Secretion and directed evolution of unspecific peroxygenases in S. cerevisiae

Yeast-based secretion systems are advantageous for engineering highly interesting enzymes that are not or barely producible in E. coli. The herein-presented production setup facilitates high-throughput screening as no cell lysis is required. All techniques are described in detail, with access to freely available online tools and all vectors have been made available on the non-profit plasmid repository AddGene. We describe the method for UPOs as a model enzyme, showcasing their secretion, detection, and evolution using S. cerevisiae. Additional material to transfer this to P. pastoris has been published by our group previously (Püllmann & Weissenborn, 2021).