<p>Lipid membranes define cell boundaries, acting as gatekeepers for transport and signaling. A central paradigm in biology is that all cellular membranes descend from a common ancestral membrane, as they cannot be generated in the absence of pre-existing lipid structures. It is thus unclear whether lipid membranes can arise from membrane-less precursors. Here we demonstrate the de novo generation of lipid bilayers in the absence of any pre-existing membranes, membrane-bound proteins, or lipid nanostructure templates. Using acetate and cysteine as simple metabolites, lipid tails are constructed by soluble enzymes and spontaneously form diacyl lipids that assemble into vesicles. Pore-forming peptides facilitate precursor transport into vesicles, allowing the continuous generation of new lipids. Formation of glycolipid membranes creates compartments that can maintain proton gradients. Our findings demonstrate that lipid compartments can form without pre-existing membranes, establishing a unique route linking lipid synthesis to compartment formation and function.</p>