The colors of peroxygenase activity: Colorimetric high-throughput screening assays for directed evolution

Fungal unspecific peroxygenases (UPOs) are arising as versatile biocatalysts for C–H oxyfunctionalization reactions. In recent years, several directed evolution studies have been conducted to design improved UPO variants. An essential part of this protein engineering strategy is the design of reliable colorimetric high-throughput screening (HTS) assays for mutant library exploration. Here, we present a palette of 12 colorimetric HTS assays along with their step-by-step protocols, which have been validated for directed UPO evolution campaigns. This array of colorimetric assays will pave the way for the discovery and design of new UPO variants.