ICP Instituto de Catalisis y Petroquímica

While the Pycnoporus cinnabarinus Laccase (PcL) is one of the most promising high redox potential enzymes for environmental biocatalysis, its practical use has to date remained limited due to the lack of directed evolution platforms with which to improve its features. Here, we describe the construction of a PcL fusion gene and the optimization of conditions to induce its functional expression inSaccharomyces cerevisiae, facilitating its directed evolution and semi-rational engineering. The native PcL signal peptide was replaced by the α-factor prepro-leader and this construct was subjected to six rounds of evolution coupled to a multi-screening assay based on the oxidation of natural and synthetic redox-mediators at more neutral pHs. The laccase total activity was enhanced 8000-fold: the evolved α-factor prepro-leader improved secretion levels 40-fold and several mutations in mature laccase provided a 13.7-fold increase in k_cat. Whilst the pH activity profile was shifted to more neutral values, the thermostability and the broad substrate specificity of PcL were retained. Evolved variants were highly secreted by Aspergillus niger (∼23 mg/L) which addresses the potential use of this combined-expression system for protein engineering. The mapping of mutations onto the PcL crystal structure shed new light on the oxidation of phenolic and non-phenolic substrates. Furthermore, some mutations arising in the evolved prepro-leader highlighted its potential for heterologous expression of fungal laccases in yeast.