title: Saturation mutagenesis of toluene ortho-monooxygenase of Burkholderia cepacia G4 for Enhanced 1-naphthol synthesis and chloroform degradation.
abstract: Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase alpha-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (V(max) of 9.3 versus 4.5 nmol.min(-1).mg of protein(-1) and unchanged K(m)), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 +/- 0.07 versus 1.30 +/- 0.06 nmol.min(-1).mg of protein(-1) [mean +/- standard deviation]) at 91 microM and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (V(max) of 2.61 versus 0.95 nmol.min(-1).mg of protein(-1) and unchanged K(m)) and chloride release (0.034 +/- 0.002 versus 0.012 +/- 0.001 nmol.min(-1).mg of protein(-1)). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 +/- 0.3 versus 1.30 +/- 0.06 nmol.min(-1).mg of protein(-1)). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.
authors.affiliation: Department of Chemical Engineering, University of Connecticut, Storrs, 06269-3222, USA.
authors.forename: Lingyun
authors.initials: L
authors.lastname: Rui
authors.forename: Young Man
authors.initials: YM
authors.lastname: Kwon
authors.forename: Ayelet
authors.initials: A
authors.lastname: Fishman
authors.forename: Kenneth F
authors.initials: KF
authors.lastname: Reardon
authors.forename: Thomas K
authors.initials: TK
authors.lastname: Wood
mesh_terms: D001665:Binding Sites; D001673:Biodegradation, Environmental; D016956:Burkholderia cepacia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D002725:Chloroform / Q000378:metabolism*; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D009284:Naphthols / Q000378:metabolism*; D010084:Oxidation-Reduction
chemical_list: D003062:Codon; D009284:Naphthols; C029350:1-naphthol; D002725:Chloroform; D006899:Mixed Function Oxygenases; C093480:toluene ortho-monooxygenase