Artificial organelle for energy production in artificial cell

Autonomous production of biological energy is one of the primal processes in living cells. In cells, the bio-energy presents as adenosine or guanosine triphosphate (ATP or GTP) is used for the most of cellular reactions. ATP is produced through the glycolytic cycle by a series of dedicated enzymes in cytosol, or through the oxidative phosphorylation of adenosine diphosphate (ADP), operated by ATP synthase, which is located on lipid membrane. For instance, in mitochondria, the proton potential across the membrane, generated by an electron transport chain, is eventually dissolved through FoF1-ATP synthase (FoF1). The flux of protons drives FoF1 and activates the synthesis of ATP, from ADP and Pi. On the other hand, bacteriorhodopsin (bR) is widely known as proton pump machinery that transports the protons to the other side of membrane due to light stimulation. Therefore, our idea is that if the bR and FoF1 were synthesized on a liposome membrane, the resulting liposome is able to generate ATP (see Figure 1). Figure 1. Schematic of bR-FoF1 liposome. Light induced bacteriorhodopsin pumps H into liposome and produced H gradient is used for FoF1-ATP synthase to synthesize ATP. Racker and Stoeckenius (1) have studied a model system by combing purple membrane, which contains bR, and isolated ATP synthase in phospholipid vesicles. In order to design and construct a synthetic cell in the synthetic biology context, this kind of “bioreactor” should be autonomously built up through an internal metabolic process such as gene expression. If both bR and FoF1 proteins were synthesized in the presence of organelle-sized vesicles, it would be possible to construct in vitro the bR-FoF1 liposomes as a consequence of the artificial protein synthesis and the self-organization of the synthesized proteins. Additionally, so produced bR-FoF1 liposomes can be applied as a bioenergy-producible plant that activates further biological reaction. For instance, if the produced ATP could be used for protein synthesis reaction, the whole system would represent an energetically independent autonomous system. In the ECAL11 meeting, we present some experimental achievements toward the construction of bR-FoF1 liposome (2, 3). Our recent results show that bR was synthesized in a cell-free protein synthesis system (4) in the presence of liposomes and all-trans retinal. Fo complex, the membrane integrated part of FoF1, was synthesized in situ and formed the desired FoF1 complex in combination with a supplied F1. FoF1 complex was fully functional, by showing ATPase driven H-translocation activity. These results imply that the bottom up construction of an artificial organelle, which is capable of generating the bioenergy, is experimentally feasible. We believe that our bR-FoF1 liposomes will be essential machinery for constructing artificial cells.