271996 Transcriptomic Analysis Reveals Global Regulation of Lignocellulolytic Enzymes within Anaerobic Fungi
Anaerobic gut fungi are among the most active and efficient plant-degrading microbes found in nature. Increased insight into the enzymatic mechanisms responsible for fungal hydrolysis would better inform sustainable chemical production strategies that rely on lignocellulosic depolymerization. Despite the powerful degradation capacity of gut fungi, remarkably little genetic or enzymatic sequence information exists for these microbes. Though fungal hydrolytic activity has been shown to depend on the identity of the substrate, it is unknown how relative cellulase expression levels are coordinated during growth on these substrates. These issues stem from difficulties associated with the isolation and cultivation of gut fungi under controlled conditions, as well as genetic sequencing challenges associated with their A/T-rich genome.
In order to enumerate novel biomass-degrading enzymes and characterize their coordinated expression, we have implemented methods to sustain an anaerobic fungus in batch culture and analyze its transcriptome via RNAseq under a number of growth conditions. A new species of gut fungus was isolated from the digestive tract of a horse, and its proliferation was monitored via a pressure transducer method that coupled growth to the accumulation of fermentation gases. The fungi exhibited high enzymatic reactivity against model cellulosic and lignocellulosic substrates (filter paper, Avicel, reed canary grass), which was repressed in the presence of simple sugars (glucose, fructose). Through strand-specific RNAseq and use of the TRINITY bioinformatic platform, we were able to assemble novel protein coding regions de novo without the need for genetic sequence information. We will discuss the global regulation patterns observed for important cellulolytic enzyme families in the presence of lignocellulose and glucose, and connect these regulation patterns to physical degradation behavior.