Simultaneous positive and negative selection of proteases in bacterium based on cell suicide and antibiotic resistance

Methods for engineering protease specificity and selectivity toward target substrates of therapeutic or industrial relevance are of wide interest. Herein, we report a bacterial system for the simultaneous detection and selection of protease activity on positive and negative target substrates. The system, based on Proteolysis‐triggered bActerial SuiCide and Antibiotic Resistance (PASCAR) in Escherichia coli, exploits the β‐lactamase and the human gasdermin D (GS) for the proteolysis‐responsive positive and negative selections, respectively. The applicability of the positive selection was illustrated with the directed evolution of the Tobacco etch virus protease toward the recognition of non‐native substrates. We also utilized the positive selection for the efficient evaluation of computationally redesigned protease variants. We constructed and optimized a series of GS mutants as suicide modules – with high to low selection stringencies – that would enable the use of PASCAR as a practically applicable dual selection system. This study provides a simple and easily accessible tool that facilitates the engineering of proteases with custom specificity and selectivity. Graphical and Lay Summary Methods for reprogramming protease specificity and selectivity towards target substrates of therapeutic or industrial relevance are of wide interest. This study reports a novel bacterial system for the simultaneous detection and selection of protease activity on positive and negative target substrates. The system was used to evolve new variants of the Tobacco Etch Virus protease and to verify computational design variants. This study provides a simple and easily accessible tool that facilitates the engineering of proteases with custom specificity and selectivity.