Minor, C. M.; Takayesu, A.; Arbing, M.; Ha, S.-m.; Gunsalus, R. P.; Pellegrini, M.; Sawaya, M. R.; Clubb, R. T.

Cellulosomes are large, surface-displayed enzyme complexes that enable anaerobic bacteria to degrade recalcitrant plant polysaccharides, yet cellulosome-expressing bacteria are thought to be rare in the human gut. Here we show that extensive sequence divergence obscures the detection of many ruminococcal cellulosomes by conventional sequence homology-based methods. Using proteome-scale AlphaFold2 structural predictions, we uncovered a substantially expanded set of cellulosome-producing Ruminococcus species, including six previously unrecognized human symbionts. Structure-based clustering identifies several novel cohesin families that retain conserved folds despite extreme sequence divergence and define distinct, phylogenetically conserved cellulosome architectures. The analysis reveals R. callidus and related human symbionts encode elaborate cellulosomes that are invisible to sequence-based annotation. Similarly, R. difficilis, a human gut symbiont, is found to produce an atypical cohesin-based assembly enriched in amylases and related starch-binding proteins that may enable this microbe to degrade resistant starches that evade digestion in the upper gastrointestinal tract. Together, these findings reveal that ruminococcal cellulosomes are far more prevalent and diverse than previously appreciated and demonstrate the power of structural proteomics to uncover deeply divergent functional systems in the gut microbiome.