(378c) Discovery and Regulation of Biomass-Degrading Enzymes From Anaerobic Gut Fungi Using Next-Generation Sequencing

Lignocellulosic biomass is a renewable resource capable of supplying one third of the domestic liquid fuel demand.  However, processes to extract fermentable sugars from this biomass involve expensive pretreatment and enzymatic digestion steps that rely on a limited number of characterized cellulases.  Therefore, there is a critical need to identify new technologies for the economic conversion of crude biomass into sugars.  Anaerobic fungi within the digestive tracts of large herbivores are powerful biomass degraders yet they remain a rich untapped source for new lignocellulolytic enzymes.  Despite their lignocellulolytic behavior, the enzymes responsible are largely unknown due to limitations associated with the cultivation and molecular characterization of gut fungi.  In this study, we sought to identify these enzymes through RNAseq, and capture their expression profiles to understand the hydrolytic behavior of gut fungi against lignocellulosic substrates.

We have isolated a novel gut fungus from the Piromyces genus, which we have employed as a model organism to study biomass degradation.  The isolate exhibits strong cellulolytic activity against diverse cellulosic and lignocellulosic substrates, which is repressed in the presence of simple sugars (e.g. glucose).  Using next generation strand specific RNAseq and the TRINITY bioinformatic platform, we have assembled the first reported de novo gut fungal transcriptome.  Analysis of the transcriptome reveals transcripts encoding over a hundred novel lignocelluloytic enzymes with diverse catalytic activities, which are either freely secreted or associated with fungal cellulosome complexes.  Furthermore, we have confirmed the biological presence of enzymes encoded by assembled transcripts, supporting the quality of the de novo assembly.  Also embedded within the transcriptome are quantitative transcript abundance information and non-coding RNA such as antisense RNA, which we hypothesize to directly regulate the expression of these cellulases.  In this talk, I will describe the subset of novel enzymes that we have identified, and discuss the global regulatory patterns observed under various growth conditions.