Cloning, expression, characterization and application ... - Springer Link

Biotechnol Lett (2012) 34:1101–1106 DOI 10.1007/s10529-012-0878-7

ORIGINAL RESEARCH PAPER

Cloning, expression, characterization and application of atcA, atcB and atcC from Pseudomonas sp. for the production of L-cysteine Jingjing Duan • Qi Zhang • Hongzhi Zhao Jun Du • Fang Bai • Gang Bai

•

Received: 22 December 2011 / Accepted: 8 February 2012 / Published online: 19 February 2012 Ó Springer Science+Business Media B.V. 2012

Abstract An isolate of a Pseudomonas sp. uses the L-NCC (N-carbamoyl-L-cysteine) pathway to convert 2 DL-2-amino-D -thiazoline-4-carboxylic acid (DL-ATC) to L-cysteine. Genes encoding ATC racemase (AtcA), L-ATC hydrolase (AtcB) and L-NCC amidohydrolase (AtcC), involved in this pathway, were cloned from the

Electronic supplementary material The online version of this article (doi:10.1007/s10529-012-0878-7) contains supplementary material, which is available to authorized users. J. Duan Q. Zhang (&) H. Zhao F. Bai G. Bai College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China e-mail: [email protected]

Pseudomonas sp. and expressed in Escherichia coli BL21 via pET-28a(?). The resulting enzymes were purified, their functions identified, and their biochemical properties are described. In vitro catalysis experiments, using these enzymes, revealed that the bioconversion rate of L-cysteine from DL-ATC in the presence of AtcA was more efficient than in the absence of AtcA. This is the first report describing simultaneous cloning and expression of atcA, atcB and atcC and characterization of their enzymes for L-cysteine production from DL-ATC via the L-NCC pathway, enabling the complete L-NCC pathway to be elucidated. Keywords DL-2-amino-D2-thiazoline-4-carboxylic acid N-carbamyl-L-cysteine pathway L-cysteine Pseudomonas sp.

J. Duan e-mail: [email protected] H. Zhao e-mail: [email protected]

Introduction

F. Bai e-mail: [email protected]

L-Cysteine

G. Bai e-mail: [email protected] J. Du G. Bai College of Life Sciences, Nankai University, Tianjin 300071, China e-mail: [email protected] J. Du China Fermentation Industry Association, Beijing 100037, China

is a basic S-containing amino acid which is used in diverse research fields including drug synthesis, food additives, and cosmetics additives. It is primarily extracted from the acid hydrolysates of human hair and duck feathers. This process, however, results in a relatively low yield, as well as creating problems in waste-water treatment. As an alternative to extraction by hydrolysis, a bioconversion process has been developed by Dhillon et al. (1987). Some bacteria, in particular those from the genus

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Biotechnol Lett (2012) 34:1101–1106

Fig. 1 a The conversion pathway from DL-ATC to L-cysteine via L-NCC as intermediate, b and the schematic representation of the organization of the L-cysteine synthesis gene cluster

Pseudomonas, hydrolyze DL-2-amino-D2-thiazoline4-carboxylic acid (DL-ATC) to L-cysteine (Sano et al. 1977). Yu et al. (2006) identified L-SCC (S-carbamylL-cysteine) as an intermediate in the bioconversion of DL-ATC to L-cysteine in Pseudomonas sp. TS1138. Tamura et al. (1998) discovered that L-NCC (N-carbamoyl-L-cysteine) was an intermediate in L-cysteine production by Pseudomonas sp. ON-4a. The bioconversion process can therefore occur via two pathways, depending on the intermediate, L-SCC or L-NCC, both comprising the following three steps: (i) enzymatic racemization of D-ATC to L-ATC; (ii) a ring-opening reaction of L-ATC to L-SCC or L-NCC as an intermediate; and (iii) the hydrolysis of L-SCC or L-NCC to L-cysteine (Tamura et al. 1998; Yu et al. 2006). The L-NCC pathway is shown in Fig. 1a. Two strains isolated by our research group, Pseudomonas sp. TS1138 (Yu et al. 2006) and Pseudomonas sp. QR-101 (Supplementary Fig. 1), produce L-cysteine. The intermediate in the former strain is L-SCC, while the intermediate in the latter strain is still unknown. The genes encoding ATC racemase (AtcA) (Shiba 2001), L-ATC hydrolase (AtcB) and L-NCC amidohydrolase (AtcC) have been cloned and sequenced from the L-NCC-pathway of the Pseudomonas sp. strains BS (Shiba et al. 2002) and ON-4a (Tashima et al. 2006). As a racemase, AtcA can enable forward and reverse transitions of L-ATC to D-ATC; hence it is an important enzyme in the bioconversion process from D-ATC to L-cysteine. However, to date, more is

