Autonomous Synthesis and Scrambling of Phospholipids, Linked to Recycling of Cofactors in Synthetic Cells

Compartmentalization of reactions is essential for life and allows nonequilibrium conditions to be maintained within cells. For cell growth, the membranes need to expand through lipid synthesis and a continuous supply of ATP and building blocks. Here, we build a minimal system in vesicles that integrates ATP supply, CTP and CMP recycling, and glycerol-3-phosphate synthesis with the conversion of phosphatidic acid to phosphatidylglycerol. We use four transmembrane proteins and three soluble enzymes to enable autonomous phospholipid synthesis in both the outer and inner leaflets of the membrane. The system displays biphasic lipid synthesis kinetics: a rapid phase with phosphatidylglycerol production in the cis leaflet of the membrane and a slower phase dependent on lipid scrambling. We present previously unreported scramblase activity of two integral membrane proteins: phosphatidylglycerophosphatase A and the mitochondrial ATP/ADP carrier. This work lays the foundation for autonomous lipid biosynthesis in synthetic cells and enables the exploration of emergent properties in compartmentalized systems.