Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases

Ancestral sequence reconstruction and resurrection provides useful information for protein engineering, yet its alliance with directed evolution has been little explored. In this study, we have resurrected several ancestral nodes of fungal laccases dating back ∼500 to 250 million years. Unlike modern laccases, the resurrected Mesozoic laccases were readily secreted by yeast, with similar kinetic parameters, a broader stability, and distinct pH activity profiles. The resurrected Agaricomycetes laccase carried 136 ancestral mutations, a molecular testimony to its origin, and it was subjected to directed evolution in order to improve the rate of 1,3-cyclopentanedione oxidation, a β–diketone initiator commonly used in vinyl polymerization reactions.
This study is based upon work funded by the Spanish Government projects BIO2013-43407-R-DEWRY and BIO2016-79106-R-Lignolution and by the Bio Based Industries Joint Undertaking under the European Union’s Horizon 2020 Research and Innovation programme (grant agreement no. 886567). B.J.G.-F. was supported by FPI national fellowship BES-2014-068887.