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Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for enhanced chlorinated ethene degradation and o-xylene oxidation

  • Applied Genetics and Molecular Biotechnology
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Abstract

Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 has been shown to degrade all chlorinated ethenes individually and as mixtures. Here, DNA shuffling of the alpha hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, F205, and E214 were used to enhance the degradation of chlorinated aliphatics. The ToMO mutants were identified using a chloride ion screen and then were further examined by gas chromatography. Escherichia coli TG1/pBS(Kan)ToMO expressing TouA saturation mutagenesis variant I100Q was identified that has 2.8-fold better trichloroethylene (TCE) degradation activity (apparent V max of 1.77 nmol min−1 mg−1 protein−1 vs 0.63 nmol min−1 mg−1 protein−1). Another variant, E214G/D312N/M399V, has 2.5-fold better cis-1,2-dichloroethylene (cis-DCE) degradation activity (apparent V max of 8.4 nmol min−1 mg−1 protein−1 vs 3.3 nmol min−1 mg−1 protein−1). Additionally, the hydroxylation regiospecificity of o-xylene and naphthalene were altered significantly for ToMO variants A107T/E214A, T201G, and T201S. Variant T201S produced 2.0-fold more 2,3-dimethylphenol (2,3-DMP) from o-xylene than the wild-type ToMO, whereas variant A107T/E214A had 6.0-fold altered regiospecificity for 2,3-DMP formation. Variant A107T/E214A also produced 3.0-fold more 2-naphthol from naphthalene than the wild-type ToMO, whereas the regiospecificity of variant T201S was altered to synthesize 3.0-fold less 2-naphthol, so that it made almost exclusively 1-naphthol (96%). Variant T201G was more regiospecific than variants A107T/E214A and T201S and produced 100% 3,4-DMP from o-xylene and >99% 1-naphthol from naphthalene. Hence, ToMO activity was enhanced for the degradation of TCE and cis-DCE and for the regiospecific hydroxylation of o-xylene and naphthalene through DNA shuffling and saturation mutagenesis.

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References

  • Baggi G, Barbieri P, Galli E, Tollari S (1987) Isolation of a Pseudomonas stutzeri strain that degrades o-xylene. Appl Environ Microbiol 53:2129–2132

    PubMed  CAS  Google Scholar 

  • Bertoni G, Bolognese F, Galli E, Barbieri P (1996) Cloning of the genes for and characterization of the early stages of toluene and o-xylene catabolism in Pseudomonas stutzeri OX1. Appl Environ Microbiol 62:3704–3711

    PubMed  CAS  Google Scholar 

  • Bradley PM, Chapelle FH (1998) Effect of contaminant concentration on aerobic microbial mineralization of DCE and VC in stream-bed sediments. Environ Sci Technol 32:553–557

    Article  CAS  Google Scholar 

  • Cafaro V, Scognamiglio R, Viggiani A, Izzo V, Passaro I, Notomista E, Piaz FD, Amoresano A, Casbarra A, Pucci P, Donato AD (2002) Expression and purification of the recombinant subunits of toluene/o-xylene monooxygenase and reconstitution of the active complex. Eur J Biochem 269:5689–5699

    Article  PubMed  CAS  Google Scholar 

  • Canada KA, Iwashita S, Shim H, Wood TK (2002) Directed evolution of toluene ortho-monooxygenase for enhanced 1-naphthol synthesis and chlorinated ethene degradation. J Bacteriol 184:344–349

    Article  PubMed  CAS  Google Scholar 

  • Carter SR, Jewell WJ (1993) Biotransformation of tetrachloroethylene by anaerobic attached-films at low temperatures. Water Res 27:607–615

    Article  CAS  Google Scholar 

  • Chauhan S, Barbieri P, Wood TK (1998) Oxidation of trichloroethylene, 1,1-dichloroethylene, and chloroform by toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1. Appl Environ Microbiol 64:3023–3024

    PubMed  CAS  Google Scholar 

  • Dolfing J, Van den Wijngaard AJ, Janssen DB (1993) Microbiological aspects of the removal of chlorinated hydrocarbons from air. Biodegradation 4:261–282

    Article  PubMed  CAS  Google Scholar 

  • Elango N, Radhakrishnan R, Froland WA, Wallar BJ, Earhart CA, Lipscomb JD, Ohlendorf DH (1997) Crystal structure of the hydroxylase component of methane monooxygenase from Methylosinus trichosporium OB3b. Protein Sci 6:556–568

    Article  PubMed  CAS  Google Scholar 

  • He J, Ritalahti KM, Yang K-L, Koenigsberg SS, Löffler FE (2003) Detoxification of vinyl chloride to ethene coupled to growth of an anaerobic bacterium. Nature 424:62–65

    Article  PubMed  CAS  Google Scholar 

  • Hylckama Vlieg JET van, Koning W de, Janssen DB (1996) Transformation kinetics of chlorinated ethenes by Methylosinus trichosporium OB3b and detection of unstable epoxides by on-line gas chromatography. Appl Environ Microbiol 62:3304–3312

    Google Scholar 

  • McCarty PL (1997) Breathing with chlorinated solvents. Science 276:1521–1522

    Article  PubMed  CAS  Google Scholar 

  • McClay K, Fox BG, Steffan RJ (1996) Chloroform mineralization by toluene-oxidizing bacteria. Appl Environ Microbiol 62:2716–2722

    PubMed  CAS  Google Scholar 

  • Moore JC, Arnold FH (1996) Directed evolution of a para-nitrobenzyl esterase for aqueous–organic solvents. Nat Biotechnol 14:458–467

