Low escape-rate genome safeguards with minimal molecular perturbation ofSaccharomyces cerevisiae

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 yeastbased 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 cerevisiaebased 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.genome safety | escape mutants | Rpd3L | histone deacetylase | yeast W ith the increasing use of genetically modified organisms, there is a growing need for biocontainment to secure biosystems from outside malice as well as to prevent propagation in an open system in the event of advertent or inadvertent release to the environment. Although recombinant DNA technology was established more than four decades ago (1), the fact that genetically modified organisms have not caused any substantial deleterious incident is due, in part, to precautionary measures taken by professional genetic engineers and the high cost of genetic engineering, largely restricting its use to academic and industrial laboratories. However, in the current era of "do-ityourself" synthetic biology, with rapid technological advances in DNA synthesis and assembly (2), genome editing (3, 4) and computational tools for design (5-9), and with the decreasing cost of DNA synthesis, the need for genomic safeguards (SGs) is clear.Early biocontainment efforts focused on the use of metabolic auxotrophy dependence (10, 11), toxin/antitoxin-dependent suicide (12-15), or both (16,17). Although top performers using these strategies do comply with the NIH standard for SGs (10

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