Selective hydroxylation of alkanes remains a significant scientific challenge in the field of classical chemistry. Chemical transformations of an inactivated C-H bond hold tremendous interest in the areas of energy and industrial chemicals. Enzymes of the cytochrome P450 superfamily catalyze the hydroxylation of thousands of substrates, including alkanes. However, no natural P450 have been shown to oxidize the gaseous alkanes, propane, ethane and methane. We present recent results with P450 BM3 variants that are able to catalyze, at room temperature and pressure, the hydroxylation of propane and ethane. Our directed evolution strategy relied on the accumulation of beneficial mutations over multiple generations of random mutagenesis and screening. Through step-wise increases in the screening pressure, mutants with improved activity on the smaller alkanes were isolated. Our results show that multiple mutations in BM3 heme domain are required to achieve high catalytic rates on alkanes, while mutations in the reductase domain improve the catalytic efficiency and favor coupling between product formation cofactor consumption.

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