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

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