A few key molecules act as central electron and energy carriers inside cells. Nicotinamide adenine dinucleotide (NADH) transfers electrons and adenosine triphosphate (ATP) supplies energy. The most common method to produce ATP requires a pH gradient to be established across a membrane. The resulting flow of protons through an embedded protein regenerates ATP. This project will test the hypothesis that alternative pathways could be developed that can regenerate ATP from NADH oxidation without the need for a membrane, proton gradient, or embedded enzyme. This will require combining native and engineered enzymes to create novel cyclic pathways. These pathways will help uncover design rules required to replicate a central biological process. This technology will be critical to the development of synthetic cells. In addition, the project will involve mentoring of students and expanding existing outreach activities in the local community.
This project will design, build and test novel synthetic metabolic pathways that could regenerate ATP using energy obtained from fuel oxidation. Fuel oxidation pathways can generate reducing equivalents in the form of NADH. However other critical operations, especially those involving motion, are powered by the hydrolysis of ATP. Oxidative phosphorylation in mitochondria supplies the majority of cellular ATP from NADH oxidation and this requires the establishment of a proton gradient. Although this supplies flexibility for cellular energy processing, replicating this system presents a difficult engineering challenge. We hypothesize that oxidative phosphorylation could be replaced with novel pathways composed of kinase enzymes. These pathways will use fuel oxidation reactions to drive NADH regeneration and NAD(H) phosphorylation will drive ATP regeneration. As a proof-of-concept, an ATP-dependent reaction (firefly luciferase) powered by methanol oxidation in simple liposomes will be demonstrated. This will represent a novel energy transduction alternative to mitochondrial oxidative phosphorylation. This pathway could be easily adopted to use a wide range of potential fuels, supporting many potential future synthetic cell and biology applications.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
