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Applied and Environmental Microbiology, June 2004, p. 3246-3252, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3246-3252.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Saturation Mutagenesis of Toluene ortho-Monooxygenase of Burkholderia cepacia G4 for Enhanced 1-Naphthol Synthesis and Chloroform Degradation

Lingyun Rui,1 Young Man Kwon,1 Ayelet Fishman,1 Kenneth F. Reardon,2 and Thomas K. Wood1*

Departments of Chemical Engineering and Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269-3222,1 Department of Chemical Engineering, Colorado State University, Fort Collins, Colorado 80523-13702

Received 22 December 2003/ Accepted 24 February 2004

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 (Vmax of 9.3 versus 4.5 nmol · min–1 · mg of protein–1 and unchanged Km), 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 µM 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 (Vmax of 2.61 versus 0.95 nmol · min–1 · mg of protein–1 and unchanged Km) 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.

* Corresponding author. Mailing address: Departments of Chemical Engineering and Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3222. Phone: (860) 486-2483. Fax: (860) 486-2959. E-mail: twood{at}engr.uconn.edu.

Applied and Environmental Microbiology, June 2004, p. 3246-3252, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3246-3252.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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