Accessing Chemo- and Regioselective Benzylic and Aromatic Oxidations by Protein Engineering of an Unspecific Peroxygenase

Unspecific peroxygenases (UPOs) enable oxyfunctionalizations of a broad substrate range with unparalleled activities. Tailoring these enzymes for chemo- and regioselective transformations represents grand challenge due to the difficulties in their heterologous productions. Herein, we performed protein engineering Saccharomyces cerevisiae using MthUPO from Myceliophthora thermophila. More than 5300 transformants were screened. This led significant reshaping active site as elucidated by computational modelling. The was responsible increased oxyfunctionalization activity, improved kcat/Km values up 16.5-fold model 5-nitro-1,3-benzodioxole. Moreover, variants identified high regioselectivities aromatic benzylic carbons, respectively. hydroxylation demonstrated perform enantioselectivities 95% ee. proposed evolutionary protocol rationalization enhanced activities selectivities acquired represent step forward toward use implementation UPOs biocatalytic synthetic pathways industrial interest.