Improving the Robustness of Engineered Bacteria to Nutrient Stress Using Programmed Proteolysis

[Image: see text] The use of short peptide tags in synthetic genetic circuits allows for the tuning of gene expression dynamics and release of amino acid resources through targeted protein degradation. Here, we use elements of the Escherichia coli and Mesoplasma florum transfer-mRNA (tmRNA) ribosome rescue systems to compare endogenous and foreign proteolysis systems in E. coli. We characterize the performance and burden of each and show that, while both greatly shorten the half-life of a tagged protein, the endogenous system is approximately 10 times more efficient. On the basis of these results we then demonstrate using mathematical modeling and experiments how proteolysis can improve cellular robustness through targeted degradation of a reporter protein in auxotrophic strains, providing a limited secondary source of essential amino acids that help partially restore growth when nutrients become scarce. These findings provide avenues for controlling the functional lifetime of engineered cells once deployed and increasing their tolerance to fluctuations in nutrient availability.