Consensus Design of an Evolved High-Redox Potential Laccase
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Consensus designHigh-redox potential laccaseAncestor mutationThermostabilityActivity
Gomez-Fernandez BJ, Risso VA, Sanchez-Ruiz JM and Alcalde M (2020) Consensus Design of an Evolved High-Redox Potential Laccase. Front. Bioeng. Biotechnol. 8:354. [doi: 10.3389/fbioe.2020.00354]
SponsorshipThis study is based upon work funded by and the Spanish Government projects BIO2013-43407-R-DEWRY and BIO2016- 79106-R-Lignolution. BG-F was supported by a FPI national fellowship BES-2014-068887.
Among the broad repertory of protein engineering methods that set out to improve stability, consensus design has proved to be a powerful strategy to stabilize enzymes without compromising their catalytic activity. Here, we have applied an in-house consensus method to stabilize a laboratory evolved high-redox potential laccase. Multiple sequence alignments were carried out and computationally refined by applying relative entropy and mutual information thresholds. Through this approach, an ensemble of 20 consensus mutations were identified, 18 of which were consensus/ancestral mutations. The set of consensus variants was produced in Saccharomyces cerevisiae and analyzed individually, while site directed recombination of the best mutations did not produce positive epistasis. The best single variant carried the consensus-ancestral A240G mutation in the neighborhood of the T2/T3 copper cluster, which dramatically improved thermostability, kinetic parameters and secretion.