Laboratory evolution of fungal heme-thiolate enzymes employing Komagataella phaffii as a host system

Fungal unspecific peroxygenases (UPOs, EC 1.11.2.1) are heme thiolate enzymes categorized into two families - long and short UPOs - that combine peroxidase and peroxygenase activity. UPOs, found in Ascomycota, Basidiomycota, and Oomycota, catalyze oxyfunctionalization reactions using hydrogen peroxide as a co-substrate to activate C-H, C-C, and C=C bonds. Beyond that, they are naturally secreted and stable enzymes. These properties make them interesting for the production of chemicals, like agrochemicals, insecticides, or pharmaceuticals. However, the main issue that persists is the challenge of producing enough of the desired enzymes or variants thereof through heterologous overexpression.
One of the best-investigated enzymes so far is the evolved secretion variant PaDa-I (derived from AaeUPO) where mutations in the secretion signal enhanced recombinant protein production. Furthermore, the importance of various fungal secretion signals for the overexpression of different short UPOs (e.g. MthUPO) was shown. Nevertheless, we could show that replacing natural secretion signals with the truncated Saccharomyces cerevisiae alpha mating factor secretion signal peptide worked ideal for all our short UPOs. With this method, we could recombinantly overexpress several UPOs by K. phaffii, whereby AbrUPO, PanUPO, and HspUPO were found to be pretty active. These three enzymes were used as starting points for gene mutant libraries which were examined for enhanced peroxide/peroxygenase activity and the found variants broaden the repertoire of oxidative multi-tool enzymes available.