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 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.

Repertoire of Computationally Designed Peroxygenases for Enantiodivergent C–H Oxyfunctionalization Reactions

“…FuncLib was originally conceived to generate catalytic repertoires of enzymes with latent/promiscuous activities, and in a very short time, it has been successfully applied to very different situations, ranging from the design of regioselective pyrazole alkylating methyltransferases to the engineering of cytoplasmic-soluble N-glycosyltransferases with narrow substrate specificity, the enhancement of the catalytic efficiency in a de novo Kemp eliminase, the increase in the reactivity profile of high-redox potential laccases, or to the stabilization of the SARS-CoV-2 spike receptor binding domain . We view FuncLib as a method for computational mutagenesis that can help address complex enzyme engineering problems, and here, we demonstrate that FuncLib represents an efficient and rapid approach to generate small but highly functional libraries enriched in enantioselective peroxygenase mutants for complex C–H oxyfunctionalization reactions, which, in the future, could be assisted by machine learning-guided evolution to search for optimum beneficial epistasis among mutations. , The opportunity of having a palette of UPO variants with different enantioselectivities will pave the way for the generation of novel compounds and catalytic pathways to be used in applications ranging from lead diversification to the synthesis of fine chemicals.…”

Increasing Reduction Potentials of Type 1 Copper Center and Catalytic Efficiency of Small Laccase from Streptomyces coelicolor through Secondary Coordination Sphere Mutations

“…However, HRPLs are heavily glycosylated and their activity has been shown to be affected by the level of glycosylation [31–33] . The instability for HRPLs in recombinant system also makes it more difficult to investigate the effect of SCS mutations on E °′ T1Cu [28,34,35] . In contrast, many bacterial laccases are more amendable to heterologous expression and purification, have wider active pH range, but exhibit low (≤480 mV) E °′ T1Cu [36–39] .…”

“…[31][32][33] The instability for HRPLs in recombinant system also makes it more difficult to investigate the effect of SCS mutations on E°' T1Cu . [28,34,35] In contrast, many bacterial laccases are more amendable to heterologous expression and purification, have wider active pH range, but exhibit low (� 480 mV) E°' T1Cu . [36][37][38][39] Therefore, the question arises as to how bacterial laccases can be reengineered to exhibit HRPLs properties.…”

Robust Oxidase-Mimetic Supramolecular Nanocatalyst for Lignin Biodegradation

“…However, the intricate structure of native lignin poses challenges in terms of its solubility in aqueous solutions and compatibility with enzymes. To date, biocatalysis has mainly been applied to degrade lignin model compounds − and solvent-treated lignin, − the latter of which is obtained through energy-intensive and environmentally unfriendly pretreatment of lignocellulosic biomass.…”