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Highlights • Increase of intracellular FAD concentration resulting in electroactive E. coli. • Neutral red-mediated electrosynthesis is applied to the MES reaction. • More reductive products are achieved by E. coli 8739(pRibAB-YbjI-YigB, pRibECF). • The succinate yield is improved by E. coli T110(pRibAB-YbjI-YigB, pRibECF). Abstract In this study, the FAD synthesis pathway was manipulated to increase its cellular concentration and thereby improve the electroactivity of E. coli. In a microbial electrosynthesis (MES) system with neutral red as electron carrier and fumarate as the sole electron acceptor, the engineered strains derived from three E. coli lines displayed increased electric current in the reaction system, indicating improved electroactivity. Furthermore, the production of succinate from fumarate increased by around 60% compared with that of the parent strains, confirming the improvement of E. coli electroactivity by manipulating the FAD synthesis pathway. An MES reaction was performed with engineered E. coli 8739, and an altered metabolic profile with more reductive fermentation products was obtained. When the electroactive succinate-producing strain E. coli T110 was used in the MES, a yield of 0.97 ± 0.02 mol/mol glucose was achieved, which corresponds to an approximately 1.4-fold increase compared with the fermentation with no electricity supply or non-electroactive T110. In addition, a carbon concentration mechanism (CCM) was employed to further improve succinate production and yield in the MES, which produced a succinate yield of 1.16 mol/mol glucose, a 1.7-fold increase compared with that of the parent strain T110, indicating that the electroactive E. coli could be used in MES to produce specific fermentation products with improved yield. [ABSTRACT FROM AUTHOR]

Renalase is a recently discovered secretory protein involved in the regulation of blood pressure. Cells synthesize all known isoforms of human renalase (1 and 2) as flavoproteins. Accommodation of FAD in the renalase protein requires the presence of its N-terminal peptide. However, in secretory proteins, such peptides are usually cleaved during their export from the cell. In the present study, we have isolated human renalase from urinary samples of healthy volunteers and human recombinant renalases 1 and 2 expressed in Escherichia coli cells. In these proteins, we investigated the presence of the renalase N-terminal peptide and the FAD cofactor and performed computer-aided molecular analysis of the renalase crystal structure to evaluate possible consequences of removal of the N-terminal peptide. In contrast to human recombinant renalase isoforms 1 and 2 containing non-covalently bound FAD and clearly detectable N-terminal peptide, renalase purified from human urine lacks both the N-terminal signal peptide and FAD. The computer-aided analysis indicates that the removal of this peptide results in inability of the truncated renalase to bind the FAD cofactor. Thus, our results indicate that human renalase secreted in urine lacks its N-terminal peptide, and therefore catalytic activities of urinary renalase reported in the literature cannot be attributed to FAD-dependent mechanisms. We suggest that FAD-dependent catalytic functions are intrinsic properties of intracellular renalases, whereas extracellular renalases act in FAD- and possibly catalytic-independent manner. [ABSTRACT FROM AUTHOR]