Rapid screening system to identify unspecific peroxygenase activity

• Published in: Clinical hemorheology and microcirculation Vol. 89; no. 4; pp. 363 - 373
• Main Authors: Schramm, Marina, Carrillo Avilés, Carlos Renato, Kalmbach, Johannes, Schmidtke, Kai-Uwe, Kiebist, Jan, Kellner, Harald, Hofrichter, Martin, Scheibner, Katrin
• Format: Journal Article
• Language: English
• Published: London, England SAGE Publications 01.04.2025
• Subjects: Aspergillus niger - enzymology; Aspergillus niger - genetics; Clopidogrel; Mixed Function Oxygenases - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; enzyme expression; unspecific peroxygenase; omega-3-lipids metabolism; screening assay; ABTS; drug metabolism
• ISSN: 1386-0291; 1875-8622
• DOI: 10.1177/13860291241306566

Unspecific peroxygenases (UPO, EC 1.11.2.1) are a valuable tool for the biocatalytic synthesis of specialty chemicals such as pharmaceutical metabolites. However, the search for new UPOs that are recombinantly expressible can be tedious and dependent on expensive equipment, especially when a large number of clones has to be examined. In this study, we present a simple agar plate-based method for the screening of active, secreted UPOs heterologously expressed in Saccharomyces cerevisiae. This allows a real high-throughput of several thousand clones at once. The approach was successfully tested with a small gene library comprising putative UPO genes and resulted in the identification of two clones producing short UPOs from the filamentous fungi Dendrothele bispora (DbiUPO) and Aspergillus niger (AniUPO). Both UPOs were partly purified and characterized with respect to their catalytic properties. With differing efficiencies and product specificities, they catalyzed the formation of human drug metabolites, e.g., lipid mediators from polyunsaturated fatty acids and the active metabolite of the prodrug clopidogrel, respectively.