Spatial engineering for biocatalytic cascade control through biomolecular compartmentalization

Spatial engineering has emerged as a transformative paradigm for orchestrating metabolic flux through biomolecular compartmentalization. In cellular systems, the cytosolic dispersion of heterologous enzymes and evolutionary-driven metabolic priorities of native pathways necessitate spatial solutions that transcend conventional enzyme engineering. Concurrently, in vitro metabolons provide critical mechanistic insights into enzymatic cascade reactions through controlled assembly. This review systematically evaluates several spatial engineering platforms for biocatalytic process control-including scaffolded compartments (liposomes, DNA origami, polymersomes, and bacterial microcompartments) and scaffoldless assemblies (membraneless organelles and coacervates)-designed to reconfigure metabolic landscapes in cellular or cell-free contexts. Through critical analysis of recent advances in model construction and functionalized applications, we establish a framework for understanding different spatial control principles governing pathway efficiency and flux redistribution. Finally, we conclude with a comprehensive assessment of current limitations in mechanistic elucidation, dynamic regulation and cross-system compatibility, while projecting future developments towards multifunctional spatial organization tools and biomimetic platforms for synthetic biology and cellular engineering.