Laccase Engineering by Directed and Computational Evolution

Directed evolution is a powerful strategy to tailor enzymes with improved attributes. The use of laboratory evolution is becoming more refined, whereby computational and experimental approaches are being combined so that more effective libraries can be created, producing enzymes with greater biotechnological potential while notably reducing the demands on screening. This chapter summarizes the most recent findings from our laboratory to tailor fungal high-redox potential laccases by bringing together computational approaches with in vitro and in vivo methods for library creation. We focus on four recent case studies of laccase engineering in which different computational algorithms were applied at both the gene and protein levels: 1. A consensus design to identify consensus ancestral mutations 2. SCHEMA-RASPP computation for homologous in vivo recombination to generate laccase chimeras 3. Computer-guided evolution to enhance the laccase redox potential and the activity toward high-redox potential mediators 4. Ancestral sequence reconstruction for the directed evolution of a resurrected basidiomycete laccase toward initiator