Content area
While all cellular metabolism is based on the flow of electrons, some microbial species have evolved the ability to transfer electrons out of their cells to extracellular electron acceptors in a process called extracellular electron transfer (EET). It has been previously shown that EET can be regulated by incorporating a protein switch into multicomponent, synthetic electron transfer pathways that span the cytosol and cell membranes to the extracellular environment. However, these multicomponent pathways are metabolically burdensome and do not port easily between microbial species.
To enable simpler, single-protein regulation of electron flow for use in biosensors and synthetic biology, we created bacterial sulfite reductase (SiR) switches via ligand binding domain insertion. Bacterial SiR is a good option for simplifying electron transfer pathways because it moves electrons directly from NADPH to the reduction of sulfite independently. The SiR switches respond to the presence of certain environmental chemicals by functionalizing the metabolic activity of SiR which controls the production of hydrogen sulfide, a redox-active metabolite that can diffuse across the cell membrane.
Prior to the creation of the switches by domain insertion, an insertion tolerance profile of the SiR hemoprotein subunit was first mapped using systematic octapeptide insertion followed by a selection for retained function. Then a subset of variants enriched by the selection were chosen for domain insertion. When an estrogen receptor ligand-binding domain was inserted at locations tolerant to octapeptide insertion, more than half of the resulting variants presented activity that was enhanced by an environmental estrogen agonist.
This conditional switching activity could be monitored electrochemically in a strain of Escherichia coli using bioelectrochemical systems, illustrating how a single protein can be used in cells to convert chemical information in the environment into an electrical signal.