Engineering Anaerobic Gut Fungi for Lignocellulose Breakdown

Anaerobic gut fungi are attractive plant-degrading microbes, yet remarkably little genetic or enzymatic sequence information exists for these microbes. Gut fungi are fibrolytic, invasive microbes that secrete an array of cellulases and cellulolytic complexes (fungal cellulosomes) for synergistic hydrolysis of lignocellulose. Though fungal hydrolytic activity has been shown to depend on the identity of the substrate, the regulation mechanisms responsible for coordinating the action of cellulases and cellulosomes within these fungi remain unknown. To enumerate novel biomass-degrading enzymes and characterize their coordinated expression in fungi, we have implemented methods to sustain an anaerobic fungus in batch culture and analyze its transcriptome via RNAseq under several growth conditions. A new species of gut fungus from the Piromyces genus was isolated from the digestive tract of a horse, and its proliferation was monitored via fermentation gas production. 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. Through strand-specific RNAseq and use of the TRINITY assembly platform, we were able to assemble hundreds of novel cellulase genes de novo from >27,000 transcripts without the need for genomic sequence information. The fungal transcriptome is particularly rich in GH6 and GH43 enzymes, and we find that 27 of 54 diverse glycosyl hydrolase families are transcriptionally repressed during growth on glucose relative to reed canary grass. Within the majority of these transcripts, dockerin-tagged elements of fungal cellulosomes are abundant, and 30% of dockerin-containing transcripts are repressed in the presence of glucose. This suggests that catalytic components of fungal cellulosomes are highly regulated in response to simple sugars. We will further discuss the transcriptional regulation patterns observed for important enzyme families under catabolic regulatory conditions, and connect these regulation patterns to protein expression and lignocellulosic degradation.