A sustainable chemiosmotic strategy for driving solute transport in synthetic cells

Abstract Cellular homeostasis requires sustained provision of metabolic energy in the form of ATP and electrochemical ion gradients. Primary and secondary active transporters are prominent consumers of cellular energy, and couple ATP hydrolysis and ion gradient dissipation, respectively, to translocation of molecules across biological membranes. Active transport is essential for the translocation of most charged and/or large hydrophilic molecules, both for nutrient uptake into and waste export from living cells. Endeavours to build synthetic cells crucially depend on simulating real cell behaviour by supplying stable and sustained energy sources and deploying them for membrane transport. Here, we provide synthetic cells with long-lasting metabolic energy supply in the form of an electrochemical proton gradient. Leveraging the L-malate decarboxylation pathway from Lactococcus lactis we generate a stable proton gradient and electrical potential in lipid vesicles by electrogenic L-malate/L-lactate exchange coupled to L-malate decarboxylation. By co-reconstitution of the pathway with the Escherichia coli transporters GltP and LacY, the synthetic cells maintain accumulation of L-glutamate and lactose over periods of hours, mimicking nutrient feeding in living cells. This study underscores the potential of harnessing a proton motive force via a simple metabolic network, involving electrogenic substrate/product exchange and substrate decarboxylation, paving the way for the development of more complex synthetic systems.