Lipid membranes, nucleic acids, proteins, and metabolism are essential for modern cellular life. Synthetic systems emulating the fundamental properties of living cells must therefore be built upon these functional elements. In this work, phospholipid-producing enzymes encoded in a synthetic minigenome are cell-free expressed within liposome compartments. The de novo synthesized metabolic pathway converts precursors into a variety of lipids, including the constituents of the parental liposome. Balanced production of phosphatidylethanolamine and phosphatidylglycerol is realized, owing to transcriptional regulation of the activity of specific genes combined with a metabolic feedback mechanism. Fluorescence-based methods are developed to image the synthesis and membrane incorporation of phosphatidylserine at the single liposome level. Our results provide experimental evidence for DNA-programmed membrane synthesis in a minimal cell model. Strategies are discussed to alleviate current limitations toward effective liposome growth and self-reproduction.

The templated ligation of oligonucleotides offers a mode of replication in an RNA world. The 2′,3′-cyclic phosphate (>P) is a prebiotically available activation group for RNA and the product of backbone hydrolysis. Using gel electrophoresis and liquid chromatography, we found that the templated ligation of RNA with >P activation proceeds in alkaline (pH 9-11) low-salt aqueous solutions with 1 mM MgCl2 in temperatures ranging from 20 to 25 °C within a few days. Under the optimum conditions of pH 10 and 5 °C, the ligation yielded 40% after 7 days. No additional catalysts were required. In contrast to previous reports, we found an equimolar mixture of 2′-5′ and 3′-5′ linked oligomers in the used conditions. We probed the nucleotide specificity at the ligation site and found that one mutation reduced the ligation yield by 82-92%. We extrapolated these results to a per-nucleotide replication fidelity of 95-98% when ligating 4- to 6-mers. With splinted oligomers, five ligations created a 96 mer strand, demonstrating a possible assembly pathway for long ribozymes. With the low salt requirements, strand separation will be compatible with the ligation conditions using non-equilibrium settings. The findings suggest that templated ligation mediated by 2′,3′-cyclic phosphate in alkaline conditions offer a slow, but precise replication and elongation reaction for RNA on early Earth.

Lipid membrane, nucleic acids, proteins, and metabolism are essential ingredients for life. Synthetic cellular systems emulating the fundamental properties of living cells must therefore be built upon these functional elements. In this work, phospholipid-producing enzymes encoded in a synthetic minigenome are cell-free expressed within liposome compartments. The de novo synthesized metabolic pathway converts precursors into a variety of lipids, including the constituents of the parental liposome. Balanced production of phosphatidylethanolamine and phosphatidylglycerol is realized, owing to transcriptional regulation of specific genes activity combined with a metabolic feedback mechanism. Fluorescence-based methods are developed to image the synthesis and membrane incorporation of phosphatidylserine at the single liposome level. Our results provide experimental evidence for DNA-programmed membrane synthesis in a minimal cell model. Strategies are discussed to alleviate current limitations toward more effective liposome growth and self-reproduction.