S32: Development of Recombinant Biocatalysts Expressing Laccase Enzyme from Trametes versicolor.

Increasing demands for sustainable energy necessitate the use of biorenewable sources such as agricultural and forestry wastes.  A major challenge of using lignocellulosic biomass for biofuel production is the recalcitrant nature of the lignin structure.    The long-term goal of our research is to develop a high-performance biocatalyst in cost-effective bioenergy production from lignocellulosic feedstocks by combining pre-treatment with laccase and subsequent bioconversion through simultaneous saccharification and fermentation (SSF). Laccase is a multi-copper oxidase that catalyzes the reduction of molecular oxygen to water while oxidizing phenolic compounds such as lignin. The specific research objective is to genetically engineer a yeast strain expressing laccase enzyme that is essential for degrading lignocellulosic feedstocks to accomplish a consolidated bioprocessing operation.  In this study, we attempt to develop recombinant biocatalysts using Saccharomyces cerevisiae PJ69-4, an auxotrophic yeast strain with mutations in the uracil, histidine, tryptophan, and leucine genes.  Two Trametes versicolor laccase gene fragments were amplified creating a full length laccase protein with the native signal DNA sequence (1563 bp) and a truncated fragment (1511 bp) without the signal sequence, cloned in the Gateway™ vector system (pENTR D TOPO) using LR Clonase II mixture. These systems were subsequently transformed and expressed in S. cerevisiae PJ69-4 using a small ubiquitin-related modifier vector system with uracil as the selectable marker (pSUMO-URA) and a high expression galactose inducible vector system with uracil as the selectable marker (pYES2DEST52).  The levels of laccase activities expressed from the recombinant yeast strains are being compared to evaluate their role as biocatalysts.