Comprehensive characterization of cellulosomes from Anaerobic Gut Fungi

Conversion of plant biomass is key for a future biological-based economy, especially for the production of biofuels and value-added products. To develop new technologies there is a great need to identify novel organisms, enzymes and molecules with features that can be exploited to accelerate breakdown and conversion. For example, anaerobic gut fungi (AGF) are part of the microbiome of large herbivores, and despite their low numbers they play a key role in the degradation of ingested plant biomass using both physical and enzymatic mechanisms. Their enzymatic system contains both free enzymes and multi-enzyme complexes called “fungal cellulosomes”, which are powerful biomass-degrading machines that tether together all the required enzymes for synergistic hydrolysis of lignocellulose. AGF are the only eukaryotes known to harbor cellulosomes, which are drastically different from their bacterial counterparts. Currently, the composition of fungal cellulosomes, mechanism of cell attachment, and remodeling mechanisms remain unclear.

In order to address these questions, we combined genome, transcriptome and proteome analysis of three novel AGF strains. Genome and transcriptome analysis reveals that AGF have the largest number of CAZymes among fungi that have been sequenced to date. In addition, 134 putative scaffoldin proteins and hundreds of dockerin domain proteins (DDPs) were identified through HMM modeling. Surprisingly, only part of the scaffoldin proteins have transmembrane helices, and only one third of carbohydrate active enzymes (CAZymes) are DDPs. In addition, about half of the DDPs are not classified as CAZymes, which we hypothesize to have a role in cellulosome remodeling. Several proteomic techniques confirm that the composition and cell attachment of the cellulosomes is strain, growth phase, and substrate dependent. Our results suggest that gut fungi are excellent degraders of lignocellulosic biomass due to their arsenal of versatile mechanisms including free enzymes and cellulosomes that can be used under different environmental cues.