Liquid-Liquid Phase Separation-Enhanced Multienzyme Catalysis: Mechanisms and Applications.

Living organisms have evolved multienzyme complexes to achieve efficient and spatially ordered metabolic reactions. Recently, liquid-liquid phase separation (LLPS) has emerged as a fundamental organizational principle underlying these natural networks, providing a versatile platform for constructing artificial multienzyme catalytic systems. Owing to their reversible self-assembly and programmable nature, LLPS condensates can modulate enzyme distribution, mass transfer, and the reaction microenvironment, thereby significantly enhancing catalytic efficiency and cascade reaction throughput. This review systematically summarizes recent advances in in vitro LLPS-based multienzyme catalytic systems. We introduce the fundamental phase separation behaviors of multienzyme condensates and their molecular interaction mechanisms. Then, diverse construction strategies are discussed based on distinct intermolecular forces and their applications in cofactor recycling, biosynthesis, biodegradation, and artificial organelle fabrication are highlighted. Furthermore, we dissect the core mechanisms of catalytic enhancement, including local molecular enrichment, microenvironment modulation, and spatial confinement. Finally, we discuss current limitations and offer future perspectives for the rational design of advanced, high-efficiency multienzyme biocatalysts.