Low escape-rate genome safeguards with minimal molecular perturbation of Saccharomyces cerevisiae

Significance As the use of synthetic biology grows, there is an increasing need to ensure biocontainment both to protect engineered proprietary strains and to mitigate potential inadvertent or advertent release to the environment. Here, we screen for the best-performing construct for essential gene-dependent transcriptional safeguards (SGs) in yeast. We have engineered strains that have near-WT fitness and a low escape rate, and grow in the presence of a nanomolar concentration of essential supplement. We also improved our “safeguard” construct by promoter engineering, as well as by analyzing a series of potential “decoy molecules” that could be used to mask the existence of critical chemical ligands required to grow the SG strain.