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known about AtcB (Ryu et al. 1995; Shiba et al. 2002) and AtcC (Shiba et al. 2002) than about AtcA. In this study, atcA was first expressed and the enzymatic properties were studied, with the aim of improving the catalytic efficiency from DL-ATC to L-cysteine. All three genes involved in the production of L-cysteine by Pseudomonas sp. QR-101 were cloned, identified, expressed and characterized. Further, the function of AtcA was tested. This description of the complete L-NCC pathway could significantly advance and guide the industrial production of L-cysteine via enzymatic conversion methods.

Materials and methods Materials E. coli strains JM109 (recAl supE44 endA1 hsdR17 gyrA96 relA1 thiA(lac-proAB) F0 [traD36 proAB? lacIq lacZDM15]) and BL21(DE3) were stored in our laboratory. Pseudomonas sp. QR-101 was isolated from industrial waste water, identified to be most closely related to Pseudomonas putida W30, and similarly stored. The pMD19-T and pET-28a (?) vectors were purchased from Takara BIO INC. (Dalian, China) and Novagen (Madison, Wisconsin, USA) respectively. L-SCC (S-carbamyl-L-cysteine) and L-NCC (Ncar bamoyl-L-cysteine) were purchased from Bachem-Americas (Torrance, California, USA) and Toronto Research Chemicals (North York, Canada)


respectively. Other chemicals used in this study were analytical grade and are commercially available. Determination of L-cysteine synthesis pathway The synthesis pathway of L-cysteine in Pseudomonas sp. QR-101 was confirmed by LC/MS/MS. 1.5 ml cell lysate suspension was added to 3 ml enzymatic reaction solution (1% DL-ATC (2-amino-D2-thiazoline-4carboxylic acid) in 0.1 M K2HPO4/KH2PO4 buffer (pH 8.0)) and incubated at 35°C for 0.5 h, after which the mixture was lyophilized, dissolved in methanol, and carried on to LC/MS/MS detection. Cloning and expression of atcA, atcB and atcC Following confirmation of the L-NCC pathway in Pseudomonas sp. QR-101, suitable primers atcARB-F and atcC-R (Supplementary Table 1) were deduced from conserved gene sequences (GenBank: AB176845.2) from another strain that produces L-cysteine via the L-NCC pathway (Shiba et al. 2002), which was shown in Supplementary Fig. 2. Initially, one 4.7 kb fragment was obtained and sequenced. Within this fragment, three open reading frames were identified, and then three pairs of primers (Supplementary Table 1) were designed to amplify the atcA, atcB and atcC genes. The genomic DNA from Pseudomonas sp. QR-101 was used as template. Amplification fragments were purified and cloned into pMD19-T Simple Vector, sequenced (Takara, Dalian, China), and linked into the BamHI and SacI sites of pET28a(?). After identification, the plasmids were put into E. coli BL21 (DE3) for enzyme expression. Strains of E. coli BL21 (DE3) harboring pET28a-atcA, pET28a-atcB and pET28a-atcC were inoculated into 4 ml LB medium containing 50 lg kanamycin/ml (LB/ Kan) and were incubated at 37°C for 12 h. The inoculum (1%, v/v) was added to 100 ml LB/Kan and cultured at 37°C to an OD600 of 0.6, after which protein production was induced by adding 1 mM IPTG and incubating at 16°C for 20 h. Enzymes purification The bacterial culture (100 ml) was centrifuged (7,0009g, 4°C) for 10 min, the cells were washed twice with 0.1 M K2HPO4/KH2PO4 buffer (pH 8.0) then resuspended in 5 ml of 0.1 m K2HPO4/KH2PO4 pH 8.0 buffer and sonicated on ice for 5 min

