine auxotrophy into the recombinant microorganism so that the medium must be supplemented with glycine or serine. Glycine may be provided in the form of purified glycine, a glycine-containing yeast extract, or tryptone. Glycine is contained at a concentration of from 0.1 to 10 g/L, and preferably at a concentration of from 0.5 to 3 g/L. As for serine, it may be provided in the form of purified serine, a serine-containing yeast extract or tryptone. Its concentration in the culture medium ranges from 0.1 to 5 g/L, and preferably from 0.1 to 1 g/L.

[0023] Preferably, when cultured in a glycine- or serine-containing medium, mutant Corynebacterium glutamicum or E. coli in which the activity of endogeneous SerB was disrupted was found to produce a higher amount of OPS, compared to the wild-type (see Tables 2, 3, 4 and 5).

[0024] The recombinant microorganism of the present invention refers to any microorganism which is modified to reduce SerB activity, thus producing OPS at an elevated level. If this condition is satisfied, any microorganism, whether prokaryotic or eukaryotic, falls within the scope of the present invention. Representative among them are enterobacteria or coryneform bacteria. Examples of the microorganisms useful in the present invention include Escherichia sp., Erwinia sp., Serratia sp., Providencia sp., Corynebacterium sp., and Brevibacterium sp. Preferable are Escherichia sp. and Corynebacterium sp, with more preference given for Escherichia sp. and with the highest preference being for E. coli.

[0025] In an embodiment, the strain capable of producing OPS was named E. coli CA07-0012, and deposited with the Korean Culture Center of Microorganisms, located at 361-221, Hongje 1, Seodaemun, Seoul, Korea, on Oct. 12, 2011 under accession number KCCM11212P.

[0026] As used herein, the term "culturing" is intended to mean growing microorganisms under artificially controlled conditions. A culturing procedure may be conducted using a suitable medium and culturing conditions well known in the art. Those skilled in the art can readily control the culturing procedure to correspond to the strains employed. For example, it may be performed in a batch type, in a continuous type, or in a fed-batch type, but is not limited thereto.

[0027] In addition, the culture medium contains a carbon source. Examples of the carbon source include saccharides and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, oils and fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These carbon sources may be present solely or in combination in the culture medium. As a nitrogen source, an organic material such as peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean, and wheat protein, or an inorganic nitrogen compound such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate may be contained in the culture medium. These nitrogen sources may be used solely or in combination. The medium may contain potassium dihydrogen phosphate, potassium phosphate, or corresponding sodium salts as a phosphorous source. The medium may contain metallic salts such as magnesium sulfate or iron sulfate. The culture medium may also contain amino acids, vitamins and suitable precursors. The nutrients may be added in a batch manner or a continuous manner to the medium.

[0028] A compound such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid may be added in a suitable manner to the culture medium during culturing to adjust the pH of the culture. In addition, during culturing, an anti-foaming agent such as fatty acid polyglycol ester is used to suppress the formation of foam. Further, in order to maintain the culture medium in an aerobic condition, oxygen or oxygen-containing gas can be injected into the culture medium. For an anaerobic or microaerobic condition, nitrogen, hydrogen, or carbon dioxide is provided without aeration. The culture medium may be typically maintained at a temperature of from 27.degree. C. to 37.degree. C. and preferably at a temperature of from 30.degree. C. to 35.degree. C. As for the culture period, it may be maintained until the product of interest is obtained in a desired amount, and preferably it ranges from 10 to 100 hours.

[0029] For further collection and recovery of the OPS produced during the culturing step from the culture medium, a suitable method well known in the art may be selected depending on the type of culture, be it a batch, continuous or fed-batch culture.

[0030] In the method of the present invention, step 2) addresses the reaction of the OPS of step 1) with a sulfide in the presence of O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing OPSS, to induce the conversion of O-phosphoserine into cysteine or its derivatives.

[0031] The sulfide may be provided in a liquid or gas form as well as in a solid form typically used in the art, because of the difference in pH, pressure and/or solubility. So long as it may be converted to a thiol group (SH), any sulfur compound such as sulfide (S.sup.2-) or thiosulfate (S.sub.2O.sub.3.sup.2-) may be used in the present invention. Preferably, Na.sub.2S, NaSH, H.sub.2S, (NH.sub.4).sub.2S, NaSH and Na.sub.2S.sub.2O.sub.3, all of which can provide a thiol group for OPS, may be used. In the reaction, one thiol group is supplied to one OPS molecule to afford one molecule of cysteine or a derivative thereof. In this enzymatic conversion, a sulfide may be preferably added at a molar concentration 0.1 to 3 times and more preferably 1 to 2 times as high as that of OPS used. In light of the economy, a thiol group-providing sulfide and OPS are most preferably used at a molar ratio of 1:1. In one embodiment of the present invention, Na.sub.2S was used as the source of sulfur. Na.sub.2S was added at a molar concentration 1 to 3 times as high as that of OPS used in the conversion reaction. Preferably, it is fed at a molar concentration twice as high as that of OPS to effectively convert OPS into cysteine (Table 15).

[0032] As used herein, the term "O-phosphoserine sulfhydrylase (OPSS)" refers to an enzyme that catalyzes the transfer of a thiol group (SH) to OPS (O-phosphoserine) to convert OPS into cysteine. The enzyme was first found in Aeropyrum pernix, Mycobacterium tuberculosis, and Trichomonas vaginalis (Mino K and Ishikawa K, FEBS letters, 551: 133-138, 2003; Burns K E et al., J. Am. Chem. Soc., 127: 11602-11603, 2005). The above mentioned enzymes have the amino acid sequences of SEQ ID No: 3 and 6.

[0033] As used herein, the term "mutant" refers to a culture or an individual that shows an inheritable or non-heritable alteration in phenotype. When used in conjunction with OPSS, the term "mutant" is intended to mean an OPSS enzyme which is genetically altered such that its activity can be effectively enhanced, compared to the wild-type.

[0034] In the present invention, the OPSS mutant can be constructed by deleting, substituting or adding a part of a nucleotide sequence encoding OPSS. According to one embodiment of the present invention, an OPSS enzyme with enhanced activity was prepared by deleting five C-terminal amino acid residues of the OPSS enzyme of Mycobacterium smegmatis. The mutant enzymes have the amino acid sequences of SEQ ID NO: 4 and 5.

[0035] The OPSS mutant can be obtained in E. coli, widely used for enzyme expression, using gene synthesis techniques based on codon optimization by which enzymes of interest can be obtained in high yield. Alternatively, screening methods of useful enzyme resources based on the bioinformatics of massive amounts of genetic information about microorganisms may be used to obtain the OPSS mutant. In one embodiment of the present invention, OPSS enzymes that utilize OPS as a substrate to synthesize cysteine were selected from various microbes by screening the homology of amino acid sequences. In this regard, cell pellets obtained using a medium and culture conditions that were suitable in the art were lyzed, followed by the purification of the supernatant containing the enzyme to afford the OPSS enzyme (Table 7).

[0036] In addition, a high-yield expression system was developed for obtaining the OPSS enzyme economically. A pET vector employing a T7 promoter is well known in the art. However, the present inventors developed an enzyme expression system, named the CJ1 system (Korean Patent 10-0620092 BI), instead of employing the typical system. In one embodiment of the present invention, the expression levels of OPSS between a 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 the pET system. In addition, the overexpression of OPSS required a low temperature (18.degree. C.) and a long period of time in the pET system, but a high temperature (37.degree. C.) and a short period of time in the pCL-pCJ1 system. Preferably, the pCL-pCJ1 system is used to obtain OPSS.

[0037] The enhancement of the enzyme activity may be achieved using various well-known methods. For example, it can be performed by increasing the number of copies of a gene encoding OPSS, using a strong promoter, or introducing a genetic mutation.

