It is shown that cellulosomal principles can operate in highly reduced cohesin‐dockerin systems, and two additional candidate interacting partners for ClcC appear to bind via a non‐canonical interface.
Abstract
Cellulosomes are efficient enzymatic nanomachines which have arisen for the degradation of cellulosic biomass. They are found abundantly in soil‐dwelling microbes and bacteria which thrive in the stomachs of ruminant mammals. Two protein domains, cohesins and dockerins, characterise cellulosomes. These domains interact with each other to form, in many cases, enormous complexes with as many as 160 individual proteins. However, genome annotation of Cellulosilyticum lentocellum DSM 5427 revealed a single cohesin domain (encoded by Clole_2599) and a single dockerin domain (Clole_2598). Therefore, we recombinantly expressed ClcC and ClcD and found they form a (predicted ~ 104 kDa) heterodimeric complex. We show that this complex formation enhances cellulase activity approximately 2‐fold on insoluble microcrystalline cellulose and 1.25‐fold on soluble carboxymethyl cellulose. Moreover, we identified two additional candidate interacting partners for ClcC, one of which appears to bind via a non‐canonical interface. These findings suggest that cellulosomal principles can operate in highly reduced cohesin‐dockerin systems.
ABSTRACT 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 putative 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, has been found to possess genes for an atypical cohesin-based assembly enriched in amylases and related starch-binding proteins, which 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. IMPORTANCE Plant cell wall polysaccharides are a major dietary carbon source, yet their degradation relies on rare, highly specialized microbial enzyme assemblies known as cellulosomes, which have long been considered uncommon in the human gut. Using proteome-scale structure prediction combined with experimental validation, we show that cellulosomes are far more widespread and structurally diverse in human-associated Ruminococcus species than previously appreciated. We identify multiple new cohesin families and reveal distinct cellulosome architectures likely adapted to degrade different dietary substrates. Together, these findings redefine the distribution and evolution of cellulosomes in gut microbes and demonstrate the power of structural proteomics to uncover deeply diverged biological systems. Plant cell wall polysaccharides are a major dietary carbon source, yet their degradation relies on rare, highly specialized microbial enzyme assemblies known as cellulosomes, which have long been considered uncommon in the human gut. Using proteome-scale structure prediction combined with experimental validation, we show that cellulosomes are far more widespread and structurally diverse in human-associated Ruminococcus species than previously appreciated. We identify multiple new cohesin families and reveal distinct cellulosome architectures likely adapted to degrade different dietary substrates. Together, these findings redefine the distribution and evolution of cellulosomes in gut microbes and demonstrate the power of structural proteomics to uncover deeply diverged biological systems.
Christine Minor, Allen Takayesu, M. Arbing et al.· bioRxiv· 0 citations
BACKGROUND
The discovery of novel biocatalysts for the sustainable valorization of complex biomass feedstocks remains a significant challenge. Domain-centric exploration of characterized CAZyme families offers a promising but underexplored strategy for identifying enzymes with unusual architectures and potentially expanded substrate specificities.
RESULTS
Systematic analysis of archaeal glycoside hydrolase family 18 (GH18) chitinases using the CANDy domain annotation pipeline led to the identification of TcChi from Thermococcus chitonophagus, a multidomain enzyme combining a GH12 and a GH18 catalytic domain alongside two carbohydrate-binding modules. Given that T. chitonophagus also encodes dedicated standalone cellulases and chitinases, we hypothesized that this multidomain assembly may have evolved a broader functional range than either composing domain alone. Biochemical assays of truncated constructs confirmed this hypothesis: the GH18 domain hydrolyzed chitin, chitosan, and β-1,3-glucan, marking the first report of β-1,3-glucanase activity (EC 3.2.1.58) in a GH18 chitinase, while the GH12 domain exhibited strong cellulase activity alongside unexpected chitosanase activity (EC 3.2.1.132), extending the known functional range of this family. Both domains demonstrated high thermostability consistent with the hyperthermophilic origin of T. chitonophagus.
CONCLUSIONS
TcChi is a thermostable, multifunctional biocatalyst capable of degrading chitin, chitosan, cellulose, and β-1,3-glucan from a single protein scaffold, making it a promising candidate for consolidated biomass deconstruction and waste valorization. These findings also demonstrate that domain-centric analysis of CAZyme families is an effective strategy for uncovering hidden functional diversity in well-characterized enzyme families.
Alex Windels, S. Dhaene, Tom Desmet· Biotechnology for Biofuels a...· 0 citations
Surprisingly, the periplasmic cellulase BcsZ, encoded in the cellulose biosynthesis operon, is necessary for efficient bacterial cellulose production and functions independently of the biosynthetic complex to clear mislocalized pEtN cellulose from the periplasm.
J. Rum, Jhih-Yi Huang, E. Kitova et al.· bioRxiv· 0 citations
It is shown that GeoA cargo loading is mediated by repeating, short cargo loading peptides (CLPs), and that GeoA encapsulation provides benefits for enzyme activity and stability, and provides insights into a putative mode of cargo protein regulation via pore state modulation.
A. Fazal, Cassandra A. Dutcher, Michael P. Andreas et al.· bioRxiv· 0 citations
Lignocellulosic biomass is a heterogeneous solid matrix whose biological deconstruction is limited by cellulose accessibility, lignin exposure, pore-scale transport, and enzyme stability at solid-liquid interfaces. Although bacterial cellulosomes provide a natural strategy for organizing multiple enzymes through scaffoldin-mediated cohesin-dockerin interactions, their engineering value depends on more than enzyme colocalization. This review frames cellulosome engineering as a process-aware macromolecular assembly problem, in which catalytic balance, substrate targeting, assembly size, inter-domain spacing, and environmental robustness must be optimized together. Natural cellulosome architecture is discussed in terms of its roles in enzyme recruitment and catalytic synergy. In addition, cellulosomal systems are compared with industrial fungal enzyme cocktails regarding production, scalability, and substrate accessibility. Rather than viewing engineered cellulosomes as universally superior multienzyme complexes, we evaluate how their performance is constrained by lignin adsorption, steric exclusion, diffusion limitations, high-solids conditions, host burden, catalyst recovery, AI-guided design validation, and the mismatch between model substrates and industrial feedstocks. Future progress will require experimentally validated, substrate-specific, and process-compatible cellulosome designs that balance catalytic diversity with assembly stability, production feasibility, and techno-economic performance.
Menglan Liao, Zi-Xuan Wan, Long-Qing Wang et al.· International Journal of Bio...· 0 citations
A new class of fungal biosurfactant proteins is introduced and a simplified downstream process based on methanol/chloroform extraction is developed, reducing costs while preserving functionality.
Rossana Pitocchi, Giulia Fichera, P. Cicatiello et al.· International Journal of Bio...· 0 citations
Related blog posts
MIT News · Artificial Intelligence· news.mit.eduAug 27, 2026
A new machine-learning framework aims to improve the success rate of computational protein design while moving away from results that reproduce sequences found in nature.
