PNNL

Abstract

Cellulosomes are large, multi-protein complexes that tether plant biomass degrading enzymes together for improved hydrolysis1. These complexes were first described in anaerobic bacteria where species specific dockerin domains mediate assembly of enzymes onto complementary cohesin motifs interspersed within non-catalytic protein scaffolds1. The versatile protein assembly mechanism conferred by the bacterial cohesin-dockerin interaction is now a standard design principle for synthetic protein-scale pathways2,3. 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)4. However, the enzyme components, modular assembly mechanism, and functional role of fungal cellulosomes remain unknown5,6. Here, we describe the comprehensive set of proteins critical to fungal cellulosome assembly, including novel, conserved scaffolding proteins unique to the Neocallimastigomycota. 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. Fungal dockerin and scaffoldin domains have no similarity to their bacterial counterparts, yet several catalytic domains originated via horizontal gene transfer with gut bacteria. Though many catalytic domains are shared with bacteria, the biocatalytic activity of anaerobic fungi is expanded by the inclusion of GH3, GH6, and GH45 enzymes in the enzyme complexes. Collectively, these findings suggest that the fungal cellulosome is an evolutionarily chimeric structure – an independently evolved fungal complex that co-opted useful activities from bacterial neighbors within the gut microbiome.