[Mechanisms and applications of liquid-liquid phase separation in enzyme activity regulation]

Living systems often rely on dynamically assembled multi-enzyme complexes to achieve precise control over metabolic reactions. In recent years, liquid-liquid phase separation (LLPS) has been recognized as one of the important mechanism by which living systems organbize and regulate enzymatic reactions, and has gradually emerged as a powerful tool for constructing artificial catalytic systems. Owing to the reversible, self-organizing, and programmable properties, LLPS-driven biomolecular condensates can modulate the local concentration, spatial arrangement, and microenvironmental features of enzymes, thereby influencing catalytic efficiency and substrate selectivity. To date, a variety of enzymatic systems, from intracellular multi-enzyme pathways to in vitro artificial reactors, have been reconstructed and enhanced within condensates. This review provides a concise overview of the physiological context of LLPS and, together with the authors' recent work on LLPS-based enzymatic systems, highlights the major mechanisms by which LLPS modulates enzymatic activity. We further summarize advances in the applications of LLPS in metabolic pathway engineering and in vitro biocatalysis and discuss the potential of programmable condensates for dynamic regulation of catalytic processes. This review proides a systematic framework for understanding the mechanisms by which LLPS regulates enzymatic catalysis and offers a theoretical basis for the rational design of efficient and controllable multi-enzyme catalytic systems.