Modular Engineering Intracellular NADH Regeneration Boosts Extracellular Electron Transfer of Shewanella oneidensis MR-1

Abstract

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/m (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.

Shewanella oneidensis NADH Dehydrogenase Mutants Exhibit an Amino Acid Synthesis Defect

“…For example, it was necessary to eliminate pathways that compete for NADH in E. coli to improve 1-butanol production (Atsumi et al, 2008). Increasing NADH generation has also been used to enhance electric current production by S. oneidensis MR-1 (Li et al, 2018). These studies show the importance of NADH availability when engineering redox cofactor-dependent pathways.…”

Effects of Biofilm Transfer and Electron Mediators Transfer on Klebsiella quasipneumoniae sp.203 Electricity Generation Performance in MFCs

“…According to previous studies, the electron mediators-mediated extracellular electron transport mechanism is a circulation mechanism. The oxidized mediators are converted into the reduced mediators after being coupled intracellularly with the reduction products on the respiratory chain [33]. Then reduced mediators are discharged out of the extracellular, and the electrons are transferred to the electrode to be oxidized.…”

“…EET mechanism of EAB in MFCs can be summarized as follows: Intracellular electrons are transferred from the quinone pool on the inner membrane to the outside of the cell through the periplasm and outer membrane, during which it passes through a series of transmembrane cytochrome complexes. Electrons pass directly to the anode, which is the rst EET mechanism [9]. The second EET mechanism is that after the electrons pass through the transmembrane cytochrome complex, the electrons are not directly transferred to the anode, but are transferred to the anode through conductive pilus, that is, nanowires [10,11].…”

Understanding and engineering electrochemically active bacteria for sustainable biotechnology

“…Fdn in MR-1 is a homologue of nitrate-inducible formate dehydrogenase FDH-N in Escherichia coli (Jormakka et al 2002), suggesting that nitrate reduction would induce the expression of fdnGHI. MR-1 may also have an NAD-dependent formate dehydrogenase (SO_3922), while it has been reported that this gene does not significantly affect catabolism in MR-1 (Li et al 2018a). However, it has also been described that the introduction of NAD-dependent FDH in Moraxella sp.…”