Protein-based condensates have been proposed to accelerate biochemical reactions by enriching reactants and enzymes simultaneously. Here, we engineered those condensates into a photo-activated switch in Escherichia coli (PhASE) to regulate enzymatic reactions via tuning the spatial correlation of enzymes and substrates. In this system, scaffold proteins undergo liquid-liquid phase separation (LLPS) to form light-responsive compartments. Tethered with a light-responsive protein, enzymes of interest (EOIs) can be recruited by those compartments from cytosol within only a few seconds after a pulse of light induction and fully released in 15 min. Furthermore, we managed to enrich small molecular substrates simultaneously with enzymes in the compartments and achieved the acceleration of luciferin and catechol oxidation by 2.3and 1.6-folds, respectively. We also developed a quantitative model to guide the further optimization of this demixed regulatory system. Our tool can thus be used to study the rapid redistribution of proteins, and reversibly regulate enzymatic reactions in E. coli.
Cell surface signaling landscapes are formidably complex. Robust tools capable of manipulating the spatiotemporal distribution of cell surface proteins (CSPs) for dissecting signaling are in high demand. Some CSPs are regulated via multivalency-driven liquid-liquid phase separation (LLPS). Employing the robustness and versatility of LLPS, we decided to engineer LLPS-based tools for precisely manipulating CSPs. We generated membrane-tethering LLPS systems by fusing multivalent modular phase separation scaffold pairs with CSP binders. Phase separation of the scaffold pairs, concomitant compartmentalization of CSPs on membranes, and cluster-dependent signaling outputs of CSPs require membrane recruitment of one or both scaffolds. We also engineered orthogonal phase separation systems to segregate CSPs into mutually exclusive compartments. The engineered phase separation systems can robustly cluster individual CSPs, co-cluster two or more CSPs, or segregate different CSPs into distinct compartments on cell surfaces. These novel tools will enable the dissection of complicated cell signaling landscapes with unprecedented precision.
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