Laboratory evolution platform for aryl alcohol oxidase in Saccharomyces cerevisiae

Abstract

Among the ligninolytic oxidoreductases secreted by white-rot fungi, the aryl alcohol oxidase (AAO) plays an outstanding role as H2O2 supplying enzyme during lignin decay1. With high enantioselectivity and broad substrate specificity, this flavo-enzyme is also a promising departure point for directed evolution studies towards different biotechnological fates, but the lack of heterologous functional expression levels precludes further advances in the field. In this study, the native signal peptide of AAO from Pleurotus eryngii was replaced by those of the mating α-factor, the toxin K1 Killer, as well as combinations of pre- and pro-regions from both leaders to achieve secretion inSaccharomyces cerevisiae2,3. AAO expression in yeast was measured with the help of an ad-hoc colorimetric-dual HTS-protocol based on the detection of H2O2 with a chemical (FOX) and an enzymatic assay (ABTS-HRP). All constructs were successfully processed and secreted by yeast showing extracellular AAO activities with several aromatic alcohols, which opens new paths for future developments.