Minimal metabolic pathways for ATP and generation of a proton motive force in synthetic cells

In an effort to mimic bacterial life and to build a bacterial cell from the bottom up, we have developed metabolic routes for synthetic cells. These enzymatic pathways could eventually provide a synthetic cell with the energy required to sustain itself and ultimately proliferate. This thesis two of these pathways, as well as methods to monitor them with sensors and external methods.
The first pathway provides the synthetic cell with electrical energy. A proton motive force is generated by a pathway consisting of two proteins. A membrane transporter imports malate, which is converted into lactate inside the vesicles. This process increases the pH on the inside of the vesicles, and additionally accumulates a negative charge. The membrane potential that is generated is great enough to potentially import other substrates into the cell or to drive the generation of ATP through the F1F0-ATPsynthetase.
The second pathway presented provides the synthetic cell with the universal energy carrier ATP through four proteins in total. The membrane protein imports arginine and exports the waste product ornithine. The three internal proteins convert arginine to citrulline and subsequently ornithine, which phosphatizes ADP in the process, yielding ATP. We show that the generated ATP can be used by another transporter to transport glycine betaine into the vesicles.
To monitor the presented pathways, methods are described to monitor the ATP to ADP ratio, pH, membrane potential, production of lactate and the concentrations of arginine, citrulline, ornithine and ammonia.