The slow rate of extracellular electron transfer (EET) of electroactive microorganisms remains a primary bottleneck that restricts the practical applications of bioelectrochemical systems. Intracellular NAD(H/+) (i.e., the total level of NADH and NAD+) is a crucial source of the intracellular electron pool from which intracellular electrons are transferred to extracellular electron acceptors via EET pathways. However, how the total level of intracellular NAD(H/+) impacts the EET rate in Shewanella oneidensis has not been established. Here, we use a modular synthetic biology strategy to redirect metabolic flux towards NAD+ biosynthesis via three modules: de novo, salvage, and universal biosynthesis modules in S. oneidensis MR-1. The results demonstrate that an increase in intracellular NAD(H/+) results in the transfer of more electrons from the increased oxidation of the electron donor to the EET pathways of S. oneidensis, thereby enhancing intracellular electron flux and the EET rate.

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.

BackgroundThe microbial fuel cell (MFC) is a green and sustainable technology for electricity energy harvest from biomass, in which exoelectrogens use metabolism and extracellular electron transfer pathways for the conversion of chemical energy into electricity. However, Shewanella oneidensis MR-1, one of the most well-known exoelectrogens, could not use xylose (a key pentose derived from hydrolysis of lignocellulosic biomass) for cell growth and power generation, which limited greatly its practical applications.ResultsHerein, to enable S. oneidensis to directly utilize xylose as the sole carbon source for bioelectricity production in MFCs, we used synthetic biology strategies to successfully construct four genetically engineered S. oneidensis (namely XE, GE, XS, and GS) by assembling one of the xylose transporters (from Candida intermedia and Clostridium acetobutylicum) with one of intracellular xylose metabolic pathways (the isomerase pathway from Escherichia coli and the oxidoreductase pathway from Scheffersomyces stipites), respectively. We found that among these engineered S. oneidensis strains, the strain GS (i.e. harbouring Gxf1 gene encoding the xylose facilitator from C. intermedi, and XYL1, XYL2, and XKS1 genes encoding the xylose oxidoreductase pathway from S. stipites) was able to generate the highest power density, enabling a maximum electricity power density of 2.1 ± 0.1 mW/m2.ConclusionTo the best of our knowledge, this was the first report on the rationally designed Shewanella that could use xylose as the sole carbon source and electron donor to produce electricity. The synthetic biology strategies developed in this study could be further extended to rationally engineer other exoelectrogens for lignocellulosic biomass utilization to generate electricity power.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0881-2) contains supplementary material, which is available to authorized users.

Microbial fuel cell (MFC) is an eco-friendly bio-electrochemical sys-tem that uses microorganism as biocatalyst to convert biomass into electricity. Glycerol, as a waste in the biodiesel refinery processes, is an appealing substrate for MFC. Nevertheless, glycerol cannot be utilized as carbon source by well-known exoelectrogens such as Shewanella oneidensis. Herein, to generate electricity by rapidly harnessing glycerol, the authors rationally constructed a Klebsiella pneumoniae-Shewanella oneidensis microbial consortium to efficiently harvest electricity from glyc-erol, in which K. pneumoniae converted glycerol into lactate, fed to S. oneidensis as carbon source and electron donor. To improve electricity output, the authors systematically engineered the consortium in terms of carbon flux distribution and efficiency of extracellular electron transfer (EET). To direct more carbon flux to lactate biosynthesis in K. pneumoniae, the authors eliminated the ethanol pathway by knocking out the alcohol dehydrogenase gene (adhE), and enhanced lactate biosynthesis by heterologously expressing a lactate dehydrogen-ase gene (ldhD) from Lactobacillus bulgaricus and a lactate transporter gene (lldP) from Escherichia coli. To facilitate EET between S. oneidensis and anode surfaces, a biosynthetic flavins pathway from Bacillus subtilis is introduced into S. oneidensis. The author further optimized the glycerol concentration, thus S. oneidensis could be continuously fed with lactate synthesized from K. pneumoniae at a constant rate. Our glycerol-fed MFC generated a maximum power density of 19.9 mW/m , significantly higher than that of the wild-type consor-tium. This work suggested that engineering microbial consortia is an effi-cient strategy to expand the spectrum of usable carbon sources and promote electricity power production in MFCs.

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