Rapid screening system to identify unspecific peroxygenase activity

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.

Keywords: ABTS; drug metabolism; enzyme expression; omega-3-lipids metabolism; screening assay; unspecific peroxygenase.

Figure 1.

Maximum likelihood tree of the putative short UPO sequences selected for the screening library. Phylogeny was calculated using PhyML 3.3.20180214 with a log-likelihood score of −10174.09410. Branch support was estimated using 200 bootstrap replicates; only support >90% were shown above branches. The outgroup UPO sequence is from Rozella allomycis (Cryptomycota), the evolutionary oldest group of fungi (Eumycota).

Figure 2.

Evaluation of an ABTS-based plate screening for UPO-secreting S. cerevisiae colonies. S. cerevisiae cells containing a plasmid with the positive control AaeUPO PaDa-I were mixed with cells that contained the empty vector at a ratio of 1:50 (2%, left) or 1:10 (10%, right). UPO-positive colonies developed blue-green zones caused by the oxidation of ABTS after addition of H₂O₂.

Figure 3.

Screening for activity of extracellular UPOs recombinantly expressed in S. cerevisiae. A pool of 20 putative UPO ORFs was screened using a plate-based approach with ABTS as indicator. Color formation caused by the UPO catalyzed oxidation of ABTS developed after addition of H₂O₂. The picture on the right shows a close-up of the plate.

Figure 4.

Influence of different medium compositions on the production of rAniUPO in the host S. cerevisiae INVSc1. The volume activity in the supernatants was monitored over the cultivation period with ABTS as substrate; media composition and the abbreviations in the legends are given in table S2.

Figure 5.

Monitoring the enzymatic volume activity in the supernatants of S. cerevisiae INVSc1 producing rAniUPO or rDbiUPO. Enzyme activity was measured with ABTS as substrate. Cultivations were carried out in duplicate.