[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 <i>in vitro</i> 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 <i>in vitro</i> 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.