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*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

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
Pages (from-to)E1470-E1479
JournalProceedings of the National Academy of Sciences (PNAS)
Volume114
Issue number8
Early online date7 Feb 2017
DOIs
Publication statusE-pub ahead of print - 7 Feb 2017

Keywords / Materials (for Non-textual outputs)

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

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