[0038] Optimization of the enzymatic conversion of OPSS may be achieved using various methods known in the art. For example, the optimization may be based on a full understanding of the characteristics of OPSS, such as the optimal temperature and pH, inhibition against substrates, substrate concentration, heat stability, etc. In addition, the optimization may be determined by optimal conditions for the enzymatic conversion, such as the optimal OPSS concentration, the optimal balances of the substrates used in terms of concentrations, a preference for sulfur compounds providing SH for the enzymatic conversion, a preference for certain buffers, the influence of ions generated, and cofactors and their optimal concentrations.

[0039] In one embodiment of the present invention, the OPSS enzyme obtained using the above-mentioned method was characterized and on the basis of the determined characteristics, an economically beneficial enzymatic conversion process that has a high conversion rate of cysteine from OPS, with the guarantee of enzyme stability, was developed. In the enzymatic conversion process, the reaction temperature can be set from 37.degree. C. to 80.degree. C. In detail, Ape-OPSS (SEQ ID NO: 6), belonging to Archea, exhibits increased enzymatic activity at 60.degree. C. compared to 37.degree. C., and the enzyme itself is highly stable to heat, with optimal reactivity at 60.degree. C. On the other hand, Msm-T (SEQ ID NO: 4) exhibits optimal activity at 37.degree. C. and is relieved the activity to heat treatment at 60.degree. C. The OPSS enzyme was observed to have enzymatic activity over a pH range of 6.0 to 10.0. Ape-OPSS showed optimal activity at pH 7.4. With the appearance of optimal activity at a pH of from 8.0 to 9.0, Msm-T showed stability over a wider pH range, compared to Ape-OPSS (Tables 9 and 12, and FIGS. 2 and 3).

[0040] As a cofactor, 0.001-2 mM PLP (pyridoxal-5'-phosphate) or 0.001-100 mM DTT may be used in the enzymatic conversion. In one embodiment of the present invention, the cysteine conversion rate was 2.3-fold increased in the presence of 25 mM DTT or 0.2 mM PLP. As such, treatment with DTT or PLP brought about an improvement in the cysteine conversion rate of step 2). The addition of the cofactor was set to a reasonable level in consideration of the increased production cost and the increased conversion rate (Table 11).

[0041] The reaction conditions for OPSS may vary depending on the kinds and concentration of the OPS used. In one embodiment of the present invention, pure OPS (commercially available), OPS purified from the culture prepared in step 1), and the OPS-containing culture of step 1) were used under various conditions to provide the optimal conversion rates. As a result, the cysteine conversion rate varied depending on the kind and concentration of OPSS and the reaction temperature and the kind and concentration of OPS (FIGS. 5 and 6, and Table 16).

[0042] The method of the present invention may further comprise isolating and purifying the cysteine produced in step 2). After the enzymatic conversion, cysteine can be isolated and purified from the culture medium using a method well known in the art.

[0043] Those skilled in the art may chemically synthesize cysteine derivatives from cysteine using a well known method. Cysteine may be readily reacted with an acetylation agent to give NAC (N-acetylcysteine) and with haloacetic acid under basic conditions to give SCMC (S-carboxymetylcysteine). These cysteine derivatives are used as materials in medicines that treat coughs, bronchitis, bronchial asthma, and sore throat.

[0044] In the present invention, the OPS broth obtained through microbial fermentation is used as a substrate for synthesizing cysteine. The OPS broth obtained by microbial fermentation has economical advantages over commercially available pure OPS in that the OPS broth can be used without having to be additionally purified and the cofactor PLP necessary for the conversion can be obtained from the fermented culture.

[0045] In one embodiment of the present invention, a conversion process was developed which ensures a cysteine conversion rate of as high as 80% when 50 .mu.g/ml Msm-T was used under reaction conditions of a 50 mM OPS broth or a 60 mM purified OPS broth, 100 mM Na.sub.2S or 120 mM Na.sub.2S, and 0.2 mM PLP. It should be appreciated to those skilled in the art that the enzymatic conversion using highly active enzymes can easily be optimized and scaled up.

[0046] In accordance with another aspect thereof, the present invention provides a recombinant microorganism which is reduced the activity of SerB for the production of OPS. Preferably, the recombinant microorganism for the production of OPS is the microorganism deposited under accession No. KCCM11212P.

MODE FOR INVENTION

[0047] 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.

Preparation of O-Phosphoserine Producing Corynebacterium and Production of O-Phosphoserine Using the Same

Example 1

Preparation of Phosphoserine Phosphatase (serB) Deficient Corynebacterium Strain

[0048] Corynebacterium glutamicum 13032 was modified by deleting the serB gene (SEQ ID NO: 7, EC 3.1.3.3) encoding phosphoserine phosphatase, which catalyses the synthesis of L-serine from O-phosphoserine, therefrom. To this end, a fragment for inactivation of serB was constructed. In this regard, primers were designed for the preparation of the recombinant strain 13032-.DELTA.serB of the present invention. First, the serB sequence of Corynebacterium glutamicum 13032 was obtained with reference to the data of the NIH GenBank, and primers SEQ ID NOS: 9 to 14 were synthesized on the basis of the serB sequence. For the site-specific gene disruption, a pDC vector which cannot replicate in Corynebacterium glutamicum was employed. A pDC-.DELTA.serB plasmid in which the open reading frame of serB was internally disrupted was constructed and adopted for the preparation of a site-specific serB gene deletion in Corynebacterium glutamicum mutant strain. The internal gene disruption of the pDC-.DELTA.serB was generated by crossover PCR using primer pairs of SEQ ID NOS: 9 and 10 and SEQ ID NOS: 11 and 12, with the genomic DNA of Corynebacterium glutamicum ATCC13032 serving as a template, and introducing the PCR product into a pDC vector. The resulting recombinant plasmid was transformed into wild-type Corynebacterium glutamicum by electroporation (van der Rest et al. 1999). The plasmid was introduced into the chromosome by primary recombination (crossing over), followed by secondary recombination (crossing over) to excise the original serB from the chromosome.

[0049] After completion of the secondary recombination, the Corynebacterium glutamicum transformants containing the deletion mutation of serB was analyzed by diagnostic PCR using a pair of gene-specific primers SEQ ID NOS: 13 and 14. The recombinant strain was named CB01-0047.

Example 2

Assay for O-Phosphoserine Productivity in the Phosphoserine Phosphatase Deficient Corynebacterium Strain

[0050] The mutant strain CB01-0047, resulting from the deletion of serB from Corynebacterium glutamicum 13032, which was anticipated to accumulate O-phosphoserine, was spread over BHIS plates and incubated overnight in a 30.degree. C. incubator. Afterwards, the colonies appearing on the BHIS plates were inoculated in 25 mL of a titer medium shown in Table 1 using a platinum loop and then incubated at 30.degree. C. for 48 hours with shaking at 200 rpm. The results are summarized in Table 2, below.

TABLE-US-00001 TABLE 1 Composition Amount (per liter) Biotin 0.09 g Glucose 100 g KH.sub.2PO.sub.4 1.1 g (NH.sub.4).sub.2SO.sub.4 45 g MgSO.sub.4.cndot.7H.sub.2O 1.2 g HSM 20 g Trace elements 20 ml Calcium carbonate 30 g pH 7.2 Trace elements Biotin 0.09 g Thiamine 0.45 g Ca-Panthenate 0.45 g NCA 3 g FeSO.sub.4.cndot.7H.sub.2O 9 g MnSO.sub.4.cndot.4H.sub.2O 9 g ZnSO.sub.4.cndot.7H.sub.2O 0.045 g CuSO.sub.4.cndot.5H.sub.2O 0.045 g

TABLE-US-00002 TABLE 2 Sugar consumed O- Strain OD 562 nm (g/L) phosphoserine (g/L) C. glutamicum 25 100 0.02 13032 CB01-0047 6.5 23 0.07

The CB01-0047 strain was observed to grow very slowly in the titer medium. This growth retardation was not improved even upon the addition of an L-glycine supplement. However, the growth was increased in the presence of L-serine, but a slight increase in the production of O-phosphoserine compared to the wild-type was observed. The results are summarized in Table 3, below.

