Discovering optimally tailored enzyme cocktails using a synthetic screening tool

Novozymes has been delivering cutting edge technologies that enable cost reductions across diverse fuel production platforms from biomass.  We continue to develop robust, versatile enzyme cocktails in a variety of processes on various biomass substrates.  Since multiple classes of cellulases and hemicellulases are required for optimal conversion of biomass to sugars, it is a challenge to discover an enzyme cocktail where components have been selected primarily for optimal synergy.  Comparison of protein candidates requires pure protein samples as well as consistent and reliable protein quantification, which can be labor-intensive. 

 Here we designed a synthetic screening tool using cell-free mini-cellulosome to enable rapid assessment of unexplored natural diversity in a multi-combinatorial fashion.   This technology platform allows us to screen for enzyme synergy between different candidates of multiple classes of enzymes and streamline purification procedure. We have engineered a highly thermostable scaffoldin comprised of distinct cohesions that stably bind cognate dockerins with high specificity.  These protein-protein interactions serve as respective binding modules for covalently-linked libraries of different enzyme classes.  Saturation of protein binding sites on the scaffoldin is followed by recovery in a simple pull-down method permitting equalization of loading from sample to sample in our libraries of enzyme candidates.  Our synthetic screening platform has been adapted to an automated high throughput system where libraries of hemicellulases from several different enzyme classes are rapidly screened to assess their ability to synergistically improve hydrolysis of AFEX-pretreated corn stover.  Improved hemicellulases that work synergistically are discovered using the cell-free mini-cellulosome high throughput screening method.