Exploiting Anaerobic Gut Fungi for Lignocellulose Breakdown and Enzyme Discovery

Anaerobic gut fungi are attractive microbes to engineer for bio-based chemical production from lignocellulose, and are an untapped reservoir for the discovery of new cellulose-degrading enzymes. These fibrolytic microbes secrete an array of powerful cellulases and cellulolytic complexes (fungal cellulosomes) for synergistic hydrolysis of plant biomass, yet little sequence information exists for gut fungi. Though fungal hydrolytic activity has been shown to be substrate dependent, the underlying regulation mechanisms that coordinate the action of cellulases and cellulosomes from gut fungi remain unknown. We hypothesize that cellulose-degrading machinery is catabolite-repressed to conserve cellular energy, and our objective is to exploit this regulation mechanism for novel enzyme discovery. To address this hypothesis, we have combined next-generation sequencing and proteomic approaches to examine cellulose-degrading enzyme production in gut fungi while supported on several growth substrates. 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-containing enzymes, and we find that 27 of 54 diverse glycosyl hydrolase families are transcriptionally repressed during growth on glucose relative to reed canary grass (lignocellulose). Within the majority of these transcripts, dockerin-tagged elements of fungal cellulosomes are abundant, and 15% 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, which is supported by recently obtained proteomic data. By deciphering catabolic regulation patterns, we have identified several putative cellulose-active enzymes that are coordinately regulated with known cellulases through metatranscriptomic cluster analysis. These transcripts bear no homology to known glycosyl hydrolases, and are potentially completely divergent novel enzymes. We will further discuss the transcriptional regulation patterns observed for other important enzyme families under catabolic regulatory conditions, and connect these regulation patterns to protein secretion and lignocellulose degradation across gut fungal genera.