(198b) A Microscale Platform for Integrated Cell-Free Expression and Activity Screening of Cellulases

Cellulases play a key role in the bioconversion of lignocellulosic material to ethanol by hydrolyzing the cellulose polymers in biomass to fermentable sugars. However, the high cost of cellulases limits their utility in commercial biorefineries. Several research efforts have therefore been dedicated to the identification and generation of cost-effective, improved cellulases for the efficient hydrolysis of cellulose. The success of these efforts relies on the development of high-throughput integrated analytical approaches for rapid and efficient expression and screening of cellulases. To address this need, we have developed a miniaturized, fluorescence-based platform that integrates cell-free expression and activity profiling of cellulases. Our approach achieves rapid cellulase expression and screening (within hours) in microwell arrays with volumes down to 2-3 microliters. In addition, we demonstrate the ability of the platform to screen cellulases in microscale volumes at elevated temperatures (up to 95°C) with minimal evaporation. In this study, we utilize our platform to express and screen a panel of beta-glucosidases and cellobiohydrolases isolated from thermophilic microorganisms. Our approach is well suited for a first pass screening of large libraries to identify cellulases with desired properties which can subsequently be produced on a large scale for detailed structural and functional characterization.