Ancestral Sequence Reconstruction to Unveil Laccase Evolution in Basidiomycete Fungi and to Provide Ancestral Enzymes as Biocatalysts
Laccases are multicopper oxidases (MCOs) that play a crucial role in the degradation of lignin and carbon recycling carried out by basidiomycete fungi in nature, and have the highest number of applications reported to date among oxidoreductases. However, very little is known about their natural evol...
| Autores: | , , , , , , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2026 |
| País: | España |
| Institución: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repositorio: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:dnet:digitalcsic_::0f89fe9f959233ce83a15868af5c9c74 |
| Acceso en línea: | http://hdl.handle.net/10261/431734 |
| Access Level: | acceso abierto |
| Palabra clave: | Ancestral multicopper oxidases Ancestral sequence reconstruction Fungi Laccase Llignin Styrene |
| Sumario: | Laccases are multicopper oxidases (MCOs) that play a crucial role in the degradation of lignin and carbon recycling carried out by basidiomycete fungi in nature, and have the highest number of applications reported to date among oxidoreductases. However, very little is known about their natural evolution, the study of which could reveal unknown structure-function relationships and guide laccase engineering. By using ancestral sequence reconstruction and structural and biochemical characterization of the resurrected ancestral enzymes, we disclosed the key evolutionary events that modified the architecture of the active site in MCOs, leading to the emergence of laccases for lignin degradation in nature. We analyzed the evolutionary trajectory of basidiomycete laccases by obtaining the ancestors of four MCO groups, including laccases sensu stricto and three types of laccase-like enzymes that we recently described. The ancestor of each group was expressed in S. cerevisiae and preliminary characterized, showing noticeable differences among them in activity, stability, and architecture of their catalytic pockets. The ancestral laccase sensu stricto outperformed the other ancestral enzymes in the activity levels secreted by the yeast, stability, and capability to oxidize all tested substrates. It was therefore expressed in Aspergillus oryzae at high yield and characterized in depth, including the resolution of its crystal structure at 1.54 Å. The enzyme showed a wide substrate promiscuity, oxidizing a variety of phenols, aryl amines, and N-heterocycles, including certain synthetic organic dyes and redox mediators with high oxidation potentials. The ancestral laccase also oxidized polymeric lignin, in contrast to the other ancestral enzymes, and it was able to oxidize the monomer of polystyrene via the laccase-mediator system. All these properties make this resurrected enzyme a promising starting point for engineering to address urgent biotechnological challenges such as the enzymatic valorization and upcycling of natural and synthetic polymers |
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