Beyond Compartmentalization: Deciphering Reaction Kinetics in Liquid–Liquid Phase Separation for Rational Biotechnological Design

Liquid-liquid phase separation (LLPS) utilizes dynamic, membrane-less compartmentalization to spatially organize and control biochemical processes, which advances synthetic biology fields such as synthetic metabolic engineering and artificial cell construction, offering novel solutions to longstanding biomedical and biotechnological challenges. However, the rational design and optimization of these promising LLPS-based applications are currently hampered by an incomplete mechanistic understanding of how LLPS precisely governs reaction kinetics. To bridge this gap, we present a comprehensive review that integrates both protein and nonprotein mediated LLPS and systematically dissecting how LLPS orchestrates reaction kinetics─through mechanisms including reactant concentration, reaction-diffusion coupling, microenvironment engineering, and enzyme activity modulation─to dictate bioreaction outcomes. Our analysis begins by outlining the thermodynamic foundations and classifications underpinning LLPS, then critically examines these kinetic regulatory mechanisms, and further summarizes burgeoning applications across biocatalysis, metabolic engineering, diagnostics, therapeutics, origins of life research, and artificial cell construction. Finally, we discuss prevailing challenges and outline strategic pathways for translating LLPS into practical technologies. By synthesizing dispersed knowledge and elucidating fundamental kinetic principles, this review not only fills a critical void in understanding but also establishes essential mechanistic insights and design guidelines to empower the rational development of next-generation LLPS-driven synthetic biology.