    Article  PubMed  CAS  Google Scholar 

  • Pikus JD, Studts JM, McClay K, Steffan RJ, Fox BG (1997) Changes in the regiospecificity of aromatic hydroxylation produced by active site engineering in the diiron enzyme toluene 4-monooxygenase. Biochemistry 36:9283–9289

    Article  PubMed  CAS  Google Scholar 

  • Pikus JD, Mitchell KH, Studts JM, McClay K, Steffan RJ, Fox BG (2000) Threonine 201 in the diiron enzyme toluene 4-monooxygenase is not required for catalysis. Biochemistry 39:791–799

    Article  PubMed  CAS  Google Scholar 

  • Rosenzweig AC, Brandstetter H, Whittington DA, Nordlund P, Lippard SJ, Frederick CA (1997) Crystal structures of the methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath): implications for substrate gating and component interactions. Proteins Struct Funct Genet 29:141–152

    Article  PubMed  CAS  Google Scholar 

  • Rui L, Kwon YM, Fishman A, Reardon KF, Wood TK (2004) Saturation mutagenesis of toluene ortho-monooxygenase from Burkholderia cepacia G4 for enhanced 1-naphthol synthesis and chloroform degradation. Appl Environ Microbiol 70:3246–3252

    Article  PubMed  CAS  Google Scholar 

  • Ryoo D, Shim H, Canada K, Barbieri P, Wood TK (2000) Aerobic degradation of tetrachloroethylene by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1. Nat Biotechnol 18:775–778

    Article  PubMed  CAS  Google Scholar 

  • Ryoo D, Shim H, Arenghi FLG, Barbieri P, Wood TK (2001) Tetrachloroethylene, trichloroethylene, and chlorinated phenols induce toluene-o-monooxygenase activity in Pseudomonas stutzeri OX1. Appl Microbiol Biotechnol 56:545–549

    Article  PubMed  CAS  Google Scholar 

  • Sakamoto T, Joern JM, Arisawa A, Arnold FH (2001) Laboratory evolution of toluene dioxygenase to accept 4-picoline as a substrate. Appl Environ Microbiol 67:3882–3887

    Article  PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Article  PubMed  CAS  Google Scholar 

  • Sazinsky MH, Bard J, Donato AD, Lippard SJ (2004) Crystal structure of the toluene/o-xylene monooxygenase hydroxylase from Pseudomonas stutzeri OX1: insight into the substrate specificity, substrate channeling and active site tuning of multicomponent monooxygenases. J Biol Chem 279:30600–30610

    Article  PubMed  CAS  Google Scholar 

  • Shim H, Wood TK (2000) Aerobic degradation of mixtures of chlorinated aliphatics by cloned toluene-o-xylene monooxygenase and toluene o-monooxygenase in resting cells. Biotechnol Bioeng 70:693–698

    Article  PubMed  CAS  Google Scholar 

  • Shim H, Ryoo D, Barbieri P, Wood TK (2001) Aerobic degradation of mixtures of tetrachloroethylene, trichloroethylene, dichloroethylenes, and vinyl chloride by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1. Appl Microbiol Biotechnol 56:265–269

    Article  PubMed  CAS  Google Scholar 

  • Stemmer WPC (1994) DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci USA 91:10747–10751

    Article  PubMed  CAS  Google Scholar 

  • Tao Y, Bentley WE, Wood TK (2004a) Regiospecific oxidation of naphthalene and fluorene by toluene monooxygenases and engineered toluene 4-monooxygenase of Pseudomonas mendocina KR1. Biotechnol Bioeng (in press)

  • Tao Y, Fishman A, Bentley WE, Wood TK (2004b) Oxidation of benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by toluene 4-monooxygenase of Pseudomonas mendocina KR1 and toluene 3-monooxygenase of Ralstonia pickettii PKO1. Appl Environ Microbiol 70:3814–3820

    Article  PubMed  CAS  Google Scholar 

  • Vardar G, Wood TK (2004) Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for synthesizing 4-methylresorcinol, methylhydroquinone, and pyrogallol. Appl Environ Microbiol 70:3253–3262

    Article  PubMed  CAS  Google Scholar 

  • Vardar G, Wood TK (2005) Alpha subunit positions methionine 180 and glutamate 214 of Pseudomonas stutzeri OX1 toluene-o-xylene monooxygenase influence catalysis. J Bacteriol 187:1511–1514

    Article  PubMed  CAS  Google Scholar 

  • Vardar G, Barbieri P, Wood TK (2005a) Chemotaxis of Pseudomonas stutzeri OX1 and Burkholderia cepacia G4 toward chlorinated ethenes. Appl Microbiol Biotechnol 66:696–701

    Article  PubMed  CAS  Google Scholar 

  • Vardar G, Ryu K, Wood TK (2005b) Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for oxidizing nitrobenzene to 3-nitrocatechol, 4-nitrocatechol, and nitrohydroquinone. J Biotechnol 115:145–156

    Article  PubMed  CAS  Google Scholar 

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Acknowledgement

This study was supported by the U.S. Army Research (DAAD19-00-1-0568).

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Correspondence to Thomas K. Wood.

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Vardar, G., Wood, T.K. Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for enhanced chlorinated ethene degradation and o-xylene oxidation. Appl Microbiol Biotechnol 68, 510–517 (2005). https://doi.org/10.1007/s00253-005-1923-4

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  • DOI: https://doi.org/10.1007/s00253-005-1923-4

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