Deciphering global regulatory patterns for cellulase discovery in anaerobic fungi

The economical breakdown of plant biomass into simple sugars remains a significant challenge in the production of renewable cellulosic biofuels. Current strategies rely on cellulolytic enzyme cocktails from engineered aerobic fungi that are expensive to produce and inefficient against recalcitrant lignin-rich biomass. Thus, there is a critical need to develop new technologies to break down crude biomass into fermentable sugars for downstream fuel development. Towards this goal, much can be learned by studying how gut fungi depolymerize crude lignocellulose in the digestive tracts of large herbivores. These anaerobic fungi have evolved unique abilities to break down lignocellulosic biomass through invasive growth, and the secretion of powerful enzymes and synergistic mutli-enzyme complexes (cellulosomes). We have isolated a panel of unique gut fungi that thrive on lignocellulosic substrates, and secrete cellulosomal complexes of cellulases, hemicellulases, and cellulose binding domains that we are characterizing by next generation sequencing and proteomics. To determine the critical pathways of biomass hydrolysis within anaerobic fungi, we have used RNA-Seq to elucidate global regulatory patterns in response to catabolic repression of biomass degradation, and in response to growth on cellulosic substrates of increasing complexity. Through these efforts, we have identified more than a hundred transcripts encoding dozens of novel enzymes for biomass degradation. Many of these transcripts are strongly repressed by the addition of simple sugars and are clustered within distinct ‘regulons’ of coordinated gene expression. Gene set enrichment analysis confirms the upregulation of these regulons across cellulosic substrates and reveals how the composition of the fungal cellulosome adapts to various substrates. More importantly, the functional enrichment of these regulons suggests a critical role for the divergent unannotated transcripts that they contain, and points to a dozen interesting candidates for truly novel cellulose-degrading enzymes. Collectively, this information will establish the molecular framework for anaerobic fungal cellulose hydrolysis, ultimately allowing us to refactor this system in anaerobic fungi and beyond.