Bioelectrochemical reduction of pyruvate to d-lactate by engineered Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 possesses an extracellular electron transfer (EET) pathway that enables bidirectional electron exchange with electrodes, making it a promising host for electro-fermentation (EF). However, the intracellular redox reactions driven by MR-1 during electron uptake from the electrodes remain poorly characterized. This study investigated the metabolic fate of pyruvate, a key fermentation intermediate, during inward electron transfer from a low-potential cathode. To examine this, an MR-1 derivative lacking formate dehydrogenase (ΔFDH), which is unable to utilize formate as an electron donor for pyruvate reduction, was incubated under open-circuit (OC) conditions and closed-circuit (CC) conditions with an electrode poised at -0.36 V (vs. the standard hydrogen electrode). A comparative analysis of pyruvate-derived metabolites under these conditions revealed that ΔFDH produced significantly higher amounts of d-lactate under CC conditions, indicating cathode-derived electron utilization for pyruvate reduction to d-lactate. Further gene knockout experiments in the ΔFDH background showed that two d-lactate dehydrogenases (D-LDHs) in MR-1, Dld (a quinone-dependent inner membrane D-LDH) and LdhA (an NADH-dependent D-LDH), contributed almost equally to cathode-dependent d-lactate production. These results indicate that electron transfer from electrodes to pyruvate in MR-1 cells involves both inner membrane quinone-mediated and NADH-mediated redox reactions, highlighting the potential applicability of MR-1 in diverse EF processes.