Sustainable membrane biosynthesis for synthetic minimal cells

One of the main questions addressed in the Nationale Wetenschapsagenda (NWA) is our ability to construct a synthetic minimal cell from lifeless, individual components. In biology, this challenge can be compared to bringing a man to the moon. Various international consortia now aim to engineer such synthetic cells, among which the Dutch initiative BaSyC that is supported by a NWO gravity program. The work presented in this thesis served as a pilot study for BaSyC, and focused on the construction and growth of a boundary layer (membrane) that should surround the synthetic cell. In living cells, membranes are composed of various phospholipids that spontaneously orient in a bilayer-like structure that separates the inside from the outside of the cell. Here, we describe the construction of a biosynthetic pathway that yields two phospholipid species critical for life. By combining catabolic and anabolic enzymes purified from various bacteria, a cascade-like biosynthetic pathway could be assembled capable of forming phospholipids from simple water-soluble building blocks that are fed to the system. The synthesis of these phospholipids resulted in the growth of a pre-established membrane that not only served as a barrier, but also supported the function of membrane embedded enzymes, including a system that is responsible for the translocation of proteins across the membrane. Sustainable production of phospholipids and growth of membranes, as well as the functional integration of membrane growth and membrane protein function, presents a first but important step towards the construction of synthetic minimal cells.