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alternating on and off every 5 s. Cell debris was removed by centrifugation and then the remaining impurities were removed by filtering through a 0.2 lm membrane. The supernatant was retained for enzyme purification. A 1 ml nickel affinity column (Ni Sepharose 6 Fast Flow, His Trap FF 1 ml, GE) was pre-equilibrated with 10 vol deionized water. Five column vol binding balance buffer (100 mM glycine/ NaOH, pH 8.0) were added to the column. The prepared crude enzyme samples were added to the nickel affinity. The columns were washed with 10 column vol imidazole (100 mM glycine/NaOH, 50 mM imidazole, pH 8.0) to remove protein impurities; the target proteins were then washed with imidazole (100 mM glycine/NaOH, 300 mM imidazole, pH 8.0); the imidazole was removed by ultrafiltration and the collected solutions were stored at -80°C. Function identification of AtcA The enzymatic reaction solution comprised 3 ml 0.5% substrate (DL-ATC) and 1.5 ml enzyme. A sample lacking enzymes was used as a control. The volume was brought to 4.5 ml with the 0.1 M K2HPO4/ KH2PO4 buffer. After catalysis at 35°C for 0.5 h, the reaction mixture was lyophilized and dissolved in 300 ll 2-propanol. D-ATC and L-ATC were assayed by HPLC using hexane/2-propanol (8.5:15 v/v and containing 0.2% trifluoroacetic acid and 0.1% diethylamine) as the mobile phase in a Chiral Pak IC column (0.46 cm I.D. 9 25 cm L) (Daicel Chiral Technologies Co. Ltd, Shanghai, China). The eluate was monitored at 220 nm. Determination of molecular mass using SDS-PAGE After measuring the protein concentrations by the Lowry method, SDS-PAGE was used to assess the purification of the active fractions, and to determine the molecular mass of both the crude and the purified enzymes. SDS-PAGE was performed in a 0.75 mm slab gel consisting of a stacking gel (5% polyacrylamide) and a separating gel (12% polyacrylamide). Precision protein standards (Beyotime, Jiangsu, China) were used as molecular marker standards. Proteins were stained with Coomassie brilliant blue R-250.

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Measurement of enzymatic activity The enzymatic activities of AtcA, AtcB and AtcC were assayed by measuring the amount of synthesized Lcysteine from DL-ATC or L-NCC in the presence of purified enzymes by Gaitonde’s ninhydrin method. The enzymatic reaction comprised 3 ml 1% substrate (DLATC for AtcA and AtcB, L-NCC for AtcC) in 0.1 M K2HPO4/KH2PO4 buffer (pH 8.0) and 1.5 ml enzyme. After 0.5 h at 35°C, L-cysteine (reflecting enzyme activity) was measured. L-NCC amidohydrolase activity was assessed by formation of L-cysteine from L-NCC. One unit of L-NCC amidohydrolase or L-ATC hydrolase activity was defined as the amount of enzyme producing 1 lM L-cysteine per h at 35°C. L-ATC hydrolase activity was assayed under conditions of excess purified L-NCC amidohydrolase, which allows complete conversion of L-NCC to L-cysteine (Ohmachi et al. 2002). For the ATC racemase activity assay, an excess of purified L-ATC hydrolase and L-NCC amidohydrolase was introduced, allowing complete conversion of L-ATC to L-cysteine. The same reaction only with the same amount of L-ATC hydrolase and L-NCC amidohydrolase was measured and used as a control. One unit of ATC racemase activity is defined as the amount of enzyme producing 1 lM more L-cysteine per hour at 35°C than the control. Analysis of optimum reaction pH, temperature and thermal stability The optimum pH values of the three enzymes were determined by adding 3 ml 1% DL-ATC and L-NCC solution to the following buffers: 0.1 M citrate buffer (pH 3.0–5.0), 0.1 M phosphate buffer (pH 6.0–8.0), and 0.1 M glycine/NaOH buffer (pH 9.0–11.0), followed by 1.5 ml enzyme. Enzyme activities at various pH values were assayed at 35°C as described above. The optimum temperatures of the three enzymes were determined by maintaining standard assay conditions over the temperature range 20–60°C, and measuring enzyme activity. At the same time, the thermal stability was determined under standard assay conditions by incubating purified enzyme solutions over the same temperature range for 30 min, and again recording their activity. Production of L-cysteine in vitro L-Cysteine was produced using the purified enzymes in vitro from DL-ATC of different concentrations

