Yeast Surface Display Enables One‐Step Production and Immobilization of Unspecific Peroxygenases

Unspecific peroxygenases (UPOs) are regarded as a “dream catalyst” for selective oxyfunctionalization reactions like oxygenations. We present the display of the model UPO rAaeUPO (PaDa-I) on the cell surface of the heterologous production host Komagataella phaffii as a one-step production and immobilization process. The coding sequence for PaDa-I was combined with genes coding for cell wall proteins from Saccharomyces cerevisiae and transformed into K. phaffii. The fusion proteins were compared among each other and with secreted, free PaDa-I. One system in particular, a C-terminal fusion of PaDa-I and SAG1 yielded near identical activity per volume culture broth to the secreted PaDa-I with ~90% of the activity being at the cell wall. The surface display simplifies downstream processing and includes immobilization on a cheap, retainable and replaceable matrix, that is the production host itself. The enzymes remained active in a repeated batch process for 10 batches and 200 h of catalysis.