Engineering Thermophillic Cellulases by Domain Shuffling

Second-generation biofuels produced from renewable lignocellulosic feedstocks present an attractive alternative to traditional fossil fuels.  Developing an energy-efficient and cost effective process to deconstruct and convert plant derived cellulose and hemi-cellulose into glucose presents a significant challenge due to the recalcitrant nature of the biomass. 

Following pretreatment, cellulases are used to break down cellulosic fibers but their efficiency is low and commercial cocktails are prohibitively expensive. Furthermore, these cocktails lose their activity at high temperatures and salt concentrations typically employed in next generation pretreatment conditions and carried over into the enzyme conversion process.  We have employed a modular approach to rapidly prototype and engineer highly robust cellulases by fusing thermophillic cellulose-binding modules (CBMs) to robust catalytic domains. 

Here we demonstrate that the addition of CBMs enhances enzymatic activity compared to the catalytic domain alone at high temperatures.  Furthermore, we show that linker characteristics and fusion geometry affect the catalytic rate of these chimeric enzymes when assayed on insoluble crystalline substrate Avicel and on a model energy crop, switchgrass. We discuss some of our initial results and outline a strategy directed towards improving the activity of these chimeric cellulases under high salt conditions.