Engineering Unspecific Peroxygenases for Enantioselective α‐Hydroxylation of β‐Ketoesters

Abstract Unspecific peroxygenases (UPOs) are promising biocatalysts for selective oxyfunctionalization. Compared to cytochrome P450 enzymes (P450s), the catalytic potential of UPOs has been less investigated, largely due to their limited natural diversity and the challenges associated with their optimization through enzyme engineering. In this study, we engineered an UPO from Aspergillus niger ( Ani UPO) to catalyze the enantioselective α‐hydroxylation of β‐ketoesters, a valuable transformation yet to be realized in biocatalysis. Through enzyme engineering, two Ani UPO variants, Ani UPO‐M3 and Ani UPO‐M6, were developed to produce a wide range of enantioenriched α‐hydroxy‐β‐ketoesters, achieving up to 97% yield, 4140 total turnover number (TTN), and >99:1 enantiomeric ratio (er). The biocatalytic process operates under mild conditions and is scalable for preparative applications. This study broadens the catalytic repertoire of UPOs and enhances their potential for industrial applications.