Title: Engineering cellulose-degrading complexes from anaerobic gut fungi

Charles Haitjema, Kevin Solomon, and Michelle Oâ??Malley

Department of Chemical Engineering, University of California, Santa Barbara
Anaerobic gut fungi are the primary colonizers and decomposers of plant biomass in monogastric herbivores and ruminants. Their ability to hydrolyze complex lignocellulosic materials is of great promise to cellulosic bioenergy development, since enzymatic breakdown of biomass is a stringent bottleneck. It is hypothesized that large cellulolytic complexes, or cellulosomes, mediate cellulolytic activity within gut fungi. While bacterial cellulosomes are well characterized, the molecular architecture of fungal cellulosomes is poorly understood. We characterized the composition and structure of fungal cellulosomes from the gut fungal isolate Piromyces sp. finn. To do so, we developed a practical approach to isolate cellulosome components from fungal cultures under a variety of substrate growth conditions. Following the purification of fungal cellulosomes, we combined tandem mass spectroscopy with strand-specific transcriptomic sequence information to identify the key components of these cellulosomes. To our knowledge, these studies are the first to reveal the entire enzymatic ensemble that comprises fungal cellulosomes. In addition, we used size-exclusion chromatography to reveal complex formation of enzymes within cellulosomes, and we provide evidence that most dockerin-fused enzymes bind to high molecular weight proteins as putative scaffolding molecules, which have long remained elusive for fungal cellulosomes. Finally, we are employing these techniques to study and compare cellulosomes from other unique isolates of gut fungi. This information will be valuable for developing consolidated bioprocessing platforms to convert plant biomass to energy.