Functional expression of aryl‐alcohol oxidase in Saccharomyces cerevisiae and Pichia pastoris by directed evolution

Aryl‐alcohol oxidase (AAO) plays a fundamental role in the fungal ligninolytic secretome, acting as a supplier of H2O2. Despite its highly selective mechanism of action, the presence of this flavooxidase in different biotechnological settings has hitherto been hampered by the lack of appropriate heterologous expression systems. We recently described the functional expression of the AAO from Pleurotus eryngii in Saccharomyces cerevisiae by fusing a chimeric signal peptide (preαproK) and applying structure‐guided evolution. Here, we have obtained an AAO secretion variant that is readily expressed in S. cerevisiae and overproduced in Pichia pastoris. First, the functional expression of AAO in S. cerevisiae was enhanced through the in vivo shuffling of a panel of secretion variants, followed by the focused evolution of the preαproK peptide. The outcome of this evolutionary campaign—an expression variant that accumulated 4 mutations in the chimeric signal peptide, plus two mutations in the mature protein‐ showed 350‐fold improved secretion (4.5 mg/L) and was stable. This secretion mutant was cloned into P. pastoris and fermented in a fed‐batch bioreactor to enhance production to 25 mg/L. While both recombinant AAO from S. cerevisiae and P. pastoris were subjected to the same N‐terminal processing and had a similar pH activity profile, they differed in their kinetic parameters and thermostability. The strong glycosylation observed in the evolved AAO from S. cerevisiae underpinned this effect, since when the mutant was produced in the glycosylation‐deficient S. cerevisiae strain Δkre2, its kinetic parameters and thermostability were comparable to its poorly glycosylated P. pastoris recombinant counterpart. Fungal aryl‐alcohol oxidases (AAO) are precious biocatalysts whose presence in industrial biotechnology is being impeded by the absence of proper heterologous expression system. In the present study, the functional expression of AAO from white rot fungus Pleurotus eryngii was adapted to a tandem‐yeast expression system aimed at boosting secretion. After applying different directed evolution strategies in S. cerevisiae, the evolved AAO was successfully overproduced in Pichia pastoris in bench‐bioreactor.