Engineering Regulation in Anaerobic Fungi During Lignocellulose Breakdown

John Henske, Kevin Solomon, Sarah Maxel, Michelle Oâ??Malley

A significant struggle in the production of biofuels and other value-added products from agricultural waste is the breakdown of complex, lignin-rich material in a cost efficient manner. Our lab has targeted fungi, isolated from the anaerobic microbiome of large herbivores, as a source for a variety of unique enzymes that work together to break down this complex material. My primary interest is to investigate how the fungi regulate these enzymes, and to manipulate these processes to develop gut fungi as novel biofuel production platforms. We have investigated transcriptional regulation through the use of next generation RNAseq to determine how the expression levels of cellulose-degrading enzymes change in an isolated strain of Piromyces fungus. Initial experimental results suggest that the production of biomass-degrading enzymes is regulated through catabolite repression in the presence of simple monosaccharides, which suggests that proteins of unknown function can be identified by mapping regulation patterns in response to substrate. Additional experiments where the fungi are grown in the presence of both crystalline cellulose (filter paper) and monosaccharides show a decrease in utilization of cellulose material when simple sugars are present, which supports the hypothesized regulation mechanism. Surprisingly, this repression of cellulose-degrading behavior exists even in the presence of monosaccharides that do not support fungal growth. In light of these results, we hypothesize that non- specific sugar sensing mechanisms underlie functional reprogramming of cellulase secretion. The aims of my project are to (1) identify and verify receptor-mediated sensing mechanisms through RNAseq and biochemical studies; (2) apply differential expression analysis to identify novel proteins that are regulated by these sensing mechanisms; and (3) determine the flux of carbon through anaerobic fungi to reveal strategic targets for altering metabolism and substrate utilization. These efforts are underway for our first isolated strain (Piromyces) and are being expanded to two additional strains, representing the Neocallimastix and Anaeromyces genera of anaerobic fungi, as additional sources for regulatory machinery. With an understanding of these regulation mechanisms, they may be used to establish a more efficient process for bio-based production from agricultural that tunes enzyme production based on the most abundant nutrient sources available.