Directed and computational evolution of fungal laccases: redox potential enhancement and development of a family of thermostable chimeras

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 18-10-2018Esta tesis tiene embargado el acceso al texto completo hasta el 18-04-2020Fungal laccases are multicopper oxidases with a broad substrate specificity that is highly dependent on their redox potential of the T1Cu site (ET1). High-redox potential laccases (HRPLs) secreted by basidiomycete white rot fungi are particularly relevant in a biotechnological context given their capacity to oxidize compounds with a higher redox potential that cannot be transformed by their medium- and low-redox potential counterparts. In the first section of this Thesis, we combined computational design with directed evolution in order to tailor a HRPL variant with improved ET1 and activity towards high-redox potential mediators, as well as enhanced stability. Laccase mutant libraries were screened in vitro using high-redox potential mediators with different chemical nature, while computer-aided evolution experiments were run in parallel to guide bench-top mutagenesis. Through this strategy, the ET1 of the evolved HRPL increased from 740 mV to 790 mV, with a concomitant improvement in thermal and acidic pH stability. The kinetic parameters for high-redox potential mediators were markedly enhanced, which were then successfully tested within laccase mediator systems (LMS). Two hydrophobic substitutions surrounding the T1Cu site appeared to underlie these effects and they were rationalized at the atomic level. Due to its complex structural organization, the generation of chimeric laccases with high sequence diversity from different orthologs is difficult to achieve without compromising protein functionality. In the second section of this Thesis, using SCHEMA-RASPP structure-guided recombination in vivo, we obtained a diverse family of functional chimeras showing increased thermostability from three fungal laccase orthologs with 70 % protein sequence identity and varied redox potential. Assisted by the high frequency of homologous DNA recombination in Saccharomyces cerevisiae, computational SCHEMA blocks were spliced and cloned in a one-pot transformation. As a result of this in vivo assembly, an enriched library of laccase chimeras was rapidly generated and it was screened at high temperature. The collection of chimeras showed considerable sequence diversity, on average varying from their closest parent homolog in 46 amino acids. The most thermostable variant increased its half-life of thermal inactivation at 70°C 5-fold (up to 108 min), whereas several chimeras also displayed improved stability at acid p