Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane Perspective

Abstract

Life-like systems need to maintain a basal metabolism, which includes importing a variety of building blocks required for macromolecule synthesis, exporting dead-end products, and recycling cofactors and metabolic intermediates, while maintaining steady internal physical and chemical conditions (physicochemical homeostasis). A compartment, such as a unilamellar vesicle, functionalized with membrane-embedded transport proteins and metabolic enzymes encapsulated in the lumen meets these requirements. Here, we identify four modules designed for a minimal metabolism in a synthetic cell with a lipid bilayer boundary: energy provision and conversion, physicochemical homeostasis, metabolite transport, and membrane expansion. We review design strategies that can be used to fulfill these functions with a focus on the lipid and membrane protein composition of a cell. We compare our bottom-up design with the equivalent essential modules of JCVI-syn3a, a top-down genome-minimized living cell with a size comparable to that of large unilamellar vesicles. Finally, we discuss the bottlenecks related to the insertion of a complex mixture of membrane proteins into lipid bilayers and provide a semiquantitative estimate of the relative surface area and lipid-to-protein mass ratios (i.e., the minimal number of membrane proteins) that are required for the construction of a synthetic cell.

A Hitchhiker’s Guide to Supplying Enzymatic Reducing Power into Synthetic Cells

“…In a cell-mimicking system, the phospholipid membrane delineates a selectively open system . The NAD­(P) + cofactors and the enzymes remain confined within the lumen of the liposomal compartment once encapsulated due to their membrane-impermeable nature.…”

“…In a cell-mimicking system, the phospholipid membrane delineates a selectively open system. 42 The NAD(P) + cofactors and the enzymes remain confined within the lumen of the liposomal compartment once encapsulated due to their membrane-impermeable nature. Only small uncharged molecules can pass through the membrane unassisted, while charged and larger molecules require specific membrane proteins to allow the transport of solutes.…”

“…Although pore-forming proteins have found some useful applications in cell-like systems, 45 − 47 they are not ideal for sustaining the out of equilibrium status over the long-term, hindering the retention and internal recycling of the small molecules necessary for metabolic homeostasis. 42 We therefore refer specifically to transporters as membrane proteins of choice in bottom-up synthetic cells to mediate the translocation of membrane-impermeable solutes.…”

“…Assuming the size of metabolically active synthetic cells in a range between 1 and 10 μm in diameter, 42 we consider the homo-octameric SthA from E. coli more suitable than the filamentous ones to guarantee the shuttle of hydrides between cofactors in a compartmentalized metabolism; in this way it would be possible to minimize the effects of excluded volume, and consequently preserve useful luminal space to encapsulate further metabolic modules. 51 We therefore focus on the biochemical features of SthA from E. coli as a representative model for the biochemistry of sTHs.…”

“…) can be employed to enable the passage of multiple reactants within a certain size depending on the cutoff of the specific pore, most membrane transporters are characterized by substrate-specificity that ensures a high degree of selectivity and control of membrane-impermeable reactants entering or leaving the liposomal lumen. Although pore-forming proteins have found some useful applications in cell-like systems, − they are not ideal for sustaining the out of equilibrium status over the long-term, hindering the retention and internal recycling of the small molecules necessary for metabolic homeostasis . We therefore refer specifically to transporters as membrane proteins of choice in bottom-up synthetic cells to mediate the translocation of membrane-impermeable solutes.…”