TABLE-US-00003 TABLE 3 A.A. (amino Sugar O- acids) consumed phosphoserine Strain added OD 562 nm (g/L) (g/L) CB01-0047 -- 6.3 21 0.09 L-Glycine 6.9 22 0.09 L-Serine 24.5 100 0.05

Preparation of O-Phosphoserine Producing E. coli

Example 3

Preparation of E. coli Strain Having the Reduced Activity of Phosphoserine Phosphatase (SerB)

[0051] E. coli was modified by deleting the serB gene (SEQ ID NO: 8) encoding phosphoserine phosphatase, which catalyses the synthesis of L-serine from O-phosphoserine, therefrom. The deletion mutant E. coli K12 was prepared using the one-step inactivation method (Datsenko K A and Wanner B L, Proc. Natl. Acad. Sci., 97: 6640-6645, 2000) to delete an antibiotic-resistant maker gene. To prepare the serB deletion strain, first, PCR was performed on a pKD3 plasmid (Datsenko K A and Wanner B L, Proc. Natl. Acad. Sci., 97: 6640-6645, 2000; GenBank No. AY048742) using a pair of primers of SEQ ID NOS: 15 and 16. The PCR product was (introduced into competent cells of pKD46 containing E. coli K12 (Datsenko K A and Wanner B L, Proc. Natl. Acad. Sci., 97: 6640-6645, 2000; GenBank No. AY048746) by electroporation. Thereafter, strains that showed resistance to chloramphenicol were subjected to PCR to confirm the deletion of serB, and then transformed with pCP20 (Datsenko K A and Wanner B L, Proc. Natl. Acad. Sci., 97: 6640-6645, 2000) to remove the antibiotic-resistant marker. The resulting mutant strain was named CA07-0012.

[0052] In addition, the initiation codon of serB was modified to lower phosphoserine phosphatase activity as follows. The wild-type serB gene with ATG as an initiation codon was obtained by PCR with the genomic DNA of E. coli W3110 serving as a template. A mutant serB with CTG as an initiation codon was constructed by sewing PCR. A pair of primes of SEQ ID NOS: 17 and 18 was used in the PCR for amplifying the wild-type serB while pairs of primers of SEQ ID NOS: 19 to 20 were employed for PCR amplification of the mutant serB. The PCR products was treated with HindIII and cloned into pccBAC1 (Epicentre) at the HindIII restriction site to construct pccBAC1-Pself-ATG-serB, and pccBAC1-Pself-CTG-serB respectively. The wild-type and the mutant serB vector was introduced into CA07-0012 to compare the phosphoserine phosphatase activity.

Example 4

Assay of Strain Having the Reduced Activity of SerB for O-Phosphoserine Productivity

[0053] The phosphoserine phosphatase deficient mutant strain CA07-0012 that was anticipated to accumulate O-phosphoserine, was spread over LB plates and incubated overnight in a 33.degree. C. incubator. Afterwards, the colonies appearing on the LB plates were inoculated in 25 mL of a titer medium shown in Table 4 using a platinum loop and then incubated at 33.degree. C. for 48 hours with shaking at 200 rpm. The results are summarized in Table 5, below.

TABLE-US-00004 TABLE 4 Composition Amount (per liter) Glucose 40 g KH.sub.2PO.sub.4 2 g (NH.sub.4).sub.2SO.sub.4 17 g MgSO.sub.4.cndot.7H.sub.2O 1 g FeSO.sub.4.cndot.7H.sub.2O 10 mg MnSO.sub.4.cndot.4H.sub.2O 10 mg ZnSO.sub.4.cndot.7H.sub.2O 10 mg Yeast extract 2 g Calcium carbonate 30 g pH 6.8

TABLE-US-00005 TABLE 5 Sugar O-phosphoserine Strain OD 562 nm consumed (g/L) (g/L) E. coli W3110 16 40 0.03 CA07-0012 9.8 16 0.5 CA07-0012/ 20 40 0 pccBAC1-Pself-ATG- serB CA07-0012/ 15 40 0.7 pccBAC-Pself-CTG- serB

[0054] To enhance the growth and O-phosphoserine productivity thereof, CA07-0012 was cultured for 48 hours in the titer medium of Table 4 additionally contained 1 g/L L-glycine.

[0055] The results are summarized in Table 6, below.

TABLE-US-00006 TABLE 6 Sugar Strain OD 562 nm consumed (g/L) O-phosphoserine (g/L) E. coli W3110 16 40 0.03 CA07-0012 18 40 1.5

[0056] As shown in Table 6, the addition of L-glycine to the culture medium allowed the strain to increase the growth rate and the O-phosphoserine productivity.

Development and Characterization of O-Phosphoserine (OPS) Sulfhydrylase (OPSS)

Example 5

Development of OPS Sulfhydrylase (OPSS)

[0057] Aeropyrum pernix, Mycobacterium tuberculosis, and Trichomonas vaginalis are reported to have O-phosphoserine sulfhydrylase (OPSS), an enzyme that employs O-phospho-L-serine (OPS), instead of O-acetyl serine (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 OPS sulfhydrylase, which converts OPS into cysteine, from Aeropyrum pernix and Mycobacterium tuberculosis H37Rv. Of them, the Mycobacterium tuberculosis H37Rv-derived OPSS enzyme was used for screening amino acid homology. As a result, three types of OPSS were secured from Mycobacterium smegmatis str. MC2 155, Rhodococcus jostii RHA1, and Nocardia farcinica IFM 10152.

[0058] 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 genes and the resulting recombinant plasmids are summarized in Table 7, below. Suitable combinations of the templates and the primers, as given in Table 7, were used for PCR for amplifying respective OPSS genes. The PCR products and the pET28a vector were digested with NdeI and HindIII (37.degree. 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 7, below.

TABLE-US-00007 TABLE 7 Enzyme Vector Template Primer Ape-OPSS pET28a-Ape- Synthetic DNA SEQ ID NOS: 21 and 22 OPSS Mtb-OPSS pET28a-Mtb- Mtb Genomic SEQ ID NOS: 23 and 24 OPSS DNA Msm-OPSS pET28a-Msm- Msm Genomic SEQ ID NOS: 25 and 26 OPSS DNA Rjo-OPSS pET28a-Rjo- Rjo Genomic SEQ ID NOS: 27 and 28 OPSS DNA Nfa-OPSS pET28a-Nfa- Nfa Genomic SEQ ID NOS: 29 and 30 OPSS DNA

[0059] 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.degree. 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 OD.sub.600 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.degree. 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 thus 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 6

Assay of OPS Sulfhydrylase (OPSS) for Cysteine Synthesis Activity

[0060] The OPS sulfhydrylase enzymes obtained from the four microorganism strains were assayed for ability to catalyze the conversion of O-phosphoserine (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 8, below.

TABLE-US-00008 TABLE 8 Stock soln Final Conc. Blank OPS sulfhydrylase 6x his-enzyme -- 40 (50 mg) 1M 100 mM HEPES 100 100 HEPES(pH 7.4) 0.5M Na.sub.2S 10 mM Na.sub.2S 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

[0061] Reaction solutions excepting of the enzymes were incubated at 37.degree. C. for 5 min, after which 50 mg of purified OPS sulfhydrylase was added to the reaction solution. At predetermined times during incubation at 37.degree. C., 100 mL of the enzyme reactions 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 OD.sub.560 according to the Gaitonde method. Cysteine synthesis activities of the four different OPS sulfhydrylase enzymes are summarized in Table 9, below. The cysteine synthesis titers of the OPSS enzymes are expressed as cysteine conversion rates with reaction time.

