Designed High-Redox Potential Laccases Exhibit High Functional Diversity.

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 the chemical industry 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 high-redox 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. These stable and versatile HRPLs are a step toward generating an effective lignin-degrading consortium of enzymes that can be secreted from yeast. The stabilize-and-diversify strategy can be applied to other challenging enzyme families to study and expand the utility of natural enzymes.