Controlling Energy Flow in Bacteria Using Engineered Ligand-Responsive Protein Electron Carriers
- Conference: Synthetic Biology Engineering Evolution Design SEED
- Year: 2017
- Proceeding: 2017 Synthetic Biology: Engineering, Evolution & Design (SEED)
- Group: Poster Session
Energy flow remains hard to manipulate in cells because we have not yet discovered biological parts that can dynamically control electron flow in response to changes in environmental and metabolic conditions. To develop proteins that can be used to rapidly switch on and off electron transfer between defined oxidoreductase partner proteins, we have engineered a first generation of ligand-responsive protein electron carriers. For these studies, we have targeted the low potential, iron-sulfur cluster containing ferredoxin (Fd) electron carriers because these proteins conserve energy and can transfer electrons across diverse metabolic pathways by leveraging interactions with nearly one hundred different partner proteins. Our initial efforts have leveraged protein family sequence information to guide the design of two classes of Fd switches. We have used protein fragmentation to create split Fds whose fragments require assistance from a ligand-dependent protein-protein interaction for electron transfer. In addition, we have used domain insertion to create a ligand-dependent Fd. As a proof-of-concept application for each design, we analyzed whether our engineered Fds can complement the growth of an Escherichia coli sulfide auxotroph that only grows in minimal medium containing sulfate as the sole sulfur source if a Fd is present that can transfer electrons from a Fd-NADP+ reductase to sulfite reductase. We have found that both of our engineered Fds are able to complement the growth of this auxotroph in a ligand-dependent manner. When the fragments of the split Fd were fused to FKBP12 and the rapamycin binding domain of mTOR, complementation of the auxotroph required rapamycin in the growth medium, which stabilizes the FKBP12-mTOR complex. When the ligand-binding domain of the estrogen receptor was inserted into the Fd, the resulting protein only complemented bacterial growth when 4-hydroxytamoxifen was added to the growth medium. Ongoing efforts are focused on isolating purified recombinant Fd switches, investigating current flow in the on and off states, and establishing the mechanism by which these ligand-dependent Fds function. Our results demonstrate how low potential protein electron carriers can be engineered to enable post-translational control over electron flow in cells. These new ligand-responsive protein electron carriers are expected to have applications in biosensing, bioelectronics, and metabolic engineering.