Rational assembly of multi-enzyme cascades: A paradigm shift from stochastic immobilization to precision assembly...

Multi-enzyme cascade catalysis has become a pivotal technology in green biomanufacturing and the synthesis of fine chemicals, facilitating efficient and highly selective transformations through the mimicry of natural metabolic pathways. Despite considerable advances in single-enzyme engineering, substantial challenges persist in enhancing the overall catalytic efficiency of multi-enzyme systems. Limitations such as inadequate inter-enzyme synergy and low operational stability significantly impede their scalable implementation. Thus, achieving precise spatial organization of multi-enzyme systems to emulate synergistic functions and improve stability within artificial environments has emerged as a crucial strategy to overcome existing bottlenecks. This review systematically categorizes multi-enzyme cascade reactions into five modular types (linear, parallel, orthogonal, cyclic, and triangular) and classifies assembly materials into non-biological (synthetic polymer and inorganic material) and biological categories (polysaccharide, nucleic acid, protein, and organism). Drawing on current literature, we systematically examine diverse enzyme assembly strategies, highlight state-of-the-art engineering methodologies, and introduce "ordered assembly" as a novel paradigm for constructing advanced multi-enzyme systems. In consideration of the distinct features of various cascade modules, we further elaborate on design principles for customized assembly strategies and illustrate their implementation through representative case studies. Finally, the current challenges and future directions of multi-enzyme assembly technology are discussed. This study aims to establish a theoretical foundation and propose innovative strategies for enhancing the application of multi-enzyme assembly in biocatalysis, thereby contributing to the sustainable manufacturing of fine chemicals.