Membraneless Compartmentalization of Cell‐Free Transcription‐Translation by Polymer‐Assisted Liquid–Liquid Phase Separation

Abstract Living cells use liquid–liquid phase separation (LLPS) to compartmentalize metabolic functions into mesoscopic‐sized droplets. Deciphering the mechanisms at play in LLPS is therefore critical to understanding the structuration and functions of cells at the subcellular level. Although observed and achieved to a significant degree of control in vivo, the reconstitution of LLPS integrating advanced biological functions, such as gene expression, has been so far limited in vitro. LLPS of cell‐free transcription‐translation (TXTL) reactions require multi‐step experimental approaches that lack biomimetic and have relatively poor efficacy, thus limiting their usage in cell‐free engineered systems such as synthetic cells. Here the polymer‐assisted LLPS of TXTL reactions are reported as the single‐pot one‐step compartmentalization of a model complex metabolic system obtain without using solvents or surfactants. LLPS occurs by adding the biocompatible polymers poly(ethylene glycol), poly(vinyl alcohol), and dextran to a TXTL reaction, that remains highly active. These polymers serve as partitioning agents that localize TXTL in mesoscopic‐sized droplets rich in dextran. Cytoplasmic and membrane‐interacting proteins are synthesized preferentially inside these droplets, and localize either uniformly or preferentially at the interface, depending on their nature. The LLPS‐TXTL system presented in this work is a step toward the design of synthetic membraneless active organelles.