Modular Engineering Strategy to Redirect Electron Flux into the Electron-Transfer Chain for Enhancing Extracellular Electron Transfer in Shewanella oneidensis

Efficient extracellular electron transfer (EET) of exoelectrogens is critical for practical applications of various bioelectrochemical systems. However, the low efficiency of electron transfer remains a major bottleneck. In this study, a modular engineering strategy, including broadening the sources of the intracellular electron pool, enhancing intracellular nicotinamide adenine dinucleotide (NADH) regeneration, and promoting electron release from electron pools, was developed to redirect electron flux into the electron transfer chain in Shewanella oneidensis MR-1. Among them, four genes include gene SO1522 encoding a lactate transporter for broadening the sources of the intracellular electron pool, gene gapA encoding a glyceraldehyde-3-phosphate dehydrogenase and gene mdh encoding a malate dehydrogenase in the central carbon metabolism for enhancing intracellular NADH regeneration, and gene ndh encoding NADH dehydrogenase on the inner membrane for releasing electrons from intracellular electron pools into the electron-transport chain. Upon assembly of the four genes, electron flux was directly redirected from the electron donor to the electron-transfer chain, achieving 62% increase in intracellular NADH levels, which resulted in a 3.5-fold enhancement in the power density from 59.5 ± 3.2 mW/m2 (wild type) to 270.0 ± 12.7 mW/m2 (recombinant strain). This study confirmed that redirecting electron flux from the electron donor to the electron-transfer chain is a viable approach to enhance the EET rate of S. oneidensis.