P25: Generation of orthogonal cohesin-dockerin pairs for the controlled assembly of cellulosome

Microbial degradation of plant cellulosic materials is an essential step in nature’s carbon cycle.  It represents the first step to convert carbon fixed by autotrophs into sugar forms that can be efficiently used by heterotrophs.  Enzymes that catalyze the degradation of plant cell walls, thus, have attractive potential to be applied as valuable tools to generate biomass-derived feedstocks for biofuel and bioenergy production.  Many anaerobic bacteria and fungi secrete a multi-protein complex, namely cellulosome, for the highly efficient degradation of cellulosic material.  Due to the complexities and heterogeneous nature of cellulosome, a controlled assembly is required in order to study and manipulate its function.  Currently, the controlled assembly of cellulosome relies completely on naturally existing orthogonal cohesin-dockerin pairs, which limits the size of the designer cellulosome to four catalytic subunits.  To address the issue, we have developed a structure-guided, semi-rational protein engineering-based strategy to quickly generate orthogonal cohesin-dockerin pairs.  The application of the devised positive and negative selection system led to the generation of unnatural cohesin-dockerin pairs that are orthogonal to their wild-type cohesin-dockerin pair parent.  Upon the successful expansion of the repertoire of orthogonal cohesin-dockerin pairs, attention will be turned to the study of synergy among cellulosomal enzymes by controlling the composition and architecture of the cellulosome assemblies.  Synthetic cellulosomes with superior cellulosic material degradation activity will also be constructed via directed evolution.