TABLE-US-00009 TABLE 9 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

[0062] The OPS sulfhydrylase enzymes derived from Aeropyrum pernix and Mycobacterium tuberculosis H37Rv, which were previously reported (Mino K and Ishikawa K, FEBS letters, 551: 133-138, 2003; Westrop G D, Goodall G, Mottram J C and Coombs G H, J. Biol. Chem., 281: 25062-25075, 2006), 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 OPS sulfhydrylase, which was obtained by screening amino acid homology with the Mtb-OPSS enzyme, was first found. As seen in the data of Table 9, 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% that was 4.3 times as high as that of Mtb-OPSS. On the other hand, the novel Nocardia farcinica IFM 10152-derived OPS sulfhydrylase, obtained by the homology screening, exhibited insufficient activity of converting O-phosphoserine into cysteine.

Example 7

Preparation of Mtb-T and Msm-T that Encode C-Terminally 5 Amino Acid Residues Truncated Mtb-OPSS and Msm-OPSS

[0063] 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 reported to be able to use an S.sup.2- 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 (Agren D, Schnell R and Schneider G, FEBS letters, 583: 330-336, 2009). On the basis of this report, Mtb-T (SEQ ID NO: 5), which can rapidly convert OPS in the presence of S.sup.2- as a sulfur source, was obtained. Msm-T was also obtained from Msm-OPSS (SEQ ID NO: 3) that shares an amino acid homology with Mtb-OPSS. Expression vectors carrying the two enzyme mutants were constructed. In this regard, pfu PCR was performed on the genomic DNA of Mycobacterium tuberculosis H37Rv or Mycobacterium smegmatis in the presence of a pair of primers of SEQ ID NOS: 31, 32, 33 and 34. 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 mutant OPSS enzymes was confirmed by 14% SDS PAGE. The two mutant OPSS enzymes are purified and expressed in the same conditions as in Example 5. As a result, Mtb-T (SEQ ID NO: 5) and Msm-T (SEQ ID NO: 4) were obtained.

Example 8

Assay of Mtb-T and Msm-T for Cysteine Conversion Activity

[0064] On the basis of the report that Mycobacterium tuberculosis H37Rv-derived OPSS mutants devoid of five C-terminal amino acid residues show increased affinity for an S.sup.2- group-containing sulfur source even in the absence of subsidiary enzymes (Agren D, Schnell R and Schneider G, FEBS letters, 583: 330-336, 2009), Mtb-T and Msm-T were obtained. They were evaluated for enzymatic activity by measuring final cysteine conversion rates. Enzymatic activity was assayed in the same condition and manner as in Example 6. The produced cysteine was quantitatively analyzed using the Gaitonde method.

TABLE-US-00010 TABLE 10 Cysteine Conversion Rate (%) 10 min 30 min 60 min Mtb-T 9.5 18.6 37.1 Msm-T 20.3 54.6 100

[0065] As seen in Table 10, Msm-T, being devoid of the five C-terminal amino acid residues of Mycobacterium smegmatis str. MC2 155-derived OPSS allowed the conversion of cysteine from the substrate at a rate of 100% in one hour.

[0066] When its amino acid sequence was modified, the O-phosphoserine sulfhydrylase (OPSS) can more effectively catalyze the biosynthesis of L-cysteine.

Example 9

Requirement of Cofactor for OPS Sulfhydrylase Activity

[0067] 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 (pyridoxal-5'-phosphate) and DTT (dithiothreitol). In this regard, the substrates of 50 mM OPS broth and 100 mM Na.sub.2S were reacted at 37.degree. 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. As seen in Table 11, 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.

TABLE-US-00011 TABLE 11 Msm-T Cysteine Conversion Rate (%) (-) PLP, (-) DTT 23.62 (+) PLP, (-) DTT 33.21 (-) PLP, (+) DTT 40.08 (+) PLP, (+) DTT 54.65

Example 10

The Influence of Temperature on the Activity of OPS Sulfhydrylase

[0068] The cysteine conversion rates of Ape-OPSS and Msm-T according to temperatures were examined. The enzymatic activity at 37.degree. C. and 60.degree. C. was measured 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 Na.sub.2S, 0.2 mM PLP, and CysM 50 .mu.g/mL. The amount of produced cysteine was determined using the Gaitonde method. In the condition of a buffer, as shown in FIG. 2, Ape-OPSS showed a faster initial reaction rate at 37.degree. C. as well as higher reactivity at 60.degree. C. than did Msm-T.

Example 11

Heat Stability of OPS Sulfhydrylase

[0069] 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.degree. C. and 60.degree. C. for 10, 30, 60, 120, and 240 min, followed by reaction at 37.degree. C. for 30 min under the condition of 5 mM OPS, 10 mM Na.sub.2S, 0.2 mM PLP, and 100 mM HEPES (pH 7.4). For this reaction, 10 .mu.g/mL Ape-OPSS and 50 .mu.g/mL Msm-T were employed. The amounts of the produced cysteine were measured using the Gaitonde method. Ape-OPSS was observed to retain its intact activity in spite of heat treatment at 60.degree. C. for 4 hours while the activity of Msm-T was maintained at 37.degree. C., but decreased by 50% upon heat treatment at 60.degree. C. for 30 min. The results are given in Table 12, below.

TABLE-US-00012 TABLE 12 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

[0070] An examination was made of the retention of enzymatic activity at 37.degree. C. when Msm-T was used in an amount of 50 .mu.g/mL, which is a practical concentration in OPS broth. In the absence of Na.sub.2S, 50 .mu.g/mL Msm-T was treated, together with 50 mM OPS broth and 0.2 mM PLP, at 37.degree. C. for 0.5, 1, 2, 4, and 6 hours, after which Na.sub.2S 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. As a result, the activity of Msm-T was decreased below 50% 2 hours after reaction at 37.degree. C. in OPS broth (Table 13).

TABLE-US-00013 TABLE 13 Time 0 30 min 60 min 120 min 240 min 360 min Cysteine 100 88 73 47 11 3 conversion rate (%)

Example 12

The Influence of pH on the OPS Sulfhydrylase

[0071] The cysteine conversion rates of Ape-OPSS and Msm-T according to pH were measured. In 100 mM buffer, Ape-OPSS and Msm-T, each having a concentration of 50 .mu.g/mL, were subjected to reaction at 37.degree. C. for 10 min. In this regard, K-phosphate buffer with a pH of 6.4/7.0/7.4/8.0, Tris-HCl buffer with a pH of 7.0/7.4/8.0/8.5/8.8, and 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. As seen in FIG. 3, 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 13

Effect of Ions on the Activity of OPS Sulfhydrylase

[0072] Effects of ions on the activity of the OPSS enzymes were examined as follows. In a reaction mixture containing 5 mM OPS, 10 mM Na.sub.2S, 0.2 mM PLP, and 100 mM HEPES (pH 7.4), the enzymes were subjected to reaction at 37.degree. C. for 30 min in the presence of (NH.sub.4).sub.2SO.sub.4 (1, 3, 5, 10, 20 g/L), KH.sub.2PO.sub.4 (0.5, 1, 2, 4, 8 g/L), or NH.sub.4Cl (0.2, 0.5, 1, 2 g/L). Ape-OPSS and Msm-T were used at a concentration of 10 .mu.g/mL and 50 .mu.g/mL, respectively. The amounts of the produced cysteine were determined using the Gaitonde method.

[0073] No changes were detected in the cysteine conversion rate when (NH.sub.4).sub.2SO.sub.4 or KH.sub.2PO.sub.4 was added to the reaction mixture. On the other hand, as seen in Table 14, the cysteine conversion rate was decreased with an increase in NH.sub.4Cl concentration. Particularly, the maximal enzyme activity was decreased by more than 70% when 2 g/L NH.sub.4Cl was added. Therefore, NH.sub.4Cl was observed to have a negative effect on the conversion activity of OPS sulfhydrylase.

TABLE-US-00014 TABLE 14 Relative activity (%) NH.sub.4Cl 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

Example 14

Effect of Sulfur Source on the Cysteine Synthesis Activity of OPS Sulfhydrylase

[0074] 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 .mu.g/mL Ape-OPSS, 50 .mu.g/mL Msm-T) was subjected to reaction at 37.degree. C. for 1 hour in the presence of 10 mM Na.sub.2S, NaSH, or Na.sub.2S.sub.2O.sub.3. The amounts of the produced cysteine were measured using the Gaitonde method. Ape-OPSS was observed to prefer Na.sub.2S.sub.2O.sub.3 as a sulfur source, whereas Msm-T prefers Na.sub.2S. The results are summarized in Table 15, below.

