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

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