Designing chimeric enzymes inspired by fungal cellulosomes.

Cellulosomes are synthesized by anaerobic bacteria and fungi to degrade lignocellulose via synergistic action of multiple enzymes fused to a protein scaffold. Through templating key protein domains (cohesin and dockerin), designer cellulosomes have been engineered from bacterial motifs to alter the activity, stability, and degradation efficiency of enzyme complexes. Recently a parts list for fungal cellulosomes from the anaerobic fungi (Neocallimastigomycota) was determined, which revealed sequence divergent fungal cohesin, dockerin, and scaffoldin domains that could be used to expand the available toolbox to synthesize designer cellulosomes. In this work, multi-domain carbohydrate active enzymes (CAZymes) from 3 cellulosome-producing fungi were analyzed to inform the design of chimeric proteins for synthetic cellulosomes inspired by anaerobic fungi. In particular, Piromyces finnis was used as a structural template for chimeric carbohydrate active enzymes. Recombinant enzymes with retained properties were engineered by combining thermophilic glycosyl hydrolase domains from Thermotoga maritima with dockerin domains from Piromyces finnis. By preserving the protein domain order from P. finnis, chimeric enzymes retained catalytic activity at temperatures over 80 °C and were able to associate with cellulosomes purified from anaerobic fungi. Fungal cellulosomes harbor a wide diversity of glycoside hydrolases, each representing templates for the design of chimeric enzymes. By conserving dockerin domain position within the primary structure of each protein, the activity of both the catalytic domain and dockerin domain was retained in enzyme chimeras. Taken further, the domain positioning inferred from native fungal cellulosome proteins can be used to engineer multi-domain proteins with non-native favorable properties, such as thermostability.

A parts list for fungal cellulosomes revealed by comparative genomics.

Cellulosomes are large, multiprotein 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 the assembly of enzymes onto cohesin motifs interspersed within protein scaffolds1. The versatile protein assembly mechanism conferred by the bacterial cohesin-dockerin interaction is now a standard design principle for synthetic biology2,3. For decades, analogous structures have been reported in anaerobic fungi, which are known to assemble by sequence-divergent non-catalytic dockerin domains (NCDDs)4. However, the components, modular assembly mechanism and functional role of fungal cellulosomes remain unknown5,6. 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.