Rewiring heme biosynthesis for improved pollutant degradation by Shewanella oneidensis

The slow rate of extracellular electron transfer (EET) in electroactive microorganisms poses a major bottleneck for the practical application of bioelectrochemical systems, such as microbial fuel cells (MFCs) and electrochemical remediation technologies. In Shewanella oneidensis, multi-heme cytochromes, integral to the metal reduction (Mtr) transmembrane electron conduit, play a critical role in determining EET efficiency. However, heme availability is constrained by the bacterium's native expression capacity, limiting the potential for efficient EET. To address this, we employed a modular synthetic biology approach, designing four functional modules to redirect metabolic pathways toward heme synthesis and assembly. Our results demonstrate that a substantial increase in heme levels broadens the EET pathway in S. oneidensis, enhancing electron flux and transfer rates. This is evidenced by a peak current density of 1311.3 mA/m² in microbial electrochemical cells (MECs) and a maximum voltage output of 311.5 mV in MFCs. Furthermore, in anaerobic reduction experiments using methyl orange as a model azo dye, the engineered strain exhibited superior performance, achieving a first-order reaction rate constant of 0.547 h^-1. This indicates that elevated heme levels markedly improve the bacterium's capacity to degrade organic pollutants. These findings not only confirm the pivotal role of heme in amplifying the EET pathway of S. oneidensis but also offer an innovative and practical strategy to overcome rate-limiting challenges in bioelectrochemical applications.