Structural Insights and Reaction Profile of a New Unspecific Peroxygenase from <i>Marasmius wettsteinii</i> Produced in a Tandem-Yeast Expression System

Fungal unspecific peroxygenases (UPOs) are gaining momentum in synthetic chemistry. Of special interest is the UPO from <i>Marasmius rotula</i> (<i>Mro</i>UPO), which shows an exclusive repertoire of oxyfunctionalizations, including the terminal hydroxylation of alkanes, the α-oxidation of fatty acids and the C-C cleavage of corticosteroids. However, the lack of heterologous expression systems to perform directed evolution has impeded its engineering for practical applications. Here, we introduce a close ortholog of <i>Mro</i>UPO, a UPO gene from <i>Marasmius wettsteinii</i> (<i>Mwe</i>UPO-1), that has a similar reaction profile to <i>Mro</i>UPO and for which we have set up a directed evolution platform based on tandem-yeast expression. Recombinant <i>Mwe</i>UPO-1 was produced at high titers in the bioreactor (0.7 g/L) and characterized at the biochemical and atomic levels. The conjunction of soaking crystallographic experiments at a resolution up to 1.6 Å together with the analysis of reaction patterns sheds light on the substrate preferences of this promiscuous biocatalyst.