Riboswitch-Controlled Lipid Remodeling Enables Functional Membrane Asymmetry in Artificial Cells

Dynamic regulation of lipid membrane composition is fundamental to living cells; however, synthetic analogs capable of such regulation remain scarce. Here, we present an artificial cell platform in which riboswitch-mediated expression of phospholipase D (PLD) enables stimulus-responsive lipid remodeling within lipid vesicles. In this system, chemically induced activation of a fluoride-responsive riboswitch triggers cell-free synthesis of PLD, which hydrolyzes phosphatidylcholine (PC) to phosphatidic acid (PA) in the inner leaflet of the vesicles. This enzymatic reaction generates a negatively charged asymmetric membrane, enabling functionalization with mechanosensitive channels such as the mechanosensitive channel of large conductance (MscL). We characterized the kinetics of asymmetry generation by varying plasmid DNA and fluoride concentrations, and evaluated membrane behavior using lipid compositions with or without cholesterol. Our platform demonstrates a novel strategy for coupling gene expression to dynamic membrane remodeling and underscores the potential of riboswitch-regulated lipid transitions in building environment-responsive artificial cells with programmable functions.