Effective metabolic pathways are essential for the construction of in vitro systems mimicking the biochemical complexity of living cells. Such pathways require the inclusion of a metabolic branch that ensures the availability of reducing equivalents. Here, we built a minimal enzymatic pathway confinable in the lumen of liposomes, in which the redox status of the nicotinamide cofactors NADH and NADPH is controlled by an externally provided formate. Formic acid permeates the membrane where a luminal formate dehydrogenase uses NAD + to form NADH and carbon dioxide. Carbon dioxide diffuses out of the liposomes, leaving only the reducing equivalents in the lumen. A soluble transhydrogenase subsequently utilizes NADH for reduction of NADP + thereby making NAD + available again for the first reaction. The pathway is functional in liposomes ranging from a few hundred nanometers in diameter (large unilamellar vesicles) up to several tens of micrometers (giant unilamellar vesicles) and remains active over a period of 7 days. We demonstrate that the downstream biochemical process of reduction of glutathione disulfide can be driven by the transfer of reducing equivalents from formate via NAD(P)H, thereby providing a versatile set of electron donors for reductive metabolism.
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.
The construction from scratch of synthetic cells by assembling molecular building blocks is unquestionably an ambitious goal from a scientific and technological point of view. To realize functional life-like systems, minimal enzymatic modules are required to sustain the processes underlying the out-ofequilibrium thermodynamic status hallmarking life, including the essential supply of energy in the form of electrons. The nicotinamide cofactors NAD(H) and NADP(H) are the main electron carriers fueling reductive redox reactions of the metabolic network of living cells. One way to ensure the continuous availability of reduced nicotinamide cofactors in a synthetic cell is to build a minimal enzymatic module that can oxidize an external electron donor and reduce NAD(P) + . In the diverse world of metabolism there is a plethora of potential electron donors and enzymes known from living organisms to provide reducing power to NAD(P) + coenzymes. This perspective proposes guidelines to enable the reduction of nicotinamide cofactors enclosed in phospholipid vesicles, while avoiding high burdens of or cross-talk with other encapsulated metabolic modules. By determining key requirements, such as the feasibility of the reaction and transport of the electron donor into the cell-like compartment, we select a shortlist of potentially suitable electron donors. We review the most convenient proteins for the use of these reducing agents, highlighting their main biochemical and structural features. Noting that specificity toward either NAD(H) or NADP(H) imposes a limitation common to most of the analyzed enzymes, we discuss the need for specific enzymes�transhydrogenases�to overcome this potential bottleneck.
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