Direct conversion of ethane to ethanol by engineered cytochrome P450 BM3.

Most natural gas sources remain untapped as energy and chemical feedstocks. A catalyst for the selective oxidation of gaseous alkanes into more easily transported alcohols could allow these reserves to be exploited. Although catalytic alkane hydroxylation has been reported by many groups, selective conversion of ethane and methane mainly to their corresponding alcohols has yet to be demonstrated. For example, limited (i.e.<500 total turnovers) catalytic ethane oxidation is supported by a variety of transition metal catalysts, but these systems produce mixtures containing significant amounts of acetaldehyde and acetic acid in addition to ethanol. Harsh oxidants such as hydrogen peroxide or sulfuric acid are usually required, although catalytic systems that make use of dioxygen have also been reported. The ideal catalyst should convert ethane to ethanol with high selectivity and productivity, be easily prepared from relatively common, nontoxic materials, function at low temperature and pressure, use dioxygen from the air as the oxidant, and produce little or no hazardous waste. Biological systems have evolved metalloenzymes that convert alkanes into alcohols with many of the features of the ideal catalyst. The well-studied methane monooxygenase (MMO), for example, catalyzes the conversion of methane to methanol and has long been a source of inspiration for catalyst designers. Unfortunately, these structurally complex enzymes have never been functionally expressed in a heterologous organism suitable for bioconversion and process optimization, and therefore have proven to be of little practical use for producing alcohols. We chose cytochrome P450 BM-3 as our target for generating an enzyme that can convert ethane and methane to alcohols because it possesses properties that make it both an ideal catalyst and straightforward to engineer: BM-3 is highly soluble, exhibits high catalytic rates on preferred substrates (thou-