Rapid screening system to identify unspecific peroxygenase activity

TL;DR

This paper introduces a fast and cost-effective method to screen for active peroxygenase enzymes, enabling the discovery of new biocatalysts for drug metabolite synthesis.

Contribution

A high-throughput agar plate-based screening system for identifying functional peroxygenases from fungal genes.

Findings

The method successfully identified two active UPOs from Dendrothele bispora and Aspergillus niger.

Both UPOs catalyzed the formation of human drug metabolites with distinct efficiencies and product specificities.

The system allows screening of thousands of clones simultaneously, improving the discovery of recombinantly expressible UPOs.

Abstract

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.