Hydrogen peroxide-independent production of α-alkenes by OleT(JE) P450 fatty acid decarboxylase

BACKGROUND: Cytochrome P450 OleT(JE) from Jeotgalicoccus sp. ATCC 8456, a new member of the CYP152 peroxygenase family, was recently found to catalyze the unusual decarboxylation of long-chain fatty acids to form α-alkenes using H(2)O(2) as the sole electron and oxygen donor. Because aliphatic α-alkenes are important chemicals that can be used as biofuels to replace fossil fuels, or for making lubricants, polymers and detergents, studies on OleT(JE) fatty acid decarboxylase are significant and may lead to commercial production of biogenic α-alkenes in the future, which are renewable and more environmentally friendly than petroleum-derived equivalents. RESULTS: We report the H(2)O(2)-independent activity of OleT(JE) for the first time. In the presence of NADPH and O(2), this P450 enzyme efficiently decarboxylates long-chain fatty acids (C(12) to C(20)) in vitro when partnering with either the fused P450 reductase domain RhFRED from Rhodococcus sp. or the separate flavodoxin/flavodoxin reductase from Escherichia coli. In vivo, expression of OleT(JE) or OleT(JE)-RhFRED in different E. coli strains overproducing free fatty acids resulted in production of variant levels of multiple α-alkenes, with a highest total hydrocarbon titer of 97.6 mg·l(-1). CONCLUSIONS: The discovery of the H(2)O(2)-independent activity of OleT(JE) not only raises a number of fundamental questions on the monooxygenase-like mechanism of this peroxygenase, but also will direct the future metabolic engineering work toward improvement of O(2)/redox partner(s)/NADPH for overproduction of α-alkenes by OleT(JE).