Directing Inward Electron Transport to the Cytosol in Mtr-Escherichia coli Using Fumarate Reductase

The use of exoelectrogens in microbial electrosynthesis offers the benefit of high Faradaic efficiency due to the electron transfer through direct contact between their transmembrane electron transfer (ET) system and the electrode. Currently, the only molecularly-defined inward ET pathway is found in Shewanella oneidensis, wherein the electrons passing through the Mtr-CymA ET system are been urilize by the periplasmic fumarate reductase FccA for fumarate reduction. Nonetheless, the reducing equivalents in this system do not pass through the inner membrane barrier to cytosolic carriers, where they would be of most value for microbial electrosynthesis. Our group has transplanted the S. oneidensis inward ET system into the host organism Escherichia coli, enabling outward ET through a heterologous system. However, it was unknown if these proteins are sufficient to confer inward electron transfer as well in E. coli. Using a three-electrode electrochemical system, we monitored electron flow from cathodes to unmodified and Mtr-expressing E. coli in response to fumarate addition. We found only the Mtr- E. coli consumed significant levels of current and sustained this consumption over 5 days. By using a knockout mutant and complementation, we showed that Mtr-modified E. coli can use the native fumarate reductase FrdABCD for cathode-driven fumarate reduction. We also found that over-expression of FrdABCD significantly increases current production in Mtr-E. coli. In contrast to FccA, FrdABCD is localized to the cytoplasmic face of the inner membrane and the reduction reaction occurs in the cytoplasm, making this a complete inward ET pathway. Thus, we have described for the first time a molecularly-defined inward ET pathway to the cytosol which opens the door for the delivery of electrode-derived reducing equivalents to cytosolic metabolic pathways.