A synthetic metabolic network for physicochemical homeostasis

Abstract

One of the grand challenges in chemistry is the construction of functional out-of-equilibrium networks, which are typical of living cells. Building such a system from molecular components requires control over the formation and degradation of the interacting chemicals and homeostasis of the internal physical-chemical conditions. The provision and consumption of ATP lies at the heart of this challenge. We report the in vitro construction in vesicles of a pathway for sustained ATP production that is maintained away from equilibrium by control of energy dissipation. We maintain a constant level of ATP with varying load on the system. The pathway enables us to control the transmembrane fluxes of osmolytes and to demonstrate basic physicochemical homeostasis. Our work demonstrates metabolic energy conservation and cell volume regulatory mechanisms in a cell-like system at a level of complexity minimally needed for life.

Cell Fuelling and Metabolic Energy Conservation in Synthetic Cells

“…[24] This property of coupling substrate and product fluxes is also possible in many other pathways and aids in keeping the reactionn etworks away from equilibrium. [25] The system can sustain a constantl evel of ATPf or many hours even when the load on the systemi sv aried by the consumption of ATPf or the uptake of solutes. [25] The system can sustain a constantl evel of ATPf or many hours even when the load on the systemi sv aried by the consumption of ATPf or the uptake of solutes.…”

“…[25] Bowie and colleagues have described am olecular rheostat that accounts for the ATPd emand throughs witchingb etween an ATP-generating and non-ATP-generating pathway according to the concentration of inorganic phosphate (Figure4). [25] Bowie and colleagues have described am olecular rheostat that accounts for the ATPd emand throughs witchingb etween an ATP-generating and non-ATP-generating pathway according to the concentration of inorganic phosphate (Figure4).…”

“…We typically form LUVs by detergent-mediated reconstitution, [51] whichi s based on am ethod originally developedb yR igaud. [25] In this way, one can study synthetic metabolic networks under various conditions of crowding,ionic strength and osmotic pressure. [59] Increasing the outside osmolality causest he vesicles to shrink due to water efflux, and the luminalc ontentsa re concentrated.…”

“…[25] When vesicles are exposed to an increasing medium osmolality,t hey shrink,t he ionic strength increases, and the concentrations of the internal components are increased. Hence, the importance of obtaining physicochemical homeostasis in cell-likes ystems.…”

“…The arginine breakdownp athway has been reconstitutedi nl iposomes with ATP/ADP and pH sensors (Table 1) in the vesicle lumen to report the synthesis of ATPa nd to monitor the changes in internal pH. [25] The system can sustain a constantl evel of ATPf or many hours even when the load on the systemi sv aried by the consumption of ATPf or the uptake of solutes.…”