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

Neta Agmon, Zuojian Tang, Kun Yang, Ben Sutter, Shigehito Ikushima, Yizhi Cai, Katrina Caravelli, James A. Martin, Xiaoji Sun, Woo Jin Choi, Allen Zhang, Giovanni Stracquadanio, Haiping Hao, Benjamin P. Tu, David Fenyo, Joel S. Bader, Jef D. Boeke

    Research output: Contribution to journalArticlepeer-review

    Abstract

    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.

    Original languageEnglish (US)
    Pages (from-to)E1470-E1479
    JournalProceedings of the National Academy of Sciences of the United States of America
    Volume114
    Issue number8
    DOIs
    StatePublished - Feb 21 2017

    Keywords

    • Escape mutants
    • Genome safety
    • Histone deacetylase
    • Rpd3L
    • Yeast

    ASJC Scopus subject areas

    • General

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