Dataset Information

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

SUBMITTER: Agmon N

PROVIDER: S-EPMC5338387 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Low escape-rate genome safeguards with minimal molecular perturbation of <i>Saccharomyces cerevisiae</i>.

Agmon Neta N Tang Zuojian Z Yang Kun K Sutter Ben B Ikushima Shigehito S Cai Yizhi Y Caravelli Katrina K Martin James A JA Sun Xiaoji X Choi Woo Jin WJ Zhang Allen A Stracquadanio Giovanni G Hao Haiping H Hao Haiping H Tu Benjamin P BP Fenyo David D Bader Joel S JS Boeke Jef D JD

Proceedings of the National Academy of Sciences of the United States of America 20170207 8

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 s ...[more]

PMID: 28174266

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Project description:Fluorescence-based chelators are commonly used to probe labile low-molecular-mass (LMM) metal pools in the cytosol of eukaryotic cells, but such chelators destroy the complexes of interest during detection. The objective of this study was to use chromatography to directly detect such complexes. Towards this end, 47 batches of cytosol were isolated from fermenting S. cerevisiae yeast cells and passed through a 10 kDa cut-off membrane. The metal contents of the cytosol and resulting flow-through solution (FTS) were determined. FTSs were applied to a size-exclusion LC column located in an anaerobic refrigerated glove box. The LC system was coupled to an online inductively-coupled-plasma mass spectrometer (ICP-MS) for detection of individual metals. Iron-detected chromatograms of cytosolic FTSs from WT cells exhibited 2-4 major species with apparent masses between 500-1300 Da. Increasing the iron concentration in the growth medium 40-fold increased the overall intensity of these peaks. Approximately 3 LMM cytosolic copper complexes with apparent masses between 300-1300 Da were also detected; their LC intensities were weak, but these increased with increasing concentrations of copper in the growth medium. Observed higher-mass copper-detected peaks were tentatively assigned to copper-bound metallothioneins Cup1 and Crs5. FTSs from strains in which Cup1 or the Cox17 copper chaperone were deleted altered the distribution of LMM copper complexes. LMM zinc- and manganese-detected species were also present in cytosol, albeit at low concentrations. Supplementing the growth medium with zinc increased the intensity of the zinc peak assigned to Crs5 but the intensities of LMM zinc complexes were unaffected. Phosphorus-detected chromatograms were dominated by peaks at apparent masses 400-800 Da, with minor peaks at 1000-1500 Da in some batches. Sulfur chromatograms contained a low-intensity peak that comigrated with a glutathione standard; quantification suggested a GSH concentration in the cytosol of ca. 13 mM. A second LMM sulfur peak that migrated at an apparent mass of 100 Da was also evident.

Dataset Information

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

Publications

Low escape-rate genome safeguards with minimal molecular perturbation of <i>Saccharomyces cerevisiae</i>.

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