Modular engineering to increase intracellular NAD (H/(+)) promotes rate of extracellular electron transfer of Shewanella oneidensis

The slow rate of extracellular electron transfer (EET) of electroactive microorganisms remains a primary bottleneck that restricts the practical applications of bioelectrochemical systems. Intracellular NAD(H/(+)) (i.e., the total level of NADH and NAD(+)) is a crucial source of the intracellular electron pool from which intracellular electrons are transferred to extracellular electron acceptors via EET pathways. However, how the total level of intracellular NAD(H/(+)) impacts the EET rate in Shewanella oneidensis has not been established. Here, we use a modular synthetic biology strategy to redirect metabolic flux towards NAD biosynthesis via three modules: de novo, salvage, and universal biosynthesis modules in S. oneidensis MR-1. The results demonstrate that an increase in intracellular NAD(H/(+)) results in the transfer of more electrons from the increased oxidation of the electron donor to the EET pathways of S. oneidensis, thereby enhancing intracellular electron flux and the EET rate.