A parts list for fungal cellulosomes revealed by comparative genomics

Abstract

Cellulosomes are large, multiprotein complexes that tether plant biomass-degrading enzymes together for improved hydrolysis. These complexes were first described in anaerobic bacteria, where species-specific dockerin domains mediate the assembly of enzymes onto cohesin motifs interspersed within protein scaffolds. The versatile protein assembly mechanism conferred by the bacterial cohesin-dockerin interaction is now a standard design principle for synthetic biology. For decades, analogous structures have been reported in anaerobic fungi, which are known to assemble by sequence-divergent non-catalytic dockerin domains (NCDDs). However, the components, modular assembly mechanism and functional role of fungal cellulosomes remain unknown. Here, we describe a comprehensive set of proteins critical to fungal cellulosome assembly, including 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. Genomic analysis coupled with proteomic validation revealed an average of 312 NCDD-containing proteins per fungal strain, which were overwhelmingly carbohydrate active enzymes (CAZymes), with 95 large fungal scaffoldins identified across four genera that bind 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. However, the biocatalytic activity of anaerobic fungal cellulosomes is expanded by the inclusion of GH3, GH6 and GH45 enzymes. These findings suggest that the fungal cellulosome is an evolutionarily chimaeric structure-an independently evolved fungal complex that co-opted useful activities from bacterial neighbours within the gut microbiome.

Proteome specialization of anaerobic fungi during ruminal degradation of recalcitrant plant fiber

“…C1A), Piromyces finnis, and Piromyces sp. E2 4,18,21,29 ] were included in the database. The RUS-refDB was further complemented with 103 metagenome-assembled genomes (MAGs) and 913 metagenome-assembled viral scaffolds (MAVS) recovered from a rumen metagenome we generated previously using a comparable experimental design of rumen-incubated switchgrass 30,31 .…”

“…Many of the corresponding protein-coding genes were also replicated within each fungal genome, demonstrating that individual rumen fungi hold several sets of functionally important genes. Despite a reportedly high degree of horizontal gene transfer (HGT) in the rumen microbiome 4,34,35 , only a few detected proteins mapped to both fungi and prokaryotes, suggesting that the overall sequences of these particular enzymes are evolutionary divergent across these two kingdoms.…”

“…In general, while the bacterial cellulosome signature sequences encompassed a single Type-I dockerin (DOC1), the fungal counterparts frequently occurred as double or triple dockerins domains (here classified as type-II; DOC2). Dockerin domains in tandem repeats are indeed associated with fungal cellulosomes, and it is believed that this construction facilitates the involvement of more binding sites, thus binding potential substrates more efficiently, than single dockerins 4,52 .…”

“…This group of enzymes is categorized further into different classes and families, which include carbohydrate-binding modules (CBMs), carbohydrate esterases (CEs), glycoside hydrolases (GHs), glycosyltransferases (GTs), and polysaccharide lyases (PLs) 3 . Previous studies have mostly been dedicated to CAZymes from rumen bacteria, although it is becoming increasingly clear that fungi and viruses also possess key roles in the carbon turnover within the rumen 4,5 . Over the last decade, targeted efforts to isolation and cultivate novel rumen microorganisms have resulted in a more detailed understanding of the physiology of anaerobic rumen archaea and bacteria and their contribution to the overall function of the rumen ecosystem 6 .…”

“…Recent studies have also shed light on the viral rumen population and although work in this area is still nascent, it suggests that the rumen virome modulates carbon cycling within the rumen ecosystem through cell lysis or re-programming of the metabolism of the host microbiome 5,7,8 . Anaerobic rumen ciliate protozoa and fungi have largely remained recalcitrant to both cultivation and molecular exploration efforts 9 , and although recent cultivation efforts have provided important insight into the lifestyle and enzymatic capacity 4,10 , their quantitative metabolic contributions to the greater rumen ecosystem are still unclear.…”