(792d) Correlating Biomass Degradation With the Dynamic Composition of Fungal Cellulosomes

Anaerobic gut fungi thrive in the digestive tract of large herbivores, where they have evolved to break down plant biomass through filamentous growth and the secretion of powerful enzymes. In particular, gut fungi secrete complexes of tethered cellulases, hemicellulases, and carbohydrate binding modules to facilitate synergistic biomass hydrolysis. These scaffolded complexes, referred to as fungal cellulosomes, differ considerably from their bacterial counterparts as they display a unique array of glycosyl hydrolases. In addition, catalytic enzymes are fused to tandem repeat fungal dockerin domains, which are speculated to mediate assembly of the large multi-protein complex. However, the mechanisms that govern the structure, enzymatic reactivity, and adaptability of fungal cellulosomes remain elusive.

We hypothesize that fungal cellulosomes are regulated at the post-transcriptional and post-translational level, leading to dynamic, tunable cellulosomes that optimize biomass breakdown. In order to correlate expression and cellulosome architecture with substrate availability, we have isolated a panel of novel gut fungi from the Piromyces, Neocallimastix, and Anaeromyces genera. Proliferation of the fungal isolates was monitored via fermentation gas production, and cellulosomes from each species were isolated through cellulose-precipitation. All fungi exhibited high enzymatic reactivity against a range of cellulosic and lignocellulosic substrates (filter paper, Avicel, reed canary grass), which was repressed in the presence of simple sugars. Within isolated cellulosomes, striking similarities are observed for certain dockerin-fused glycosyl hydrolases, and these proteins are not secreted from fungi when simple sugars are present. This suggests that the secretion of cellulosomal enzymes is highly regulated in response to simple sugars. We will further link this behavior to the transcriptional regulation patterns observed for important enzyme families under catabolic regulatory conditions via RNAseq. In addition, we will discuss a new model for fungal cellulosome assembly and evolution during lignocellulose degradation.