Promiscuous fungal peroxygenase from Chaetomium globosum by directed evolution

Fungal unspecific peroxygenases (UPOs) are appealing enzymes for C–H oxyfunctionalization reactions, and, among them, the short UPO type from Chaetomium globosum ( Cgl UPO) is of special interest due to its diverse substrate scope and selectivity. Despite its significance, Cgl UPO shows low stability and poor heterologous expression which has hitherto impeded its use for practical applications. In this study, Cgl UPO was engineered by directed evolution to produce a highly active and stable secreted variant that performs selective oxidations on a panel of organic molecules, including ethylbenzene, styrene, 1,2,3,4-tetrahydronaphthalene (THN), isophorone, pinene, and limonene. Molecular dynamic simulations demonstrate how mutations F154L and M210V in the heme access channel are responsible for dramatic changes in the enzyme performance, including inverted enantioselectivities and anti-Markovnikov oxidation reactions.