{Reference Type}: Journal Article
{Title}: A robust yeast biocontainment system with two-layered regulation switch dependent on unnatural amino acid.
{Author}: Chang T;Ding W;Yan S;Wang Y;Zhang H;Zhang Y;Ping Z;Zhang H;Huang Y;Zhang J;Wang D;Zhang W;Xu X;Shen Y;Fu X;
{Journal}: Nat Commun
{Volume}: 14
{Issue}: 1
{Year}: 2023 10 14
{Factor}: 17.694
{DOI}: 10.1038/s41467-023-42358-4
{Abstract}: Synthetic auxotrophy in which cell viability depends on the presence of an unnatural amino acid (unAA) provides a powerful strategy to restrict unwanted propagation of genetically modified organisms (GMOs) in open environments and potentially prevent industrial espionage. Here, we describe a generic approach for robust biocontainment of budding yeast dependent on unAA. By understanding escape mechanisms, we specifically optimize our strategies by introducing designed "immunity" to the generation of amber-suppressor tRNAs and developing the transcriptional- and translational-based biocontainment switch. We further develop a fitness-oriented screening method to easily obtain multiplex safeguard strains that exhibit robust growth and undetectable escape frequency (<~10-9) on solid media for 14 days. Finally, we show that employing our multiplex safeguard system could restrict the proliferation of strains of interest in a real fermentation scenario, highlighting the great potential of our yeast biocontainment strategy to protect the industrial proprietary strains.