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Proc Natl Acad Sci U S A. 2017 Feb 21;114(8):E1470-E1479. doi: 10.1073/pnas.1621250114. Epub 2017 Feb 7.

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

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Institute for Systems Genetics, New York University Langone Medical Center, New York, NY 10016.
School of Medicine, Johns Hopkins University, Baltimore, MD 21205.
Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, TX 75390-9038.
Central Laboratories for Key Technologies, Kirin Company Limited, Yokohama, Kanagawa 236-0004, Japan.
School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom.
School of Computer Science and Electronic Engineering, University of Essex, Colchester CO4 3SQ, United Kingdom.
Institute for Systems Genetics, New York University Langone Medical Center, New York, NY 10016;


As the use of synthetic biology both in industry and in academia grows, there is an increasing need to ensure biocontainment. There is growing interest in engineering bacterial- and yeast-based safeguard (SG) strains. First-generation SGs were based on metabolic auxotrophy; however, the risk of cross-feeding and the cost of growth-controlling nutrients led researchers to look for other avenues. Recent strategies include bacteria engineered to be dependent on nonnatural amino acids and yeast SG strains that have both transcriptional- and recombinational-based biocontainment. We describe improving yeast Saccharomyces cerevisiae-based transcriptional SG strains, which have near-WT fitness, the lowest possible escape rate, and nanomolar ligands controlling growth. We screened a library of essential genes, as well as the best-performing promoter and terminators, yielding the best SG strains in yeast. The best constructs were fine-tuned, resulting in two tightly controlled inducible systems. In addition, for potential use in the prevention of industrial espionage, we screened an array of possible "decoy molecules" that can be used to mask any proprietary supplement to the SG strain, with minimal effect on strain fitness.


Rpd3L; escape mutants; genome safety; histone deacetylase; yeast

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