Bioelectrochemical systems (BESs) span environmentally friendly applications including bioelectricity generation, bioremediation, biosensing, electrosynthesis, etc. Engineering an efficient electroactive Escherichia coli to leverage its enormous synthetic biology toolkit opens up the boundless potential for BES. After the initial screening, we first designed and constructed electroactive E. coli with multiple electron transfer pathways, which combined the direct Mtr pathway from Shewanella oneidensis MR-1 and the indirect phenazine-1-carboxylate (PCA) pathway from Pseudomonas aeruginosa PAO1. The dual pathways exhibited excellent electron transfer performance and complementarity. Subsequently, electron transfer efficiency was improved from the perspective of transmembrane electron transfer and the cell–electrode interface by coordinating the Mtr and PCA pathways and enhancing the biofilm formation ability. Meanwhile, molecular dynamics simulations and dissociation constant analyses revealed an interaction of PCA and the outer membrane cytochrome MtrC in the Mtr pathway. Finally, the engineered electroactive E. coli was applied in BES, where its current density in microbial fuel cells increased to 1994.9 mA m−2, and the inward current reached 120.4 μA cm−2. The bidirectional electron transfer capability was better than that of natural wild-type electroactive microbes, such as P. aeruginosa and S. oneidensis. In addition, the engineered electroactive E. coli promoted the fixation of CO2 in a microbial electrosynthesis system of succinate production. Furthermore, upon introducing a thiosulfate response module into the electroactive E. coli, the biosensor achieved real-time monitoring of thiosulfate. This work provides valuable reference points for the rational design and integration of different EET pathways in non-electroactive microorganisms to endow them with efficient electroactivity and also offers a possible and effective chassis cell for exploring bioelectrochemical processes and opening up further opportunities in BESs.
