Reversing an extracellular electron transfer pathway for electrode-driven NADH generation

Microbial electrosynthesis is an emerging technology with the potential to simultaneously store renewably generated energy, fix carbon dioxide, and produce high-value organic compounds. However, limited understanding of the route of electrons into the cell remains an obstacle to developing a robust microbial electrosynthesis platform. To address this challenge, we engineered an inward electron transfer pathway in Shewanella oneidensis MR-1. The pathway uses native Mtr proteins to transfer electrons from an electrode to the inner membrane quinone pool. Subsequently, electrons are transferred from quinones to NAD+ by native NADH dehydrogenases. This reverse functioning of NADH dehydrogenases is thermodynamically unfavorable, therefore we have added a light-driven proton pump (proteorhodopsin) to generate proton-motive force to drive this activity. Finally, we use reduction of acetoin to 2,3-butanediol via a heterologous butanediol dehydrogenase (Bdh) as an electron sink. Bdh is an NADH-dependent enzyme, therefore, observation of acetoin reduction supports our hypothesis that cathodic electrons are transferred to intracellular NAD+. Multiple lines of evidence indicate proper functioning of the engineered electrosynthesis system: electron flux from the cathode is influenced by both light and acetoin availability; and 2,3-butanediol production is highest when both light and a poised electrode are present. Using a hydrogenase-deficient S. oneidensis background strain resulted in a stronger correlation between electron transfer and 2,3-butanediol production, suggesting that hydrogen production is an off-target electron sink in the wild-type background. This system represents a promising genetically engineered microbial electrosynthesis platform and will enable a new focus on synthesis of specific compounds using electrical energy.