Regioselective Hydroxylation in the Production of 25‐Hydroxyvitamin D by Coprinopsis cinerea Peroxygenase

Abstract Monohydroxylated metabolites of vitamin D 3 (cholecalciferol) and vitamin D 2 (ergocalciferol), generically known as 25‐hydroxycalciferol, are better for several diseases, and other applications, than vitamin D (calciferol). This work describes a novel biotechnological approach for the preparation of 25‐hydroxycalciferols, starting from readily available cholecalciferol and ergocalciferol. This approach enables the regioselective (100 %) hydroxylation of these compounds (at the C‐25 position) under mild and environmentally friendly conditions by using a peroxidase from the fungus Coprinopsis cinerea (gene model CC1G_08427T0 from the sequenced genome), which catalyzes monooxygenation with H 2 O 2 as the only co‐substrate (peroxygenase). Hydroxylation of cholecalciferol and ergocalciferol is a true peroxygenation, as demonstrated by incorporation of 18 O from H 2 18 O 2 into the products. The peroxygenase has additional advantages related to its recombinant nature, enabling enzyme engineering and low‐cost overexpression in an industrial host. Therefore, the peroxygenase is a promising biocatalyst for the production of vitamin D active metabolites.