@misc{10481/64103,
year = {2020},
month = {9},
url = {http://hdl.handle.net/10481/64103},
abstract = {ABSTRACT 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  –diketone initiator commonly used in vinyl polymerization
reactions.
IMPORTANCE The broad variety of biotechnological uses of fungal laccases is beyond
doubt (food, textiles, pulp and paper, pharma, biofuels, cosmetics, and bioremediation),
and protein engineering (in particular, directed evolution) has become
the key driver for adaptation of these enzymes to harsh industrial conditions. Usually,
the first requirement for directed laccase evolution is heterologous expression,
which presents an important hurdle and often a time-consuming process. In this
work, we resurrected a fungal Mesozoic laccase node which showed strikingly high
heterologous expression and pH stability. As a proof of concept that the ancestral
laccase is a suitable blueprint for engineering, we performed a quick directed evolution
campaign geared to the oxidation of the  -diketone 1,3-cyclopentanedione, a
poor laccase substrate that is used in the polymerization of vinyl monomers.},
publisher = {American Society for Microbiology},
keywords = {Ancestral reconstruction},
keywords = {Directed evolution},
keywords = {Laccase},
title = {Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases},
doi = {10.1128/AEM .00778-20.},
author = {Gomez Fernandez, Bernardo J and Risso, Valeria Alejandra and Rueda, Andrés and Sánchez Ruiz, José Manuel and Alcaldea, Miguel},
}