ATP Recycling Fuels Sustainable Glycerol 3-Phosphate Formation in Synthetic Cells Fed by Dynamic Dialysis

[Image: see text] The bottom-up construction of an autonomously growing, self-reproducing cell represents a great challenge for synthetic biology. Synthetic cellular systems are envisioned as out-of-equilibrium enzymatic networks encompassed by a selectively open phospholipid bilayer allowing for protein-mediated communication; internal metabolite recycling is another key aspect of a sustainable metabolism. Importantly, gaining tight control over the external medium is essential to avoid thermodynamic equilibrium due to nutrient depletion or waste buildup in a closed compartment (e.g., a test tube). Implementing a sustainable strategy for phospholipid biosynthesis is key to expanding the cellular boundaries. However, phospholipid biosynthesis is currently limited by substrate availability, e.g., of glycerol 3-phosphate, the essential core of phospholipid headgroups. Here, we reconstitute an enzymatic network for sustainable glycerol 3-phosphate synthesis inside large unilamellar vesicles. We exploit the Escherichia coli glycerol kinase GlpK to synthesize glycerol 3-phosphate from externally supplied glycerol. We fuel phospholipid headgroup formation by sustainable l-arginine breakdown. In addition, we design and characterize a dynamic dialysis setup optimized for synthetic cells, which is used to control the external medium composition and to achieve sustainable glycerol 3-phosphate synthesis.