Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases

ABSTRACT Ancestral sequence reconstruction and resurrection provides useful information nfor protein engineering, yet its alliance with directed evolution has been nlittle explored. In this study, we have resurrected several ancestral nodes of fungal nlaccases dating back 500 to 250 million years. Unlike modern laccases, the resurrected nMesozoic laccases were readily secreted by yeast, with similar kinetic parameters, na broader stability, and distinct pH activity profiles. The resurrected Agaricomycetes nlaccase carried 136 ancestral mutations, a molecular testimony to its origin, nand it was subjected to directed evolution in order to improve the rate of 1,3- ncyclopentanedione oxidation, a –diketone initiator commonly used in vinyl polymerization nreactions. nIMPORTANCE The broad variety of biotechnological uses of fungal laccases is beyond ndoubt (food, textiles, pulp and paper, pharma, biofuels, cosmetics, and bioremediation), nand protein engineering (in particular, directed evolution) has become nthe key driver for adaptation of these enzymes to harsh industrial conditions. Usually, nthe first requirement for directed laccase evolution is heterologous expression, nwhich presents an important hurdle and often a time-consuming process. In this nwork, we resurrected a fungal Mesozoic laccase node which showed strikingly high nheterologous expression and pH stability. As a proof of concept that the ancestral nlaccase is a suitable blueprint for engineering, we performed a quick directed evolution ncampaign geared to the oxidation of the -diketone 1,3-cyclopentanedione, a npoor laccase substrate that is used in the polymerization of vinyl monomers.