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(5 and 10 g/l). 3 ml of enzymatic reaction solution, 0.5 ml AtcA (2.5 mg/ml), 0.5 ml AtcB (2.1 mg/ml) and 0.5 ml of AtcC (2.1 mg/ml) were mixed and incubated at 35°C for 0.5 h. In the reaction lacking AtcA, 0.5 ml of 0.1 M phosphate buffer replaced AtcA. The yields of L-cysteine and conversion ratios of DL-ATC were measured and calculated, respec tively.

Results and discussion Analysis of L-cysteine synthesis pathway Elucidating the L-cysteine synthesis pathway of Pseudomonas sp. QR-101 is dependent upon the identification of the reaction product of AtcB from DL-ATC. This was achieved through the use of ESIMS/MS, which is more reliable and accurate than TLC method used by Shiba et al. (2002). The expected cleavage patterns turned up in the MS/MS spectra (Table 1; Supplementary Fig. 3); that is, substrate DL-ATC and the end product L-cysteine were both detected (Supplementary Fig. 3a, c). The intermediate was identified as L-NCC, generated from DL-ATC by AtcB catalysis (Supplementary Fig. 3b). In summary, the ESI MS/MS results confirmed that L-cysteine synthesis in Pseudomonas sp. QR-101 occurs via the L-NCC pathway. Cloning and expression of enzymes The 4.7 kb fragment was amplified by primers atcARB-F and atcC-R, sequenced, and deposited into Genbank with the accession number of JN986826 (Fig. 1b). The three gene sequences, atcA, atcB and atcC of Pseudomonas sp. QR-101 were found to be 82.7, 88.3 and 83.3% homologous to their equivalents in Pseudomonas sp. BS (GenBank: AB176845.2), respectively (Fig. 1b). These gene sequences encode, respectively AtcA, AtcB and AtcC, which are involved in L-cysteine biosynthesis from DL-ATC. The amino acid sequences were 85.45, 91.1 and 87.9% homologous with those of Pseudomonas sp. BS. The successful expression of atcA, atcB and atcC enabled the enzymes to be characterized for the first time. The molecular masses were confirmed by SDS-PAGE to be 29 kDa for AtcA, 23 kDa for AtcB, and 48 kDa for AtcC (Fig. 2).


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Table 1 Data of (?) ESI–MS and (-) ESI–MS of enzymatic reaction product No.

Identification

1

DL-2-Amino-D

2

-thiazoline-4-carboxylic acid

tR/(min)

Formula

Positive ion (m/z)

Negative ion (m/z)

0.68

C4H6O2N2S

147 (M?)

145 (M-)

?

101 (M –HCOOH) 2

L-N-carbamoyl-L-cysteine

1.25

C4H8O3N2S

165 (M?) 87 (M?–HS–COOH)

163 (M-) 85 (M-HS–COOH)

3

L-Cysteine

0.65

C3H7O2NS

122 (M?)

120 (M-)

?

