Efficient extracellular electron transfer (EET) of exoelectrogens is essentially for practical applications of versatile bioelectrochemical systems. Intracellular electrons flow from NADH to extracellular electron acceptors via EET pathways. However, it was yet established how the manipulation of intracellular NADH impacted the EET efficiency. Strengthening NADH regeneration from NAD⁺, as a feasible approach for cofactor engineering, has been used in regulating the intracellular NADH pool and the redox state (NADH/NAD⁺ ratio) of cells. Herein, we first adopted a modular metabolic engineering strategy to engineer and drive the metabolic flux toward the enhancement of intracellular NADH regeneration. We systematically studied 16 genes related to the NAD⁺-dependent oxidation reactions for strengthening NADH regeneration in the four metabolic modules of S. oneidensis MR-1, i.e., glycolysis, C1 metabolism, pyruvate fermentation, and tricarboxylic acid cycle. Among them, three endogenous genes mostly responsible for increasing NADH regeneration were identified, namely gapA2 encoding a NAD⁺-dependent glyceraldehyde-3-phosphate dehydrogenase in the glycolysis module, mdh encoding a NAD⁺-dependent malate dehydrogenase in the TCA cycle, and pflB encoding a pyruvate-formate lyase that converted pyruvate to formate in the pyruvate fermentation module. An exogenous gene fdh* from Candida boidinii encoding a NAD⁺-dependent formate dehydrogenase to increase NADH regeneration in the pyruvate fermentation module was further identified. Upon assembling these four genes in S. oneidensis MR-1, ∼4.3-fold increase in NADH/NAD⁺ ratio, and ∼1.2-fold increase in intracellular NADH pool were obtained under anaerobic conditions without discharge, which elicited ∼3.0-fold increase in the maximum power output in microbial fuel cells, from 26.2 ± 2.8 (wild-type) to 105.8 ± 4.1 mW/m² (recombinant S. oneidensis), suggesting a boost in the EET efficiency. This modular engineering method in controlling the intracellular reducing equivalents would be a general approach in tuning the EET efficiency of exoelectrogens.

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