Construction of a Yeast Cell-surface Display System and Expression of Trametes sp. Laccase

摘要

Laccases (1.10.3.2, p-diphenol: dioxygen oxidoreductases) is a family of blue copper-containing oxidases that are commonly found in bacteria, fungi and plants. It is able to oxidize and degrade a variety of aromatic compounds and other organic compounds. Due to this ability, laccases can serve environmental bioremediation processes and industrial purposes. Cell-surface display of enzymes is one of the most attractive applications in yeast. It is a effective utilization to construct the whole cell biocatalyst. The cDNA sequence of Trametes sp. C30 LAC3 was optimized and synthesized according to the codon bias of Saccharomyces cerevisiae, because codon optimization has been proved to be effective to maximize production of heterologous proteins in yeast. The genes encoding galactokinase (GAL1) promoter, á-mating factor 1 (MFá1) pre-pro secretion signal, fully codon-optimized LAC3, the 320 amino acids of C terminal of á-agglutinin, alcohol dehydrogenase (ADH1) terminator and kanMX cassette were amplified and cloned into YEplac181 to construct a cell-surface display vector called pGMAAK-lac3 with á-agglutinin as an anchor. Then pGMAAK-lac3 was transformed into S. cerevisiae. The results show LAC3 was immobilized and actively expressed on S. cerevisiae. However, the substrate specifity and activity were obviously changed. The displayed LAC3 lost the activity to phenolic substrate (guaiacol) and its activity to non-phenolic substrate (ABTS) was greatly reduced. To our knowledge, this was the first attempt to construct and express laccase through cell-surface display technology.