Heterologous expression and random mutagenesis of Phlebia radiata laccase

Laccases have many potential areas of commercial use including e.g. pulp bleaching, textile dye decolorisation, bioglueing, detoxification, biofuel cells and biosensors. Deeper understanding of the structure-function interactions would be needed to make better use of laccases in various applications. Our aim is to study and improve the performance, especially the enzyme-substrate interactions, of fungal laccases by means of protein engineering, including also directed evolution methods. Phlebia radiata is a basidiomycetous fungi expressing a typical, non-thermotolerant laccase (PrL) with a pH-optimum in acidic region. The crystal structure of the protein is not known, but a structural model could be built based on known fungal laccase structures. In order to be able to do directed evolution studies, a heterologous expression of PrL in Saccharomyces cerevisiae under an inducible GAL1 promoter was set-up. The cultivation of the recombinant yeast strain was optimised in microtiter plate format, and a robotic screen was also set-up. Targeted random mutagenesis was applied using error-prone PCR. Two random laccase mutant libraries have now been generated and screened for mutants having altered substrate specificity. 15 mutant clones showing changed substrate specificity for the non-phenolic ABTS as compared to the phenolic DMP compound were picked for a replica robotic assay. The characterization of the 5 best mutants is now being carried out.