Cellulosome engineering as biological macromolecular assembly for lignocellulose deconstruction: Structure, interactions, and functional design.
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.