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.

Hydrogenosomes are organelles that produce ATP and hydrogen, and are found in various unrelated eukaryotes, such as anaerobic flagellates, chytridiomycete fungi and ciliates. Although all of these organelles generate hydrogen, the hydrogenosomes from these organisms are structurally and metabolically quite different, just like mitochondria where large differences also exist. These differences have led to a continuing debate about the evolutionary origin of hydrogenosomes. Here we show that the hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis, which thrives in the hindgut of cockroaches, have retained a rudimentary genome encoding components of a mitochondrial electron transport chain. Phylogenetic analyses reveal that those proteins cluster with their homologues from aerobic ciliates. In addition, several nucleus-encoded components of the mitochondrial proteome, such as pyruvate dehydrogenase and complex II, were identified. The N. ovalis hydrogenosome is sensitive to inhibitors of mitochondrial complex I and produces succinate as a major metabolic end product--biochemical traits typical of anaerobic mitochondria. The production of hydrogen, together with the presence of a genome encoding respiratory chain components, and biochemical features characteristic of anaerobic mitochondria, identify the N. ovalis organelle as a missing link between mitochondria and hydrogenosomes.

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SummaryAnaerobic chytridiomycete fungi possess hydrogenosomes, which generate hydrogen and ATP, but also acetate and formate as end-products of a prokaryotic-type mixed-acid fermentation. Notably, the anaerobic chytrids Piromyces and Neocallimastix use pyruvate:formate lyase (PFL) for the catabolism of pyruvate, which is in marked contrast to the hydrogenosomal metabolism of the anaerobic parabasalian flagellates Trichomonas vaginalis and Tritrichomonas foetus , because these organisms decarboxylate pyruvate with the aid of pyruvate:ferredoxin oxidoreductase (PFO). Here, we show that the chytrids Piromyces sp. E2 and Neocallimastix sp. L2 also possess an alcohol dehydrogenase E (ADHE) that makes them unique among hydrogenosomebearing anaerobes. We demonstrate that Piromyces sp. E2 routes the final steps of its carbohydrate catabolism via PFL and ADHE: in axenic culture under standard conditions and in the presence of 0.3% fructose, 35% of the carbohydrates were degraded in the cytosol to the end-products ethanol, formate, lactate and succinate, whereas 65% were degraded via the hydrogenosomes to acetate and formate. These observations require a refinement of the previously published metabolic schemes. In particular, the importance of the hydrogenase in this type of hydrogenosome has to be revisited.

(Vogels et al., 1980;Yarlett et al., 1981;1986;van Bruggen et al., 1983;Zwart et al., 1988;Broers et al., 1990;Gijzen et al., 1991;Marvin-Sikkema et al., 1992;1993a; reviewed by Müller, 1993;Fenchel and Finlay, 1995;Hackstein et al., 1999;2001;Roger, 1999). Hydrogenosomes are membranebound organelles that compartmentalize terminal reactions of the eukaryotic energy metabolism. However, unlike mitochondria, which fulfil this function in aerobic eukaryotes, hydrogenosomes are found exclusively in unicellular anaerobes. Hydrogenosomes generate hydrogen, acetate (or acetate and formate respectively) and carbon dioxide because they can use protons as an electron acceptor (Müller, 1993;1998). Despite the obvious differences from the mitochondrial metabolism and despite their occurrence in only distantly related taxa of anaerobic protists, a wealth of (circumstantial) evidence argues for a common ancestry of mitochondria and hydrogenosomes (Embley et al., 1997;Martin and Müller, 1998;Plümper et al., 1998;Andersson and Kurland, 1999;Hackstein et al., 1999;Dyall and Johnson, 2000; Molecular Microbiology (2002)