MOESM1 of Engineering Shewanella oneidensis enables xylose-fed microbial fuel cell

Additional file 1: Figure S1. Construction of synthetic xylose metabolic pathways in Shewanella oneidensis MR-1. (A) Schematic of the plasmid with a synthesized functional fragment of genes. The restriction sites EcoRI and XbaI with the ribosome binding site (RBS) are located upstream of each codon-optimized gene sequence, while the restrictions SpeI and PstI are located downstream of the gene. (B) Four plasmid constructs with xylose utilization pathways. To construct the multigene assembly in S. oneidensis, a Biobrick compatible expression vector pYYDT was adopted, which was previously constructed in our laboratory. Layout of the four plasmid constructs containing gene components in the xylose pathway examined in this study. Figure S2. Xylose consumption rate by E. coil (BL21) and by the recombinant S. oneidensis strain. The error bars were calculated from triplicate experiments. Figure S3. Metabolic pathway of riboflavin synthesis from xylose fermentation in S. oneidensis. A synthetic intracellular xylose metabolic pathway, i.e. the oxidoreductase pathway including genes XYL1, XYL2 and XKS1 from S. stipites, is incorporated into S. oneidensis MR-1 to enable the direct utilization of xylose. Xylulose 5-phosphate, as a metabolite in the oxidoreductase pathway, was converted to ribulose-5-P by ribulose-phosphate 3-epimerase (encoded by the rpe gene) in the pentose phosphate pathway, which was a crucial precursor for the biosynthesis of riboflavin via the riboflavin synthesis pathway. Figure S4. Xylose consumption under anaerobic conditions with 10 mM and 50 mM fumarate. The error bars were calculated from triplicate experiments

MOESM2 of Engineering Shewanella oneidensis enables xylose-fed microbial fuel cell

Additional file 2: Table S1. Summary of the reported energy output of Xylose-Fed MFCs. Table S2. Genes used in this study. Table S3. Synthesized sequences of genes in this study. Table S4. Strains and plasmids used in this study. Table S5. Main constituents for S. oneidensis basal medium (SBM). Table S6. Main constituents for M9 buffer

Modular Engineering Intracellular NADH Regeneration Boosts Extracellular Electron Transfer of <i>Shewanella oneidensis</i> MR‑1

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 <i>via</i> 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 <i>S. oneidensis</i> MR-1, <i>i.e.</i>, glycolysis, C1 metabolism, pyruvate fermentation, and tricarboxylic acid cycle. Among them, three endogenous genes mostly responsible for increasing NADH regeneration were identified, namely <i>gapA2</i> encoding a NAD⁺-dependent glyceraldehyde-3-phosphate dehydrogenase in the glycolysis module, <i>mdh</i> encoding a NAD⁺-dependent malate dehydrogenase in the TCA cycle, and <i>pflB</i> encoding a pyruvate-formate lyase that converted pyruvate to formate in the pyruvate fermentation module. An exogenous gene <i>fdh</i>* from <i>Candida boidinii</i> encoding a NAD⁺-dependent formate dehydrogenase to increase NADH regeneration in the pyruvate fermentation module was further identified. Upon assembling these four genes in <i>S. oneidens</i>is 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 <i>S. oneidensis</i>), 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