Laboratory evolution of a fungal unspecific peroxygenase to elucidate the structural and functional relationship

Fungal unspecific peroxygenases (UPOs, EC 1.11.2.1) are heme thiolate enzymes classified into two families - long and short UPOs - that combine peroxidase and peroxygenase activity. UPOs, which are naturally secreted by Ascomycota, Basidiomycota, and Oomycota, can be used in biocatalytic oxyfunctionalization reactions using hydrogen peroxide as a co-substrate for the production of specialty chemicals. However, despite the basic robustness of these secreted heme enzymes, higher process stability and activity for preferred substrates are still desired. The best-studied enzyme to date, PaDa-I (a large UPO derived from AaeUPO by laboratory evolution), was generated by various mutations throughout the enzyme and showed increased peroxidase and peroxygenase activity. In our study, we randomly introduced several uncharacterized mutations into a short UPO derived from Podospora anserina (PanUPO). Our high-throughput screening methods using standard colorimetric UPO assays revealed ten interesting variants. Two of them (11B4 and 19D10) showed increased peroxidase activity (substrate: ABTS, Figure 1 A). Variant 11B4 showed increased activity and 19D10 was more active but also more stable at hydrogen peroxide concentrations of 1.0, 2.0, and 5.0 mM. A mutation identified in variant 19B8 increased peroxygenase activity (substrate: naphthalene, Figure 1 B). These three newly identified PanUPO variants expand the repertoire of UPOs for biocatalysis in order to evaluate their industrial use and also improve our understanding of the structure/function relationship for this diverse toolbox of multipurpose oxidative enzymes.