Deciphering Menaquinone-Ndh axis and combinatorial engineering of Shewanella oneidensis for boosting formate electrosynthesis

Microbial electrosynthesis (MES) is currently hindered by an incomplete understanding of electron-uptake mechanisms and a lack of specialized genetic parts for electroactive microorganisms. Here, a formate-oxidation-coupled visualization strategy was used to map electron transfer pathways in Shewanella oneidensis, revealing an association between the menaquinone (MQ)-linked respiratory network and Ndh-2 in formate-associated electron routing toward intracellular redox metabolism. Building on this finding, an indirect electron-transfer strategy was implemented to drive reductive metabolism and SoFdh2 was identified as an efficient catalytic module for electro-driven CO₂ reduction. Through enzyme engineering and metabolic rewiring, an electrosynthesis platform was established that enabled high-titer formate production. Altogether, this rational engineering of electron transport in S. oneidensis provides a mechanistic framework for advancing sustainable microbial electrosynthesis.