TABLE-US-00015 TABLE 15 Relative activity (%) Enzyme Na.sub.2S NaSH Na.sub.2S.sub.2O.sub.3 Ape-OPSS 100.0 95.2 142.3 Msm-T 106.7 98.3 66.2

Example 15

Construction of the Expression Vector Carrying OPS Sulfhydrylase (pCL-Pcj1 System) and Expression in E. coli

[0075] PCR was performed using primers of SEQ ID NOS: 35 and 36, with the pET28a-Msm-T vector serving as a template. The PCR product thus obtained was treated with EcoRV and HindIII and cloned into pCL-P(CJ1) to construct a recombinant vector named pCL-P(CJ1)-Msm-T. To examine a difference in the expression level of Msm-T 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 used the K12G strain. Single colonies taken from LB plates were inoculated into 5 mL of LB broth and cultured at 37.degree. 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 OD.sub.600 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.degree. 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 (FIG. 4).

Example 16

Cysteine Synthesis by OPS Sulfhydrylase with the Purified OPS Fermentation Broth

[0076] The conversion rates from purified OPS to cysteine of Msm-T and Ape-OPSS were determined. In the presence of 75 .mu.g/mL of each of the enzymes and 0.2 mM PLP, 60 mM OPS purified from OPS fermentation broth was reacted with 120 mM Na.sub.2S at 37.degree. C. or 70.degree. C. for 30, 60, 90, and 120 min. The reaction was conducted only at 37.degree. C. for Msm-T, but at both 37.degree. C. and 70.degree. C. for Ape-OPSS. The amounts of the produced cysteine were measured using the Gaitonde method. As seen in FIG. 5, a purified OPS fermentation broth served well as a substrate for the enzymatic conversion into cysteine. Particularly, the conversion rate of Ape-OPSS was increased at 70.degree. C. even upon the use of the purified OPS fermentation broth.

Example 17

Cysteine Synthesis by OPS Sulfhydrylase with the OPS Fermentation Broth

[0077] 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 100 mM Na.sub.2S and 0.2 mM PLP, 50 mM of OPS fermentation broth was reacted with 5 .mu.g/mL or 50 .mu.g/mL of each of Msm-T and Ape-OPSS at 37.degree. C. The amounts of the produced cysteine were measured using the Gaitonde method. As seen in FIG. 6, the highest conversion rate was detected in 50 .mu.g/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 18

Cysteine Conversion Rate According to OPS Concentration

[0078] To examine the effect of OPS concentration on the conversion rate of Msm-T, 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 .mu.g. The amounts of cysteine in the reaction solution were measured using the Gaitonde method. Msm-T exhibited a conversion rate of as high as 100% when the concentration of OPS was about 30 g/L.

[0079] When the concentration of OPS exceeded 50 g/L, both the conversion rate and the conversion percentage were found to decrease. From these results, it is understood that when OPS fermentation broth is used as a substrate, there is an optimal concentration ratio between OPS and the enzyme.

TABLE-US-00016 TABLE 16 Cysteine Conversion Rate (Msm-T 50 ug) Time 0 min 10 min 30 min 60 min 120 min 180 min OPS measured 0 23.03 65.38 65.70 61.95 55.35 10.65 g/l OPS measured 0 1.15 10.23 28.07 97.84 100.34 36.09 g/l OPS measured 0 0 2.36 7.41 42.69 66.67 55.6 g/l

