Biological electron transfer is challenging to directly regulate using environmental conditions. To enable dynamic, protein-level control over energy flow in metabolic systems for synthetic biology and bioelectronics, we created ferredoxin logic gates that utilize transcriptional and post-translational inputs to control energy flow through a synthetic electron transfer pathway that is required for bacterial growth. These logic gates were created by subjecting a thermostable, plant-type ferredoxin to backbone fission and fusing the resulting fragments to a pair of proteins that self-associate, a pair of proteins whose association is stabilized by a small molecule, and to the termini of a ligand-binding domain. We show that the latter domain insertion design strategy yields an allosteric ferredoxin switch that acquires an oxygen-tolerant [2Fe–2S] cluster and can use different chemicals, including a therapeutic drug and an environmental pollutant, to control the production of a reduced metabolite in Escherichia coli and cell lysates.
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E. coli EW11 and the genes encoding Zea mays FNR, Zea mays SIR, and Spinacia oleracea Fd were a gift from P. Silver (Harvard University). Cellular assay development was supported by DOE grant DE-SC0014462 (G.N.B. and J.J.S.), split Fd efforts were supported by NASA grant NNX15AL28G (J.J.S. and G.N.B.), domain insertion efforts were supported by ONR grant N00014-17-1-2639 (J.J.S.), and electrochemistry was supported by DOE grant DE-SC0012598 (S.J.E.). J.T.A. was supported by NSF GRFP and DOE SGCSR fellowships.
J.J.S., J.T.A., G.N.B., and I.J.C. have submitted a patent application (No 16/186,226) covering the use of fragmented Fds as chemical-dependent electron carriers, entitled “Regulating electron flow using fragmented proteins.”
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Atkinson, J.T., Campbell, I.J., Thomas, E.E. et al. Metalloprotein switches that display chemical-dependent electron transfer in cells. Nat Chem Biol 15, 189–195 (2019). https://doi.org/10.1038/s41589-018-0192-3
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