Recalcitrant biomass is an attractive source of renewable energy; however, more efficient methods are required to break down the raw feedstock into fermentable sugars. Anaerobic fungi are among the most efficient organisms at degrading biomass and do so primarily through large protein complexes, called cellulosomes. These complexes were first described in anaerobic bacteria where species specific dockerin domains mediate assembly of enzymes onto complementary cohesin motifs of non-catalytic scaffolds. For decades, analogous structures have been reported in the early branching anaerobic fungi, which are known to assemble by sequence divergent non-catalytic dockerin domains (NCDD). However, the enzyme components, modular assembly mechanism, and functional role of fungal cellulosomes remain unknown. Leveraging the resources and expertise of the Joint Genome Institute (JGI) and Environmental Molecular Sciences Laboratory (EMSL), we conducted a multi-OMICs study to elucidate the parts of fungal cellulosomes. High quality genomes of the anaerobic fungi Anaeromyces robustus, Neocallimastix californiae and Piromyces finnis were assembled with long-read, single molecule technology to overcome their repeat-richness and extremely low GC content. Genomic analysis coupled with proteomic validation revealed an average 320 NCDD-containing proteins per fungal strain that were overwhelmingly carbohydrate active enzymes (CAZymes), with 95 large fungal scaffoldins identified across 4 genera that contain a conserved amino acid sequence repeat that binds to NCDDs. The newly discovered scaffoldins were validated biochemically using Enzyme Linked Immunosorbent Assays (ELISA) and Surface Plasmon Resonance (SPR). Fungal dockerin and scaffoldin domains have no similarity to their bacterial counterparts, yet several catalytic domains originated via horizontal gene transfer with gut bacteria. Collectively, these findings suggest that the fungal cellulosome is an evolutionarily chimeric structure – a fungal complex that co-opted useful activities from bacterial neighbors within the gut microbiome.

Parts list for fungal cellulosomes revealed by proteomics and comparative genomics

253rd National Meeting (2017)

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