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

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.