Ancestral sequence reconstruction and resurrection provides useful information protein engineering, yet its alliance with directed evolution has been explored. In this study, we have resurrected several ancestral nodes of fungal dating back 500 to 250 million years. Unlike modern laccases, the resurrected laccases were readily secreted by yeast, with similar kinetic parameters, broader stability, and distinct pH activity profiles. The resurrected Agaricomycetes carried 136 ancestral mutations, a molecular testimony to its origin, it was subjected to directed evolution in order to improve the rate of 1,3- oxidation, a –diketone initiator commonly used in vinyl polymerization . The broad variety of biotechnological uses of fungal laccases is beyond (food, textiles, pulp and paper, pharma, biofuels, cosmetics, and bioremediation), protein engineering (in particular, directed evolution) has become key driver for adaptation of these enzymes to harsh industrial conditions. Usually, first requirement for directed laccase evolution is heterologous expression, presents an important hurdle and often a time-consuming process. In this , we resurrected a fungal Mesozoic laccase node which showed strikingly high expression and pH stability. As a proof of concept that the ancestral is a suitable blueprint for engineering, we performed a quick directed evolution geared to the oxidation of the -diketone 1,3-cyclopentanedione, a laccase substrate that is used in the polymerization of vinyl monomers.
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Agans et al. (2018) studied this question.