Cloning and Expression of Laccase From Trametes Versicolor in Saccharomyces Cerevisiae Using a Novel Vector System

The long-term goal of this research is to increase efficiency and decrease cost of ethanol fermentation of lignocellulosic feedstocks by combining pre-treatment using laccase enzyme and subsequent fermentation to ethanol through simultaneous saccharification and fermentation paradigms. The first step is to develop a genetically engineered yeast strain capable of degrading lignocellulosic feedstocks to accomplish a consolidated bioprocessing operation. In the study, the Trametes versicolor laccase gene from the vector pBARLAC has been amplified, cloned using the Gateway™ vector system (pENTR DTOPO) and LR Clonase II enzyme mix, and transformed and expressed in an auxotrophic strain of Saccharomyces cerevisiae PJ69-4 diploid, using a small ubiquitin-related modifier (SUMO) vector system with histidine as the selectable marker (pSUMOduo-HIS). Laccase activity from the newly constructed recombinant yeast strain, YT2-2, has been determined in the presence of different substrates.