Designed high-redox potential laccases exhibit high functional diversity

Abstract

White-rot fungi secrete an impressive repertoire of high-redox potential laccases (HRPLs) and peroxidases for efficient oxidation and utilization of lignin. Laccases are attractive enzymes for greenchemistry applications due to their broad substrate range and low environmental impact. Since expression of functional recombinant HRPLs is challenging, however, iterative directed evolution protocols have been applied to improve their expression, activity and stability. We implement a rational, stabilize-and-diversify strategy to two HRPLs that we could not functionally express: first, we use the PROSS stability-design algorithm to allow functional expression in yeast. Second, we use the stabilized enzymes as starting points for FuncLib active-site design to improve their activity and substrate diversity. Four of the FuncLib designed HRPLs and their PROSS progenitor exhibit substantial diversity in reactivity profiles against highredox potential substrates, including lignin monomers. Combinations of 3-4 subtle mutations that change the polarity, solvation and sterics of the substrate-oxidation site result in orders of magnitude changes in reactivity profiles.