The TonB-Dependent Transport System Facilitates the Uptake of Inorganic Metal Mediators in Pseudomonas putida KT2440 in a Bioelectrochemical System

Mediator-based extracellular electron transfer (EET) in a bioelectrochemical system is a unique approach to regulate the microbial redox and energy metabolism of Pseudomonas putida KT2440, which enables a new-to-nature high product yield under anaerobic conditions. Previous studies identified respiratory complex III in the inner membrane as a key redox protein involved in mediator (ferricyanide) interactions, but the exact mechanism through which the mediator crosses the outer membrane to extract electrons from membrane-bound redox proteins and transfer them to the anode remains unclear. In this study, we demonstrated the critical role of the TonB-dependent system, a widespread transportation system in gram-negative bacteria, in the mediator-based EET process. Transcriptomic analyses revealed significant upregulation of TonB-dependent receptors in response to ferricyanide exposure, suggesting their involvement in mediator uptake. Deletion of the TonB complex resulted in a > 50% decrease in the mediator reduction rate and current output, confirming the role of the TonB-dependent system in mediator transport. Additionally, increasing passive diffusion through the overexpression of the general porin OprF increased cell permeability and the mediator reduction rate, but it failed to compensate for the absence of TonB-dependent transport. These findings suggest that both systems act in a complementary manner: the TonB-dependent system is likely the primary mechanism for periplasmic mediator uptake, whereas OprF is likely involved mainly in mediator efflux. Further bioelectrochemical system experiments demonstrated that, with a functional TonB-dependent system, OprF overexpression increased current output, glucose consumption, and 2-ketogluconate production, suggesting a viable strategy for enhancing the efficacy of mediator-based EET. This work reveals the major mediator transport mechanism in P. putida and deepens the understanding of the mediator-based EET pathway, laying the basis for future rational engineering of EET kinetics and facilitating the integration of mediator-based electron transfer into industrial biotechnology to push its process boundaries.