Recalcitrant biomass is an attractive source of renewable energy because it avoids many of the economic and environmental problems that plague traditional energy sources. In order to make this resource viable, more efficient methods are required to break down the polymer into simple sugars, which can then be fermented into high density fuels or other value-added chemicals. Anaerobic fungi are among the most efficient at degrading biomass and do so primarily through large protein complexes, called cellulosomes. This system is similar to the cellulosome found in some anaerobic bacteria, where the interactions are mediated by two domains termed the dockerin and cohesin. In fungal cellulosomes, the dockerin domain has previously been characterized and expressed, but the cohesin domain has remained elusive. In this study, the interaction of recombinant dockerin with native fungal cellulosome was quantified through an ELISA. Putative cohesins were identified using the recombinant dockerin through a “bait and prey” assay. These cohesins were expressed recombinantly and assayed for interaction with recombinant dockerin. Additionally, fusion proteins were created through the recombinant addition of dockerin domains onto exogenous proteins, with the aim of imparting novel function to native cellulosomes. These novel functions include thermostability, a wider range of substrates, and faster metabolism of released sugars, and came from several source organisms like <i>Thermotoga maritima </i>or <i>Pyrococcus furiosus.</i> Kinetic parameters were compared for these proteins with and without the fusion domains to ensure activity was retained. These proteins were then assayed for interaction with native cellulosome. Finally, several perturbation studies were performed to determine conditions required to break apart the cellulosome with pH, Temperature, SDS concentration, and reducing agent. Future goals include determining the specific domain on the cohesins required for interaction with the dockerin, and then using these domains to mediate the formation of entirely synthetic complexes.

Engineering synthetic fungal cellulose-degrading complexes

249th National Meeting (2015)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.