Deciphering Transcriptional Regulation Patterns for Novel 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 biomass-degrading enzymes. These fibrolytic microbes secrete an array of powerful cellulases and cellulolytic complexes (cellulosomes) for synergistic hydrolysis of plant biomass in herbivores, 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 to discover novel enzymes. To address this hypothesis, we have combined next-generation sequencing and proteomic approaches to examine cellulose-degrading enzyme production in a panel of gut fungi isolated from natural ecosystems. All 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 thousands of novel genes de novo from >27,000 transcripts without the need for genomic sequence information. RNA-Seq also elucidated global regulatory patterns in response to catabolite repression of biomass degradation, and in response to growth on cellulosic substrates of increasing complexity. Through these efforts, we have identified hundreds of transcripts encoding novel enzymes for biomass degradation, and the fungal transcriptome is particularly rich in GH6, GH48, and GH43-containing enzyme domains. As hypothesized, most of these transcripts are strongly repressed by the addition of simple sugars and are clustered within distinct ‘regulons’ of coordinated gene expression. Gene set enrichment analysis confirms the upregulation of these regulons across cellulosic substrates, and complementary proteomic analyses reveals that the composition the fungal cellulosomes is tuned by the presence of different substrates. More importantly, the functional enrichment of these regulons suggests a critical role for the divergent and unannotated transcripts that they contain. A dozen co-regulated transcripts from our screen bear no homology to known enzymes, and likely harbor previously undiscovered glycosyl hydrolase domains from nature, which we are currently investigating through protein crystallography. Collectively, this information will establish the molecular framework for anaerobic fungal hydrolysis, ultimately allowing us to refactor this system in anaerobic fungi and beyond.