Liquid-Liquid Phase Separation Modulated Thylakoid-Enzyme Interfacial Coupling for Enhanced Light-Powered Biocatalysis.

Thylakoid (TK) membranes serve as promising photosynthesis platforms for light-driven biocatalysis, yet their catalytic efficiency is largely restricted by poor coupling between photo-dependent NADPH regeneration and enzymatic conversion. Herein, we report a facile TK-anchored enzyme condensate strategy to overcome this bottleneck. Specifically, the UTX-derived intrinsically disordered region (IDR) fused with target enzymes mediates liquid-liquid phase separation to form enzyme-rich condensates, which then stably anchor on TK membranes and spatially confine catalysis at the NADPH-generating membrane interface. Taking carbonyl reductase SmCRV4 and ene-reductase SsERA314S as model enzymes, the TK-enzyme hybrid condensates exhibit prominently improved catalytic performance, especially under NADPH regeneration-limited conditions, relative to freely dispersed enzyme systems. The TK-UTX-SmCRV4 hybrid improves the total product yield up to 3.1-fold, and the TK-UTX-SsERA314S hybrid reaches a 2.9-fold higher conversion within 20 min. Coarse-grained molecular dynamics simulations validate a dynamic multivalent interfacial anchoring mode: the IDR domain mediates stable membrane binding via high-frequency residue-lipid interactions dominated by short-range Lennard-Jones forces (with auxiliary electrostatic contributions), while the enzyme domain remains solvent-exposed. This work establishes a feasible strategy that physically links light-driven NADPH recycling with enzymatic catalysis, offering a general approach for constructing high-efficiency artificial photosynthesis systems.