105 (M –NH3) 76 (M?–HCOOH) The main constituents of the enzymatic reaction product were obtained by means of LC/MS with a mobile phase consisting of 2% acetonitrile and 0.1% formic acid aqueous solution. The resulting protonated (M?) molecules (m/z: 147, m/z: 165 and m/z: 122) were selected as precursor ions for fragmentation to produce MS/MS product-ion spectra. The results are also presented in Supplementary Fig. 3

Fig. 2 SDS-PAGE analysis Lane M, protein molecular weight marker; lanes 1, 3 and 5: crude enzymes of AtcA, AtcB and AtcC, comprising the lysate of E. coli BL21 (DE3) containing pET28a-actA, pET28a-actB and pET28a-actC inserts, respectively; lanes 2, 4 and 6: purified enzymes of AtcA, AtcB and AtcC; lane 7: control group, comprising the lysate of E. coli BL21 (DE3) containing pET28a(?) insert alone. Lanes M, 1, 3 and 5 are from one gel, and lanes 2, 4, 6 and 7 are from another gel

Key role of AtcA AtcA plays an important role in the production of L-cysteine in vitro. The HPLC spectrum retention times of the two chiral compounds was 11 min for D-ATC and 18.5 min for L-ATC. The relative D-ATC and L-ATC content respectively, in the absence of enzymes, was 49.4 and 50.6% (Supplementary Fig. 4a). After the simultaneous addition of AtcB and AtcC, the L-ATC content decreased to 12% as most of the L-ATC was catalyzed into L-cysteine (Supplementary Fig. 4b). When all three enzymes were simultaneously added, the L-ATC content was restored to 50.3%. Since the subsequent L-ATC hydrolase reaction

is severely L-ATC specific, when the fraction of L-ATC becomes sufficiently low, AtcA catalyzes D-ATC to L-ATC to restore L-ATC levels (Supplementary Fig. 4c). However, the transformation of D-ATC to L-ATC was barely detectable in the presence of AtcA alone. In this case, the concentrations of D-ATC and L-ATC were 49.7 and 50.23% respectively (Supplementary Fig. 4d). We propose that a high concentration of L-ATC in the substrate prevents the transformation of D-ATC into L-ATC. We conclude that L-ATC is an effective substrate for AtcB, and that D-ATC is not a substrate for AtcA, unless a reduction of L-ATC has occurred. Racemases can react with both isomers of the substrates, and catalyze the either racemization. This means the D-ATC is eventually converted into the LATC, and thus the L-ATC is eventually converted into the D-ATC as well. From these results, we conclude that all three enzymes could be used in the efficient production of L-cysteine with DL-ATC as substrate. Investigation of optimum pH, temperature and thermal stability The optimal activities of all three expressed proteins were at pH 8.0 (Supplementary Fig. 5a) and 35°C (Supplementary Fig. 5b). The thermal stability of the three enzymes is shown in Supplementary Fig. 5c. The optimal pH and temperature of the three enzymes were similar, which is advantageous for co-catalysis from DLATC to L-cysteine. AtcC was stable within the pH range 5.0–9.0. At higher pH, an abrupt increase in activity was observed, perhaps because the L-NCC substrate converted to L-cysteine spontaneously at pH greater than 9.0.

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Table 2 In vitro production of L-cysteine from lyzed by the purified enzymes DL-2-amino-D

2

-

Enzymea

thiazoline4-carboxylic acid (g/l)

L-cysteine (g/l)

DL-ATC

cata-

Conversion ratio %

6.67

A?B?C

4.71 ± 0.11

71

3.33

B?C A?B?C

2.51 ± 0.13 2.02 ± 0.06

38 61

B?C

0.79 ± 0.05

24

L-cysteine. We also provided the first description of the complete L-NCC pathway, which can be used to guide industrial production of L-cysteine via enzymatic conversion methods.

Acknowledgments This study was supported by Grant No. 09ZCKFSH00900 from the Natural Science Foundation of Tianjin, China.

