Expression of the extracellular electron transfer pathway of Acidithiobacillus ferrooxidans enabling Fe(III)-citrate reduction and anodic respiration in Escherichia coli

Exoelectrogens are capable of transferring electrons through a defined route outside of the cell, making them ideal for microbial fuel cells, electrosynthesis and biosensing applications. Engineering well characterized hosts and transferring exoelectrogenic capabilities can further our understanding of these extracellular electron transfer pathways and lead to new developments in their utilization for biotechnological applications. The extreme acidophile Acidithiobacillus ferrooxidans is capable of extracellular iron reduction under anaerobic conditions. In order to confirm the functionality of the suggested iron reduction pathway, we produced the c-type cytochromes CycA2, Cyc1A, Cyc2A and the copper redox protein rusticyanin in Escherichia coli C43(DE3), BL21(DE3), T7 Express and Vibrio natriegens Vmax X2. Both Vmax X2 and C43(DE3) produced all four redox proteins in a redox-active state in the correct cell compartment. T7 Express was unable to produce holo-CycA and BL21(DE3) only reached low relative expression levels per OD₆₀₀ of holo-Cyc2. Introducing the At. ferrooxidans iron reduction pathway into E. coli lead to a 2-fold and 1.4-fold increased Fe(III)-citrate reduction per OD₆₀₀ in C43(DE3) and BL21(DE3) respectively. The inner membrane cytochrome CycA was necessary for efficient iron reduction in the E. coli strains. While Vmax X2 was able to produce the entire iron reduction pathway, it was unable to utilize it for Fe(III)-citrate reduction. The recombinant iron reduction pathway of C43(DE3) was redox active under turnover conditions and enabled 5-fold increased current production in an electrochemical H-cell while respiring with a graphite felt electrode. Overall, this study provides the first experimental validation of the suggested minimal iron reduction pathway of At. ferrooxidans consisting of CycA, Cyc1, Rus, and Cyc2.