Evolution of C-Terminal Modification Tolerance in Full-Length and Split T7 RNA Polymerase Biosensors

T7 RNA polymerase (RNAP) is a powerful protein scaffold for the construction of synthetic biology tools and biosensors. However, both T7 RNAP and its split variants are intolerant to C-terminal modifications or fusions, thus placing a key limitation on their engineering and deployment. Here, we use rapid continuous-evolution approaches to evolve both full-length and split T7 RNAP variants that tolerate modified C termini and fusions to entire other proteins. Moreover, we show that the evolved split C-terminal RNAP variants can function as small-molecule biosensors, even in the context of large C-terminal fusions. This work provides a panel of modified RNAP variants with robust activity and tolerance to C-terminal fusions, and provides insights into the biophysical requirements of the C-terminal carboxylic acid functional group of T7 RNAP.