Oliver Lentz1, Vlada Urlacher1, Anton Feenstra2, Rolf D. Schmid1
1Institut für Technische Biochemie, Universität Stuttgart, Germany
2Department of Pharmacochemistry, Vrije Universiteit Amsterdam, Netherlands
Changing the regioselectivity of cytochrome P450 CYP102A3 from Bacillus subtilis by directed evolution, in preparation (2005).

Within the Bacillus subtilis genome, a novel monooxygenase was discovered which revealed a similarity of 76% to the well-known cytochrome P450 BM-3 (CYP102A1) of B. megaterium. Both enzymes are natural fusion proteins consisting of a heme and a FAD/FMN reductase domain. We here report the laboratory evolution of B. subtilis enzyme CYP102A3 leading to a different hydroxylation pattern. The regioselectivity of this enzyme which is similar to that of B. megaterium CYP102A1 was changed to hydroxylate n-octane not only in subterminal but also in terminal position. A double mutant was prepared that produces 48% 1-octanol and 52% of secondary octanoles. For the screening procedure, a new HTS assay was developed that is able to discriminate between terminal and subterminal hydroxylation of carbon chains.