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Open AccessArticle

Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae

Microbial & Plant Biotechnology Department, Centro de Investigaciones Biológicas Margarita Salas (CIB), CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain
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Int. J. Mol. Sci. 2021, 22(3), 1157; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22031157
Received: 22 December 2020 / Revised: 18 January 2021 / Accepted: 21 January 2021 / Published: 25 January 2021
(This article belongs to the Special Issue OxiZymes)
Laccases secreted by saprotrophic basidiomycete fungi are versatile biocatalysts able to oxidize a wide range of aromatic compounds using oxygen as the sole requirement. Saccharomyces cerevisiae is a preferred host for engineering fungal laccases. To assist the difficult secretion of active enzymes by yeast, the native signal peptide is usually replaced by the preproleader of S. cerevisiae alfa mating factor (MFα1). However, in most cases, only basal enzyme levels are obtained. During directed evolution in S. cerevisiae of laccases fused to the α-factor preproleader, we demonstrated that mutations accumulated in the signal peptide notably raised enzyme secretion. Here we describe different protein engineering approaches carried out to enhance the laccase activity detected in the liquid extracts of S. cerevisiae cultures. We demonstrate the improved secretion of native and engineered laccases by using the fittest mutated α-factor preproleader obtained through successive laccase evolution campaigns in our lab. Special attention is also paid to the role of protein N-glycosylation in laccase production and properties, and to the introduction of conserved amino acids through consensus design enabling the expression of certain laccases otherwise not produced by the yeast. Finally, we revise the contribution of mutations accumulated in laccase coding sequence (CDS) during previous directed evolution campaigns that facilitate enzyme production. View Full-Text
Keywords: laccase; heterologous production; S. cerevisiae; directed evolution; signal peptide; N-glycosylation; consensus design; synonymous mutations laccase; heterologous production; S. cerevisiae; directed evolution; signal peptide; N-glycosylation; consensus design; synonymous mutations
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MDPI and ACS Style

Aza, P.; de Salas, F.; Molpeceres, G.; Rodríguez-Escribano, D.; de la Fuente, I.; Camarero, S. Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae. Int. J. Mol. Sci. 2021, 22, 1157. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22031157

AMA Style

Aza P, de Salas F, Molpeceres G, Rodríguez-Escribano D, de la Fuente I, Camarero S. Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae. International Journal of Molecular Sciences. 2021; 22(3):1157. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22031157

Chicago/Turabian Style

Aza, Pablo; de Salas, Felipe; Molpeceres, Gonzalo; Rodríguez-Escribano, David; de la Fuente, Iñigo; Camarero, Susana. 2021. "Protein Engineering Approaches to Enhance Fungal Laccase Production in S. cerevisiae" Int. J. Mol. Sci. 22, no. 3: 1157. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22031157

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