Directed Evolution of a Highly Selective Fungal Peroxygenase for the Synthesis of Indigo

The large-scale production of indigo, a widely used textile dye, currently relies on chemical processes that consume non-renewable resources and that generate toxic waste, although biocatalysis is emerging as an environmentally friendly alternative. Here, an unspecific peroxygenase from the fungus Daldinia sp. EC12 (DspUPO-I) was subjected to a directed evolution campaign for activity and expression in yeast. With four mutations in the mature protein, the resulting evolved variant showed 95% regioselectivity for indoxyl formation, the precursor of indigo, along with a striking resistance to oxidative inactivation by hydrogen peroxide. These features enable this biocatalyst to synthesize 0.70 g/L indigo on a semipreparative scale. Molecular dynamics simulations highlighted the particular geometry of the broad heme-access channel as the main driving force for the high selectivity and the oxidative stability of this robust biocatalyst during indigo production.