Sequence CWU 1

1

361446PRTCorynebacterium glutamicum 13032PEPTIDE(1)..(446)phosphoserine phosphatase, SerB 1Met Ser Cys Ser Ala Leu Arg His Glu Thr Ile Val Ala Val Thr Glu1 5 10 15 Leu Ile Gln Asn Glu Ser Gln Glu Ile Ala Glu Leu Glu Ala Gly Gln 20 25 30 Gln Val Ala Leu Arg Glu Gly Tyr Leu Pro Ala Val Ile Thr Val Ser 35 40 45 Gly Lys Asp Arg Pro Gly Val Thr Ala Ala Phe Phe Arg Val Leu Ser 50 55 60 Ala Asn Gln Val Gln Val Leu Asp Val Glu Gln Ser Met Phe Arg Gly65 70 75 80 Phe Leu Asn Leu Ala Ala Phe Val Gly Ile Ala Pro Glu Arg Val Glu 85 90 95 Thr Val Thr Thr Gly Leu Thr Asp Thr Leu Lys Val His Gly Gln Ser 100 105 110 Val Val Val Glu Leu Gln Glu Thr Val Gln Ser Ser Arg Pro Arg Ser 115 120 125 Ser His Val Val Val Val Leu Gly Asp Pro Val Asp Ala Leu Asp Ile 130 135 140 Ser Arg Ile Gly Gln Thr Leu Ala Asp Tyr Asp Ala Asn Ile Asp Thr145 150 155 160 Ile Arg Gly Ile Ser Asp Tyr Pro Val Thr Gly Leu Glu Leu Lys Val 165 170 175 Thr Val Pro Asp Val Ser Pro Gly Gly Gly Glu Ala Met Arg Lys Ala 180 185 190 Leu Ala Ala Leu Thr Ser Glu Leu Asn Val Asp Ile Ala Ile Glu Arg 195 200 205 Ser Gly Leu Leu Arg Arg Ser Lys Arg Leu Val Cys Phe Asp Cys Asp 210 215 220 Ser Thr Leu Ile Thr Gly Glu Val Ile Glu Met Leu Ala Ala His Ala225 230 235 240 Gly Lys Glu Ala Glu Val Ala Ala Val Thr Glu Arg Ala Met Arg Gly 245 250 255 Glu Leu Asp Phe Glu Glu Ser Leu Arg Glu Arg Val Lys Ala Leu Ala 260 265 270 Gly Leu Asp Ala Ser Val Ile Asp Glu Val Ala Ala Ala Ile Glu Leu 275 280 285 Thr Pro Gly Ala Arg Thr Thr Ile Arg Thr Leu Asn Arg Met Gly Tyr 290 295 300 Gln Thr Ala Val Val Ser Gly Gly Phe Ile Gln Val Leu Glu Gly Leu305 310 315 320 Ala Glu Glu Leu Glu Leu Asp Tyr Val Arg Ala Asn Thr Leu Glu Ile 325 330 335 Val Asp Gly Lys Leu Thr Gly Asn Val Thr Gly Lys Ile Val Asp Arg 340 345 350 Ala Ala Lys Ala Glu Phe Leu Arg Glu Phe Ala Ala Asp Ser Gly Leu 355 360 365 Lys Met Tyr Gln Thr Val Ala Val Gly Asp Gly Ala Asn Asp Ile Asp 370 375 380 Met Leu Ser Ala Ala Gly Leu Gly Val Ala Phe Asn Ala Lys Pro Ala385 390 395 400 Leu Lys Glu Ile Ala Asp Thr Ser Val Asn His Pro Phe Leu Asp Glu 405 410 415 Val Leu His Ile Met Gly Ile Ser Arg Asp Glu Ile Asp Leu Ala Asp 420 425 430 Gln Glu Asp Gly Thr Phe His Arg Val Pro Leu Thr Asn Ala 435 440 445 2322PRTEscherichia coli K12 W3110PEPTIDE(1)..(322)phosphoserine phosphatase, SerB 2Met Pro Asn Ile Thr Trp Cys Asp Leu Pro Glu Asp Val Ser Leu Trp1 5 10 15 Pro Gly Leu Pro Leu Ser Leu Ser Gly Asp Glu Val Met Pro Leu Asp 20 25 30 Tyr His Ala Gly Arg Ser Gly Trp Leu Leu Tyr Gly Arg Gly Leu Asp 35 40 45 Lys Gln Arg Leu Thr Gln Tyr Gln Ser Lys Leu Gly Ala Ala Met Val 50 55 60 Ile Val Ala Ala Trp Cys Val Glu Asp Tyr Gln Val Ile Arg Leu Ala65 70 75 80 Gly Ser Leu Thr Ala Arg Ala Thr Arg Leu Ala His Glu Ala Gln Leu 85 90 95 Asp Val Ala Pro Leu Gly Lys Ile Pro His Leu Arg Thr Pro Gly Leu 100 105 110 Leu Val Met Asp Met Asp Ser Thr Ala Ile Gln Ile Glu Cys Ile Asp 115 120 125 Glu Ile Ala Lys Leu Ala Gly Thr Gly Glu Met Val Ala Glu Val Thr 130 135 140 Glu Arg Ala Met Arg Gly Glu Leu Asp Phe Thr Ala Ser Leu Arg Ser145 150 155 160 Arg Val Ala Thr Leu Lys Gly Ala Asp Ala Asn Ile Leu Gln Gln Val 165 170 175 Arg Glu Asn Leu Pro Leu Met Pro Gly Leu Thr Gln Leu Val Leu Lys 180 185 190 Leu Glu Thr Leu Gly Trp Lys Val Ala Ile Ala Ser Gly Gly Phe Thr 195 200 205 Phe Phe Ala Glu Tyr Leu Arg Asp Lys Leu Arg Leu Thr Ala Val Val 210 215 220 Ala Asn Glu Leu Glu Ile Met Asp Gly Lys Phe Thr Gly Asn Val Ile225 230 235 240 Gly Asp Ile Val Asp Ala Gln Tyr Lys Ala Lys Thr Leu Thr Arg Leu 245 250 255 Ala Gln Glu Tyr Glu Ile Pro Leu Ala Gln Thr Val Ala Ile Gly Asp 260 265 270 Gly Ala Asn Asp Leu Pro Met Ile Lys Ala Ala Gly Leu Gly Ile Ala 275 280 285 Tyr His Ala Lys Pro Lys Val Asn Glu Lys Ala Glu Val Thr Ile Arg 290 295 300 His Ala Asp Leu Met Gly Val Phe Cys Ile Leu Ser Gly Ser Leu Asn305 310 315 320 Gln Lys3323PRTMycobacterium smegmatics str. MC2 155PEPTIDE(1)..(323)Msm-OPSS 3Met Thr Arg Tyr Asp Ser Leu Leu Gln Ala Leu Gly Asn Thr Pro Leu1 5 10 15 Val Gly Leu Gln Asn Leu Ser Pro Arg Trp Asp Asp Glu Asp Gly Lys 20 25 30 Pro His Val Arg Leu Trp Ala Lys Leu Glu Asp Arg Asn Pro Thr Gly 35 40 45 Ser Ile Lys Asp Arg Pro Ala Leu Arg Met Ile Glu Gln Ala Glu Arg 50 55 60 Asp Gly Leu Leu Gln Pro Gly Ala Thr Ile Leu Glu Pro Thr Ser Gly65 70 75 80 Asn Thr Gly Ile Ser Leu Ala Met Ala Ala Leu Leu Lys Gly Tyr Asn 85 90 95 Met Ile Cys Val Met Pro Glu Asn Thr Ser Ile Glu Arg Arg Gln Ile 100 105 110 Leu Glu Leu Tyr Gly Ala Arg Ile Ile Phe Ser Pro Ala Glu Gly Gly 115 120 125 Ser Asn Thr Ala Val Ala Thr Ala Lys Glu Leu Ala Ala Gln Asn Pro 130 135 140 Ser Trp Val Met Leu Tyr Gln Tyr Gly Asn Pro Ala Asn Ser Asp Ala145 150 155 160 His Tyr Phe Gly Thr Gly Pro Glu Leu Leu Ala Asp Leu Pro Glu Ile 165 170 175 Thr His Phe Val Ala Gly Leu Gly Thr Thr Gly Thr Leu Met Gly Thr 180 185 190 Gly Arg Phe Leu Arg Glu His Val Pro Gly Val Gln Ile Val Ala Ala 195 200 205 Glu Pro Arg Tyr Gly Glu Gly Val Tyr Ala Leu Arg Asn Ile Asp Glu 210 215 220 Gly Phe Ile Pro Glu Leu Tyr Asp Ala Asp Val Leu Thr Thr Arg Phe225 230 235 240 Ser Val Gly Ser Phe Asp Ala Val Arg Arg Thr Arg Glu Leu Val Thr 245 250 255 Arg Glu Gly Ile Phe Ala Gly Ile Ser Thr Gly Ala Val Leu His Ala 260 265 270 Ala Leu Gly Met Ala Ala Lys Ala Val Lys Ala Gly Glu Arg Ala Asp 275 280 285 Ile Ala Phe Val Val Ala Asp Ala Gly Trp Lys Tyr Leu Ser Thr Gly 290 295 300 Ala Tyr Ala Gly Ser Leu Asp Asp Ala Glu Asp Ala Leu Glu Gly Gln305 310 315 320 Leu Trp Ala4318PRTMycobacterium smegmatics str. MC2 155PEPTIDE(1)..(318)Msm-T 4Met Thr Arg Tyr Asp Ser Leu Leu Gln Ala Leu Gly Asn Thr Pro Leu1 5 10 15 Val Gly Leu Gln Asn Leu Ser Pro Arg Trp Asp Asp Glu Asp Gly Lys 20 25 30 Pro His Val Arg Leu Trp Ala Lys Leu Glu Asp Arg Asn Pro Thr Gly 35 40 45 Ser Ile Lys Asp Arg Pro Ala Leu Arg Met Ile Glu Gln Ala Glu Arg 50 55 60 Asp Gly Leu Leu Gln Pro Gly Ala Thr Ile Leu Glu Pro Thr Ser Gly65 70 75 80 Asn Thr Gly Ile Ser Leu Ala Met Ala Ala Leu Leu Lys Gly Tyr Asn 85 90 95 Met Ile Cys Val Met Pro Glu Asn Thr Ser Ile Glu Arg Arg Gln Ile 100 105 110 Leu Glu Leu Tyr Gly Ala Arg Ile Ile Phe Ser Pro Ala Glu Gly Gly 115 120 125 Ser Asn Thr Ala Val Ala Thr Ala Lys Glu Leu Ala Ala Gln Asn Pro 130 135 140 Ser Trp Val Met Leu Tyr Gln Tyr Gly Asn Pro Ala Asn Ser Asp Ala145 150 155 160 His Tyr Phe Gly Thr Gly Pro Glu Leu Leu Ala Asp Leu Pro Glu Ile 165 170 175 Thr His Phe Val Ala Gly Leu Gly Thr Thr Gly Thr Leu Met Gly Thr 180 185 190 Gly Arg Phe Leu Arg Glu His Val Pro Gly Val Gln Ile Val Ala Ala 195 200 205 Glu Pro Arg Tyr Gly Glu Gly Val Tyr Ala Leu Arg Asn Ile Asp Glu 210 215 220 Gly Phe Ile Pro Glu Leu Tyr Asp Ala Asp Val Leu Thr Thr Arg Phe225 230 235 240 Ser Val Gly Ser Phe Asp Ala Val Arg Arg Thr Arg Glu Leu Val Thr 245 250 255 Arg Glu Gly Ile Phe Ala Gly Ile Ser Thr Gly Ala Val Leu His Ala 260 265 270 Ala Leu Gly Met Ala Ala Lys Ala Val Lys Ala Gly Glu Arg Ala Asp 275 280 285 Ile Ala Phe Val Val Ala Asp Ala Gly Trp Lys Tyr Leu Ser Thr Gly 290 295 300 Ala Tyr Ala Gly Ser Leu Asp Asp Ala Glu Asp Ala Leu Glu305 310 315 5318PRTMycobacterium tuberculosis H37RvPEPTIDE(1)..(318)Mtb-T 5Met Thr Arg Tyr Asp Ser Leu Leu Gln Ala Leu Gly Asn Thr Pro Leu1 5 10 15 Val Gly Leu Gln Arg Leu Ser Pro Arg Trp Asp Asp Gly Arg Asp Gly 20 25 30 Pro His Val Arg Leu Trp Ala Lys Leu Glu Asp Arg Asn Pro Thr Gly 35 40 45 Ser Ile Lys Asp Arg Pro Ala Val Arg Met Ile Glu Gln Ala Glu Ala 50 55 60 Asp Gly Leu Leu Arg Pro Gly Ala Thr Ile Leu Glu Pro Thr Ser Gly65 70 75 80 Asn Thr Gly Ile Ser Leu Ala Met Ala Ala Arg Leu Lys Gly Tyr Arg 85 90 95 Leu Ile Cys Val Met Pro Glu Asn Thr Ser Val Glu Arg Arg Gln Leu 100 105 110 Leu Glu Leu Tyr Gly Ala Gln Ile Ile Phe Ser Ala Ala Glu Gly Gly 115 120 125 Ser Asn Thr Ala Val Ala Thr Ala Lys Glu Leu Ala Ala Thr Asn Pro 130 135 140 Ser Trp Val Met Leu Tyr Gln Tyr Gly Asn Pro Ala Asn Thr Asp Ser145 150 155 160 His Tyr Cys Gly Thr Gly Pro Glu Leu Leu Ala Asp Leu Pro Glu Ile 165 170 175 Thr His Phe Val Ala Gly Leu Gly Thr Thr Gly Thr Leu Met Gly Thr 180 185 190 Gly Arg Phe Leu Arg Glu His Val Ala Asn Val Lys Ile Val Ala Ala 195 200 205 Glu Pro Arg Tyr Gly Glu Gly Val Tyr Ala Leu Arg Asn Met Asp Glu 210 215 220 Gly Phe Val Pro Glu Leu Tyr Asp Pro Glu Ile Leu Thr Ala Arg Tyr225 230 235 240 Ser Val Gly Ala Val Asp Ala Val Arg Arg Thr Arg Glu Leu Val His 245 250 255 Thr Glu Gly Ile Phe Ala Gly Ile Ser Thr Gly Ala Val Leu His Ala 260 265 270 Ala Leu Gly Val Gly Ala Gly Ala Leu Ala Ala Gly Glu Arg Ala Asp 275 280 285 Ile Ala Leu Val Val Ala Asp Ala Gly Trp Lys Tyr Leu Ser Thr Gly 290 295 300 Ala Tyr Ala Gly Ser Leu Asp Asp Ala Glu Thr Ala Leu Glu305 310 315 6389PRTArtificial SequenceApe-OPSS 6Met Ala Leu Ala Asp Ile Ser Gly Tyr Leu Asp Val Leu Asp Ser Val1 5 10 15 Arg Gly Phe Ser Tyr Leu Glu Asn Ala Arg Glu Val Leu Arg Ser Gly 20 25 30 Glu Ala Arg Cys Leu Gly Asn Pro Arg Ser Glu Pro Glu Tyr Val Lys 35 40 45 Ala Leu Tyr Val Ile Gly Ala Ser Arg Ile Pro Val Gly Asp Gly Cys 50 55 60 Ser His Thr Leu Glu Glu Leu Gly Val Phe Asp Ile Ser Val Pro Gly65 70 75 80 Glu Met Val Phe Pro Ser Pro Leu Asp Phe Phe Glu Arg Gly Lys Pro 85 90 95 Thr Pro Leu Val Arg Ser Arg Leu Gln Leu Pro Asn Gly Val Arg Val 100 105 110 Trp Leu Lys Leu Glu Trp Tyr Asn Pro Phe Ser Leu Ser Val Lys Asp 115 120 125 Arg Pro Ala Val Glu Ile Ile Ser Arg Leu Ser Arg Arg Val Glu Lys 130 135 140 Gly Ser Leu Val Ala Asp Ala Thr Ser Ser Asn Phe Gly Val Ala Leu145 150 155 160 Ser Ala Val Ala Arg Leu Tyr Gly Tyr Arg Ala Arg Val Tyr Leu Pro 165 170 175 Gly Ala Ala Glu Glu Phe Gly Lys Leu Leu Pro Arg Leu Leu Gly Ala 180 185 190 Gln Val Ile Val Asp Pro Glu Ala Pro Ser Thr Val His Leu Leu Pro 195 200 205 Arg Val Met Lys Asp Ser Lys Asn Glu Gly Phe Val His Val Asn Gln 210 215 220 Phe Tyr Asn Asp Ala Asn Phe Glu Ala His Met Arg Gly Thr Ala Arg225 230 235 240 Glu Ile Phe Val Gln Ser Arg Arg Gly Gly Leu Ala Leu Arg Gly Val 245 250 255 Ala Gly Ser Leu Gly Thr Ser Gly His Met Ser Ala Ala Ala Phe Tyr 260 265 270 Leu Gln Ser Val Asp Pro Ser Ile Arg Ala Val Leu Val Gln Pro Ala 275 280 285 Gln Gly Asp Ser Ile Pro Gly Ile Arg Arg Val Glu Thr Gly Met Leu 290 295 300 Trp Ile Asn Met Leu Asp Ile Ser Tyr Thr Leu Ala Glu Val Thr Leu305 310 315 320 Glu Glu Ala Met Glu Ala Val Val Glu Val Ala Arg Ser Asp Gly Leu 325 330 335 Val Ile Gly Pro Ser Gly Gly Ala Ala Val Lys Ala Leu Ala Lys Lys 340 345 350 Ala Ala Glu Gly Asp Leu Glu Pro Gly Asp Tyr Val Val Val Val Pro 355 360 365 Asp Thr Gly Phe Lys Tyr Leu Ser Leu Val Gln Asn Ala Leu Glu Gly 370 375 380 Ala Gly Asp Ser Val385 71341DNACorynebacterium glutamicum 13032gene(1)..(1341)serB 7atgtcgtgtt ccgcgctcag acatgagaca attgttgccg tgactgaact catccagaat 60gaatcccaag aaatcgctga gctggaagcc ggccagcagg ttgcattgcg tgaaggttat 120cttcctgcgg tgatcacagt gagcggtaaa gaccgcccag gtgtgactgc cgcgttcttt 180agggtcttgt ccgctaatca ggttcaggtc ttggacgttg agcagtcaat gttccgtggc 240tttttgaact tggcggcgtt tgtgggtatc gcacctgagc gtgtcgagac cgtcaccaca 300ggcctgactg acaccctcaa ggtgcatgga cagtccgtgg tggtggagct gcaggaaact 360gtgcagtcgt cccgtcctcg ttcttcccat gttgttgtgg tgttgggtga tccggttgat 420gcgttggata tttcccgcat tggtcagacc ctggcggatt acgatgccaa cattgacacc 480attcgtggta tttcggatta ccctgtgacc ggcctggagc tgaaggtgac tgtgccggat 540gtcagccctg gtggtggtga agcgatgcgt aaggcgcttg ctgctcttac ctctgagctg 600aatgtggata ttgcgattga gcgttctggt ttgctgcgtc gttctaagcg tctggtgtgc 660ttcgattgtg attccacgtt gatcactggt gaggtcattg agatgctggc ggctcacgcg 720ggcaaggaag ctgaagttgc ggcagttact gagcgtgcga tgcgcggtga gctcgatttc 780gaggagtctc tgcgtgagcg tgtgaaggcg ttggctggtt tggatgcgtc ggtgatcgat 840gaggtcgctg ccgctattga gctgacccct ggtgcgcgca ccacgatccg tacgctgaac 900cgcatgggtt accagaccgc tgttgtttcc ggtggtttca tccaggtgtt ggaaggtttg 960gctgaggagt tggagttgga ttatgtccgc gccaacactt tggaaatcgt tgatggcaag 1020ctgaccggca acgtcaccgg aaagatcgtt gaccgcgctg cgaaggctga gttcctccgt 1080gagttcgctg cggattctgg cctgaagatg taccagactg tcgctgtcgg tgatggcgct 1140aatgacatcg atatgctctc cgctgcgggt ctgggtgttg ctttcaacgc gaagcctgcg 1200ctgaaggaga ttgcggatac ttccgtgaac cacccattcc tcgacgaggt tttgcacatc 1260atgggcattt cccgcgacga gatcgatctg gcggatcagg aagacggcac tttccaccgc 1320gttccattga ccaatgccta a 13418969DNAEscherichia coli K12 W3110gene(1)..(969)serB 8atgcctaaca ttacctggtg cgacctgcct gaagatgtct ctttatggcc gggtctgcct 60ctttcattaa gtggtgatga agtgatgcca ctggattacc acgcaggtcg tagcggctgg 120ctgctgtatg gtcgtgggct ggataaacaa cgtctgaccc aataccagag caaactgggt 180gcggcgatgg tgattgttgc cgcctggtgc gtggaagatt atcaggtgat tcgtctggca 240ggttcactca ccgcacgggc tacacgcctg gcccacgaag cgcagctgga tgtcgccccg 300ctggggaaaa

