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Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases

Gomez-Fernandez, Bernardo J, Risso, Valeria A, Rueda, Andres, Sanchez-Ruiz, Jose M et Alcalde, Miguel (2020). Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases Applied and Environmental Microbiology , vol. 86 , nº 14. p. 1-15. DOI: 10.1128/AEM.00778-20.

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Résumé

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 approximately 500-250 million years. Unlike modern laccases, the resurrected Mesozoic laccases were readily secreted by yeast, with similar kinetic parameters, a broader stability and distinct pH profiles of activity. 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 beta-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), as well as the fact that protein engineering (in particular, directed evolution) has become the key driver in their adaptation to harsh industrial conditions. Usually, the first requirement for directed laccase evolution, heterologous expression, is an important hurdle that led to a time-consuming process. In this work we resurrected a fungal Mesozoic laccase node which showed a striking 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 beta-diketone 1,3-cyclopentanedione, a poor laccase substrate that is used in the polymerization of vinyl monomers.

Type de document: Article
Informations complémentaires: document e00778-20
Mots-clés libres: ancestral reconstruction; directed evolution; laccase
Centre: Centre INRS-Institut Armand Frappier
Date de dépôt: 20 juill. 2021 03:55
Dernière modification: 20 juill. 2021 03:55
URI: https://espace.inrs.ca/id/eprint/11593

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