Hydroxylation of non-substituted polycyclic aromatic hydrocarbons by cytochrome P450 BM3 engineered by directed evolution.

Chrysene and pyrene are known toxic compounds recalcitrant to biodegradation. Here directed evolution allowed us to identify two new mutants of cytochrome P450 BM3 that are able to hydroxylate both compounds. Random mutagenesis has been used to generate libraries of mutants of P450 BM3 active toward polycyclic aromatic hydrocarbons (PAHs) PAHs. After two rounds of error-prone PCR and backcross with parental DNA, three mutants were identified for improved activity toward pyrene and for the first time a new activity toward chrysene in comparison to the wild type enzyme. The mutants show higher affinity and coupling efficiency for chrysene with faster rates of product formation compared to the wild type. Furthermore, the mutants are able to hydroxylate chrysene in different positions, producing four metabolites, 1-, 3-, 4-, and 6-hydroxychrysene, and to hydroxylate pyrene to 1-hydroxypyrene. The majority of the mutation sites are found to be far from the active site, demonstrating the power of directed evolution in identifying mutations difficult to predict with a rational design approach. The different product profiles obtained for the different P450 BM3 mutants indicate that substrate orientation in the catalytic pocket of the protein can be modified by protein engineering. The mutants can be used for metabolic engineering for safe and cost-effective sustainable production of hydroxylated PAHs for industrial purposes as well as for the assessment of their carcinogenic activity in mammals.