Light/NADH Bistimulation ATP Synthesis Artificial Organelles for Nitrogen Assimilation and RNA Transcription in...

Energy supply is a fundamental requirement for maintaining a complex metabolic network within the artificial cells. RNA transcription is a key step in the genetic inheritance of artificial cells. Targeting these two key issues in the field of artificial cells, a light/NADH bisimulation adenosine triphosphate (ATP) synthesis artificial organelle (LNAO) capable of producing ATP through two distinct pathways is built to power reactions within artificial cells. The LNAO, derived from Escherichia coli expressing rhodopsin, mimics photosynthetic phosphorylation by using rhodopsin and ATP synthase to produce ATP; meanwhile, it mimics oxidative phosphorylation by employing the electron-transport chain modules of E. coli membranes to synthesize ATP from NADH. The produced ATP is used to produce CTP with inorganic nitrogen (NH₄ ⁺) assimilation catalyzed by CTP synthase (CTPS). The synthesized ATP and CTP are subsequently used as substrates for RNA transcription. The LNAO, CTPS, and iSpinach RNA transcription pathways are encapsulated into giant unilamellar vesicles (GUVs) to construct artificial cells capable of inorganic nitrogen assimilation and RNA transcription. The LNAOs provide flexible energy supply methods for constructing complex metabolic networks in artificial cells. The successful coupling of LNAO, CTPS, and the RNA transcription pathway inside artificial cells steps forward toward autonomous artificial cells.