(446d) Biochemical Insight Into Fungal Cellulosome Architecture and Regulation

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 cellulolytic activity within gut fungi is mediated by large cellulolytic complexes called fungal cellulosomes. While bacterial cellulosomes are well-characterized, the molecular architecture of fungal cellulosomes is quite poorly understood. We characterized the composition and structure of fungal cellulosomes from the gut fungal isolate Piromyces sp. finn. A practical approach was developed 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 comprise fungal cellulosomes. In addition, we will 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. We will connect these results to the regulation patterns observed for fungal cellulosomes in the presence of cellulosic and non-cellulosic substrates.