Tuning Extracellular Electron Transfer byShewanella oneidensisUsing Transcriptional Logic Gates

Abstract

AbstractExtracellular electron transfer pathways, such as those in the bacteriumShewanella oneidensis, interface cellular metabolism with a variety of redox-driven applications. However, designer control over EET flux inS. oneidensishas proven challenging since a functional understanding of its EET pathway proteins and their effect on engineering parameterizations (e.g., response curves, dynamic range) is generally lacking. To address this, we systematically altered transcription and translation of single genes encoding parts of the primary EET pathway ofS. oneidensis, CymA/MtrCAB, and examined how expression differences affected model-fitted parameters for Fe(III) reduction kinetics. Using a suite of plasmid-based inducible circuits maintained by appropriateS. oneidensisknockout strains, we pinpointed construct/strain pairings that expressedcymA, mtrA, andmtrCwith maximal dynamic range of Fe(III) reduction rate. These optimized EET gene constructs were employed to create Buffer and NOT gate architectures, that predictably turn on and turn off EET flux, respectively, in response to IPTG. Furthermore, we found that response functions generated by these logic gates (i.e., EET activity vs. inducer concentration) were comparable to those generated by conventional synthetic biology circuits, where fluorescent reporters are the output. Our results provide insight on programming EET activity with transcriptional logic gates and suggest that previously developed transcriptional circuitry can be adapted to predictably control EET flux.