tcccgcacct gcgcacgccg ggtttgctgg tgatggatat ggactccacc 360gccatccaga ttgaatgtat tgatgaaatt gccaaactgg ccggaacggg cgagatggtg 420gcggaagtaa ccgaacgggc gatgcgcggc gaactcgatt ttaccgccag cctgcgcagc 480cgtgtggcga cgctgaaagg cgctgacgcc aatattctgc aacaggtgcg tgaaaatctg 540ccgctgatgc caggcttaac gcaactggtg ctcaagctgg aaacgctggg ctggaaagtg 600gcgattgcct ccggcggctt tactttcttt gctgaatacc tgcgcgacaa gctgcgcctg 660accgccgtgg tagccaatga actggagatc atggacggta aatttaccgg caatgtgatc 720ggcgacatcg tagacgcgca gtacaaagcg aaaactctga ctcgcctcgc gcaggagtat 780gaaatcccgc tggcgcagac cgtggcgatt ggcgatggag ccaatgacct gccgatgatc 840aaagcggcag ggctggggat tgcctaccat gccaagccaa aagtgaatga aaaggcggaa 900gtcaccatcc gtcacgctga cctgatgggg gtattctgca tcctctcagg cagcctgaat 960cagaagtaa 969926DNAArtificial Sequenceprimer for deletion cassette of serB 9gcgatatcat gaccttagaa tggtgg 261023DNAArtificial Sequenceprimer for deletion cassette of serB 10gctctagatc acgcatgcct cgc 231130DNAArtificial Sequenceprimer for deletion cassette of serB 11gcgatatcat gtcacccctg tgaaaatgac 301229DNAArtificial Sequenceprimer for deletion cassette of serB 12gctctagatc agttcgatac ctggggtat 291324DNAArtificial Sequenceprimer for deletion cassette of serB 13atcatgttac tggcaggcgc tatc 241429DNAArtificial Sequenceprimer for deletion cassette of serB 14gctctagatt acaaagtgaa agagagacg 291570DNAArtificial Sequenceprimer for deletion cassette of serB 15atgcctaaca ttacctggtg cgacctgcct gaagatgtct ctttatggcc gtgtaggctg 60gagctgcttc 701670DNAArtificial Sequenceprimer for deletion cassette of serB 16ggatggcggg ccaccaatta cttctgattc aggctgcctg agaggatgca catatgaata 60tcctccttag 701727DNAArtificial Sequenceprimer for amplification of pself-serB to construct pBAC-pself-serB 17cccaagcttc ttccaccctt tgaaaat 271827DNAArtificial Sequenceprimer for amplification of pself-serB to construct pBAC-pself-serB 18cccaagcttt tacttctgat tcaggct 271919DNAArtificial Sequenceprimer for amplification of pself-CTG-serB to construct pBAC-pself-CTG-serB 19ggagccttac tgcctaaca 192019DNAArtificial Sequenceprimer for amplification of pself-CTG-serB to construct pBAC-pself-CTG-serB 20tgttaggcag taaggctcc 192129DNAArtificial Sequenceforward primer for Ape-OPSS 21gtcatatgat ggctctggct gacatctct 292229DNAArtificial Sequencereverse primer for Ape-OPSS 22gtaagctttt aaacagagtc accagcacc 292329DNAArtificial Sequenceforward primer for Mtb-OPSS 23gtcatatgat gacacgatac gactcgctg 292429DNAArtificial Sequencereverse primer for Mtb-OPSS 24gtaagctttc atgcccatag ttgcccttc 292529DNAArtificial Sequenceforward primer for Msm-OPSS 25ataagctttc atgcccatag ctgcccttc 292629DNAArtificial Sequencereverse primer for Msm-OPSS 26ataagctttc attccagcgc gtcctcggc 292729DNAArtificial Sequenceforward primer for Rjo-OPSS 27gtcatatgat ggcgcggttc gattcgctg 292831DNAArtificial Sequencereverse primer for Rjo-OPSS 28tagcggccgc tcatgcccac aactgccctt c 312929DNAArtificial Sequenceforward primer for Nfa-OPSS 29gtcatatgat ggcacgctac gaatcgctg 293028DNAArtificial Sequencereverse primer for Nfa-OPSS 30gtaagctttc aggcccagag ctggcctt 283129DNAArtificial Sequenceforward primer for Mtb-T 31gtcatatgat gacacgatac gactcgctg 293229DNAArtificial Sequencereverse primer for Mtb-T 32gtaagctttc attccagagc ggtctcggc 293329DNAArtificial Sequenceforward primer of Msm-T 33gtcatatgat gacgcgctac gactccctg 293429DNAArtificial Sequencereverse primer of Msm-T 34ataagctttc attccagcgc gtcctcggc 293521DNAArtificial Sequenceforward primer of pCL-P(CJ1)-Msm-T 35gatatcgcag cagccatcat c 213628DNAArtificial Sequencereverse primer of pCL-P(CJ1)-Msm-T 36cccaagcttt cattccagcg cgtcctcg 28

