Directed evolution of a cytochrome P450 monooxygenase for alkane oxidation

Cytochrome P450 monooxygenase BM-3 (EC 1.14.14.1) hydroxylates fatty acids with chain lengths between C-12 and C-18. It is also known to oxidize the corresponding alcohols and amides. However, it is not known to oxidize alkanes. Here we report that P450 BM-3 oxidizes octane, which is four carbons shorter and lacks the carboxylate functionality of the shortest fatty acid P450 BM-3 is known to accept, to 4-octanol, 3-octanol, 2-octanol, 4-octanone, and 3-octanone. The rate is much lower than for oxidation of the preferred fatty acid substrates. In an effort to explore the plasticity and mechanisms of substrate recognition in this powerful biocatalyst, we are using directed evolution - random mutagenesis, recombination, and screening - to improve its activity towards saturated hydrocarbons. A spectrophotometric assay has been validated for high throughput screening, and two generations of laboratory evolution have yielded variants displaying up to five times the specific activity of wild-type P450 BM-3.