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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 moder...

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Autores principales: Gomez-Fernandez, Bernardo J., Risso, Valeria A., Rueda, Andres, Sanchez-Ruiz, Jose M., Alcalde, Miguel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357490/
https://www.ncbi.nlm.nih.gov/pubmed/32414792
http://dx.doi.org/10.1128/AEM.00778-20
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author Gomez-Fernandez, Bernardo J.
Risso, Valeria A.
Rueda, Andres
Sanchez-Ruiz, Jose M.
Alcalde, Miguel
author_facet Gomez-Fernandez, Bernardo J.
Risso, Valeria A.
Rueda, Andres
Sanchez-Ruiz, Jose M.
Alcalde, Miguel
author_sort Gomez-Fernandez, Bernardo J.
collection PubMed
description 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. 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 β-diketone 1,3-cyclopentanedione, a poor laccase substrate that is used in the polymerization of vinyl monomers.
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spelling pubmed-73574902020-07-31 Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases Gomez-Fernandez, Bernardo J. Risso, Valeria A. Rueda, Andres Sanchez-Ruiz, Jose M. Alcalde, Miguel Appl Environ Microbiol Biotechnology 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. 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 β-diketone 1,3-cyclopentanedione, a poor laccase substrate that is used in the polymerization of vinyl monomers. American Society for Microbiology 2020-07-02 /pmc/articles/PMC7357490/ /pubmed/32414792 http://dx.doi.org/10.1128/AEM.00778-20 Text en Copyright © 2020 Gomez-Fernandez et al. https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Biotechnology
Gomez-Fernandez, Bernardo J.
Risso, Valeria A.
Rueda, Andres
Sanchez-Ruiz, Jose M.
Alcalde, Miguel
Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases
title Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases
title_full Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases
title_fullStr Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases
title_full_unstemmed Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases
title_short Ancestral Resurrection and Directed Evolution of Fungal Mesozoic Laccases
title_sort ancestral resurrection and directed evolution of fungal mesozoic laccases
topic Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7357490/
https://www.ncbi.nlm.nih.gov/pubmed/32414792
http://dx.doi.org/10.1128/AEM.00778-20
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