Claims

1. A method for producing cysteine or a derivative thereof, comprising: 1) culturing a recombinant microorganism in which the activity of endogeneous phosphoserine phosphatase (SerB) is reduced, to produce O-phosphoserine (OPS); and 2) reacting the OPS of step 1) with a sulfide in presence of O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing OPSS, to produce cysteine or a derivative thereof.

2. The method of claim 1, wherein the phosphoserine phosphatase has an amino acid sequence of SEQ ID NO: 1 or 2.

3. The method of claim 1, wherein the recombinant microorganism in which the activity of endogenous SerB is disrupted is cultured in a medium containing glycine or serine.

4. The method of claim 3, wherein the medium contains glycine in an amount of from 0.1 to 10 g/L.

5. The method of claim 3, wherein the medium contains serine in an amount of from 0.1 to 5 g/L.

6. The method of claim 1, wherein the recombinant microorganism is Escherichia sp. or Coryneform bacteria.

7. The method of claim 1, wherein the sulfide of step 2) is selected from the group consisting of Na.sub.2S, NaSH, (NH.sub.4).sub.2S, H.sub.2S, Na.sub.2S.sub.2O.sub.3 and a combination thereof.

8. The method of claim 1, wherein the OPSS of step 2) is from at least one species selected from the group consisting of Aeropyrum pernix, Mycobacterium tuberculosis, Mycobacterium smegmatis and Trichomonas vaginalis.

9. The method of claim 8, wherein the OPSS is a further modified to increase a conversion rate of step 2).

10. The method of claim 1, wherein the conversion of step 2) is carried out in presence of a cofactor selected from 0.001 to 2 mM PLP (pyridoxal-5-phosphate), 0.001 to 100 mM DTT (dithiothreitol), and a combination thereof.

11. The method of claim 1, further comprising isolating and purifying the cysteine or its derivatives.

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