(693d) Development and Assembly of a Synthetic Membrane-Less Enzymatic ATP Regeneration Cascade Powered By Fuel Oxidation

ATP regeneration is an important bioengineering capability that can be used to support for cell-free protein synthesis (CFPS), pseudocell development, and other in vitro applications. Methods for regenerating ATP include expensive phosphate donors and ATP-synthase enzymes that require a proton gradient across a membrane. In this talk, we propose a membrane-less ATP-generating synthetic enzyme cascade that is driven by NADH oxidation. We demonstrate the cascade using the oxidation of formate, a cheap fuel source, and polyphosphate, as a cheap phosphate donor. At the center of the proposed cascade is the ATP-NAD⁺ kinase (NADK). All of the commonly investigated isoforms of NADK (human, S. cerevisiae, Arabidopsis and E. coli) have been measured, or assumed, to have irreversible reactions and can only accept ATP as a reactant. One group, in 1977, has reported NADK obtained from pigeon livers (C. livia) with reverse activity. First, we confirmed a reversible reaction for the recombinant pigeon NADK. Additionally, we identified 2 other isoforms (duck and cat) that can use ATP as a substrate and as a product. We determined that the human NADK has an affinity for NAD⁺ that is ~600 times higher than the pigeon, duck and cat isoforms, and we concluded that NAD⁺ serves as a potent product inhibitor in the reverse direction for the human NADK, which accounts for the observed irreversible behavior. Then, we constructed the membrane-less enzyme cascade with an NADK with reversible activity, a polyphosphate NAD⁺ kinase, a formate dehydrogenase, and an NADPH oxidase. This cascade continuously produced ATP for several hours when coupled with firefly luciferase. To show the utility of the cascade, the purified enzymes were incorporated into a CFPS system and we demonstrate formate-powered recombinant protein expression of sfGFP and HRP. We believe this enzyme cascade is a valuable new biosynthetic system for regenerating ATP in emerging biotechnology applications.