Utilization of a heterologous exocellular electron transfer pathway in Escherichia coliand Vibrio natriegens

The Gram-negative acidophile Acidithiobacillus ferrooxidans is capable of iron reduction through exocellular electron transfer. Its electron transfer chain spans across both membranes and the periplasm and consists of three c-type cytochromes and the copper redox protein rusticyanin. To circumvent the low biomass production of acidophilic microorganisms, the proteins were heterologously produced to accelerate protein characterization and interaction studies. The heterologous production of proteins from acidophilic microorganisms in neutrophilic expression hosts presents a challenge mainly due to the difference in extracellular and periplasmic pH which can affect protein folding and co-factor integration. Posttranslational modifications as well as the synthesis and incorporation of heme into the recombinant c-type cytochromes pose an additional challenge for the host. We reconstructed the iron reduction pathway from At. ferrooxidans in four different E. coli strains and Vibrio natriegens, an emerging biotechnology chassis. By screening the periplasmic, cytoplasmic and membrane fractions we showed that V. natriegens was able to produce more mature c-type cytochromes and rusticyanin than the E. coli hosts. The periplasmic cytochrome c Cyc1 and rusticyanin were correctly translocated into the periplasm of the neutrophilic hosts while the inner and outer membrane-bound c-type cytochromes were present in the membrane fraction exclusively. The recombinant c-type cytochromes heterologously produced by V. natriegens and E. coli were redox-active. We were able to demonstrate that E. coli C43(DE3) is able to utilize the recombinant proteins from At. ferrooxidans for ferric iron reduction. To our knowledge, this is the first demonstration of electron transfer functionality of recombinant redox proteins from an acidophile in neutrophiles.