Chemo- and Regioselective Dihydroxylation of Benzene to Hydroquinone Enabled by Engineered Cytochrome P450 Monooxygenase.

Abstract

Hydroquinone (HQ) is produced commercially from benzene by multi-step Hock-type processes with equivalent amounts of acetone as side-product. We describe an efficient biocatalytic alternative using the cytochrome P450-BM3 monooxygenase. Since the wildtype enzyme does not accept benzene, a semi-rational protein engineering strategy was developed. Highly active mutants were obtained which transform benzene in a one-pot sequence first into phenol and then regioselectively into HQ without any overoxidation. A computational study shows that the chemoselective oxidation of phenol by the P450-BM3 variant A82F/A328F leads to the regioselective formation of an epoxide intermediate at the C3=C4 double bond, which departs from the binding pocket and then undergoes fragmentation in aqueous medium with exclusive formation of HQ. As a practical application, an E. coli designer cell system was constructed, which enables the cascade transformation of benzene into the natural product arbutin, which has anti-inflammatory and anti-bacterial activities.