LAUSR

Shewanella oneidensis MR‐1, as a model exoelectrogen with divergent extracellular electron transfer (EET) pathways, has been widely used in microbial fuel cells (MFCs). The electron transfer rate is largely determined by riboflavin (RF) and c‐type cytochromes (c‐Cyts). However, relatively low RF production and inappropriate amount of c‐Cyts substantially impede the capacity of improving the EET rate. In this study, coupling of riboflavin de novo biosynthesis and c‐Cyts expression was implemented to enhance the efficiency of EET in S. oneidensis. First, the upstream pathway of RF de novo biosynthesis was divided into four modules, and the expression level of 22 genes in above four modules was fine‐tuned by employing promoters with different strengths. Among them, genes zwf*, glyA, and ybjU which exhibited optimal RF production were combinatorially overexpressed, leading to the enhancement of maximum output power density by 166%. Second, the diverse c‐Cyts genes were overexpressed to match high RF production, and omcA was selected for further combination. Third, RF de novo biosynthesis and c‐Cyts expression were combined, resulting in 2.34‐fold higher power output than the parent strain. This modular and combinatorial manipulation strategy provides a generalized reference to advance versatile practical applications of electroactive microorganisms.