References

n=3 a

A AtcA, B AtcB, C AtcC

In vitro production of L-cysteine The ability of the purified enzymes to produce in vitro was assessed. As seen in Table 2, using a three-enzyme co-catalysis, 61% (2.02 g L-cysteine/l) of the 3.33 g DL-ATC/l (final concentration) was obtained. Similarly, 71% (4.71 g L-cysteine/l) was obtained when the final concentration of DL-ATC was doubled to 6.67 g/l. This revealed that a high concentration of DL-ATC could facilitate substrate conversion. This enzymatic conversion ratio was higher than that (lower than 48%) of Pseudomonas sp. QR-101 when the whole cells were used as catalyzer (data not shown). The lower bioconversion rate in the wild type strain may due to the existence of L-cysteine desulfhydrase in this strain which would convert L-cysteine to pyruvic acid, H2S and NH3 (Zheng et al. 1994). However, more optimal conditions for the bioconversion from DL-ATC to L-cysteine using enzymes need to be investigated such as the addition of metal ions and the ratio of each of the enzymes in the reaction mixture. In addition, the increases in the yield of L-cysteine were 2.2 g/l and 1.23 g/l when 6.67 g DL-ATC/l and 3.33 g DL-ATC/l, respectively were used as substrate. These respective increases in conversion ratios were 33 and 37% which indicate that D-ATC had been efficiently converted to L-ATC. It is clear that AtcA can increase the yield and conversion ratio of L-cysteine at both DL-ATC concentrations. In conclusion, the enzymatic properties of AtcA were investigated and characterized for the first time. These results offer significant opportunities for improving the catalytic efficiency from DL-ATC to L-cysteine

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Dhillon GS, Nagasawa T, Yamada H (1987) Microbial process for L-cysteine production. Enzyme Microb Tech 9:277–280 Ohmachi T, Mizuka N, Maki K, Namiko E, Hiroko F, Megumi N, Kazuyuki M, Yoshiharu T, Yoshihiro A (2002) Identification, cloning and sequencing of the genes involved in the conversion of D, L-2-amino-D2-thiazoline-4-carboxylic acid to L-cysteine in Pseudomonas sp. strain ON-4a. Biosci Biotech Bioch 66:1097–1104 Ryu OH, Oh SW, Yoo SK, Shin CS (1995) The stability of L-ATC hydrolase participating in L-cysteine production. Biotechnol Lett 17:275–280 Sano K, Yokozeki K, Tamura F, Yasuda N, Noda I, Mitsugi K (1977) Microbial conversion of D, L-2-amino-D2-thiazoline-4-carboxylic acid to L-cysteine and L-cystine: screening of microorganisms and identification of products. Appl Environ Microb 34:806–810 Shiba T (2001) 2-Aminothiazoline-4-carboxylate racemase and gene encoding therefore. U.S. patent 6214590 B1 Shiba T, Takeda K, Yajima M, Tadano M (2002) Genes from Pseudomonas sp. strain BS Involved in the conversion of L-2-amino-2-thiazolin-4-carbonic acid to L-cysteine. Appl Environ Microb 68:2179–2187 Tamura Y, Nishino M, Ohmachi T, Asada Y (1998) N-carbamoyl-L-cysteine as an intermediate in the bioconversion from D, L-2-amino-D2-thiazoline-4-carboxylic acid to L-cysteine by Pseudomonas sp. ON-4a. Biosci Biotech Bioch 62:2226–2229 Tashima I, Yoshida T, Asada Y, Ohmachi T (2006) Purification and characterization of a novelL-2-amino-D2-thiazoline-4carboxylic acid hydrolase from Pseudomonas sp. strain ON-4a expressed in E. coli. Appl Microbiol Biot 72: 499–507 Yu YS, Liu Z, Liu CQ, Li Y, Jin YJ, Yang WB, Bai G (2006) Cloning, expression and identification of genes involved in the conversion of DL-2-amino-D2-thiazoline-4-carboxylic acid to L-cysteine via S-carbamyl-L-cysteine Pathway in Pseudomonas sp. TS1138. Biosci Biotech Bioch 70: 2262–2267 Zheng LM, White RH, Cash VL, Dean DR (1994) Mechanism for the desulfurization of L-cysteine catalyzed by the nifs gene product. Biochemistry 33:4714–4720