S72: Engineering C-H oxidation chemistry in bacteria

Aliphatic C-H oxidation, hydroxylation reactions in complex molecule synthesis, is one of the most intriguing problems in synthetic organic chemistry. Activating inert C-H bonds and achieving stereo selectivity in C-H oxidation reactions using synthetic catalysts are particularly daunting tasks. In nature however, enzyme mediated C-H oxidation chemistry often occurs in the biosynthesis of natural products. Many of these natural products are active therapeutic molecules or scaffolds for drug discovery, but low accumulation of these functionalized scaffolds in the native hosts limits the exploitation of these highly malleable chemical species. Alternatively, engineering of natural products biosynthesis in a fast-growing microorganism may enable large scale production. In this context functional reconstruction of the Cytochrome P450 (CYP) enzyme-mediated C-H oxidation reactions is a technically challenging task. Here we present our recent success on engineering an E. coli bacteria for functional expression of P450 enzymes and high level production of hydroxylated taxanes. We developed various engineered enzymes to increase the functional activity and flux through the biosynthetic pathway and achieved more than 500 mg/L production of hydroxylated taxanes from an engineered E coli. This result not only provides insights into performing oxidation chemistry in bacteria but debunks the generally accepted notion that prokaryotic bacterial cells are not amenable hosts for complex hydroxylation reactions. In addition, the productivity achieved is unprecedented compared to the state of art 25 µg/L hydroxylated taxane production using P450 enzyme compatible eukaryotic yeast.