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Modular engineering to increase intracellular NAD(H/+) promotes rate of extracellular electron transfer of Shewanella oneidensis.

ABSTRACT: 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.

SUBMITTER: Li F

PROVIDER: S-EPMC6128845 | biostudies-other | 2018 Sep

REPOSITORIES: biostudies-other

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Modular engineering to increase intracellular NAD(H/+) promotes rate of extracellular electron transfer of Shewanella oneidensis.

Li Feng F Li Yuan-Xiu YX Cao Ying-Xiu YX Wang Lei L Liu Chen-Guang CG Shi Liang L Song Hao H

Nature communications 20180907 1

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/+) ...[more]

PMID: 30194293

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Project description:UnlabelledShewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe (bacterial flavin adenine dinucleotide [FAD] exporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular flavins, while overexpression of bfe increased the concentration of extracellular flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the ?bfe mutant. With the same concentration of supplemented flavins, the two strains produced similar amounts of current. However, when exogenous flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria.ImportanceExtracellular electron transfer by microbes is critical for the geochemical cycling of metals, bioremediation, and biocatalysis using electrodes. A controversy in the field was addressed by demonstrating that flavin electron shuttling, not direct electron transfer or nanowires, is the primary mechanism of extracellular electron transfer employed by the bacterium Shewanella oneidensis. We have identified a flavin adenine dinucleotide transporter conserved in all sequenced Shewanella species that facilitates export of flavin electron shuttles in S. oneidensis. Analysis of a strain that is unable to secrete flavins demonstrated that electron shuttling accounts for ~75% of the insoluble extracellular electron transfer capacity in S. oneidensis.

Project description:Extracellular iodate reduction by Shewanella spp. contributes to iodide generation in the biogeochemical cycling of iodine. However, there is a disagreement on whether Shewanella spp. use different extracellular electron transfer pathways with dependence on electron donors in iodate reduction. In this study, a series of gene deletion mutants of Shewanella oneidensis MR-1 were created to investigate the roles of dmsEFABGH, mtrCAB, and so4357-so4362 operons in iodate reduction. The iodate-reducing activity of the mutants was tested with lactate, formate, and H2 as the sole electron donors, respectively. In the absence of single-dms gene, iodate reduction efficiency of the mutants was only 12.9%-84.0% with lactate at 24 hours, 22.1%-85.9% with formate at 20 hours, and 19.6%-57.7% with H2 at 42 hours in comparison to complete reduction by the wild type. Progressive inhibition of iodate reduction was observed when the dms homolog from the so4357-so4362 operon was deleted in the single-dms gene mutants. This result revealed complementation of dmsEFABGH by so4357-so4362 at the single-gene level, indicating modularity of the extracellular electron transfer pathway encoded by dmsEFABGH operon. Under the conditions of all electron donors, significant inhibition of iodate reduction and accumulation of H2O2 were detected for ΔmtrCAB. Collectively, these results demonstrated that the dmsEFABGH operon encodes an essential and modular iodate-reducing pathway without electron donor dependence in S. oneidensis MR-1. The mtrCAB operon was involved in H2O2 elimination with all electron donors. The findings in this study improved the understanding of molecular mechanisms underlying extracellular iodate reduction.IMPORTANCEIodine is an essential trace element for human and animals. Recent studies revealed the contribution of microbial extracellular reduction of iodate in biogeochemical cycling of iodine. Multiple reduced substances can be utilized by microorganisms as energy source for iodate reduction. However, varied electron transfer pathways were proposed for iodate reduction with different electron donors in the model strain Shewanella oneidensis MR-1. Here, through a series of gene deletion and iodate reduction experiments, we discovered that the dmsEFABGH operon was essential for iodate reduction with at least three electron donors, including lactate, formate, and H2. The so4357-so4362 operon was first demonstrated to be capable of complementing the function of dmsEFABGH at single-gene level.

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Modular engineering to increase intracellular NAD(H/+) promotes rate of extracellular electron transfer of Shewanella oneidensis.

Publications

Modular engineering to increase intracellular NAD(H/<sup>+</sup>) promotes rate of extracellular electron transfer of Shewanella oneidensis.

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