Membraneless organelles formed by liquid–liquid phase separation of proteins or nucleic acids are involved in diverse biological processes in eukaryotes. However, such cellular compartments have yet to be discovered or created synthetically in prokaryotes. Here, we report the formation of liquid protein condensates inside the cells of prokaryotic Escherichia coli upon heterologous overexpression of intrinsically disordered proteins such as spider silk and resilin. In vitro reconstitution under conditions that mimic intracellular physiologically crowding environments of E. coli revealed that the condensates are formed via liquid–liquid phase separation. We also show functionalization of these condensates via targeted colocalization of cargo proteins to create functional membraneless compartments able to fluoresce and to catalyze biochemical reactions. The ability to form and functionalize membraneless compartments may serve as a versatile tool to develop artificial organelles with on-demand functions in prokaryotes for applications in synthetic biology.

蛋白质或核酸的液相液相分离形成的无膜细胞器参与了真核生物的多种生物过程。然而，尚未在原核生物中合成发现或产生这种细胞区室。在这里，我们报道了原发性无序蛋白如蜘蛛丝和弹性蛋白的异源过表达后，原核大肠杆菌细胞内液体蛋白冷凝物的形成。在模拟大肠杆菌细胞内生理拥挤环境的条件下进行体外重构揭示冷凝物是通过液相分离形成的。我们还显示了通过货物蛋白质的目标共定位来创建冷凝物的功能化，以创建能够发荧光并催化生化反应的功能性无膜隔室。形成无膜隔室并使其功能化的能力可以用作开发用于原核生物中具有按需功能的人工细胞器以用于合成生物学的通用工具。