%0 Journal Article
%T A robust yeast biocontainment system with two-layered regulation switch dependent on unnatural amino acid.
%A Chang T
%A Ding W
%A Yan S
%A Wang Y
%A Zhang H
%A Zhang Y
%A Ping Z
%A Zhang H
%A Huang Y
%A Zhang J
%A Wang D
%A Zhang W
%A Xu X
%A Shen Y
%A Fu X
%J Nat Commun
%V 14
%N 1
%D 2023 10 14
%M 37838746
%F 17.694
%R 10.1038/s41467-023-42358-4
%X 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.