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. 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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Free PMC article

Low Escape-Rate Genome Safeguards With Minimal Molecular Perturbation of Saccharomyces cerevisiae

Neta Agmon et al. Proc Natl Acad Sci U S A. .
Free PMC article

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.

Keywords: Rpd3L; escape mutants; genome safety; histone deacetylase; yeast.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Outline of steps to create shuffle strains. Genes were amplified from the yeast genome with 500 bp upstream and 200 bp downstream, as well as 50 bp in each end, for recombination with the pRS416 vector. SmaI-digested pRS416 and each amplified essential gene were transformed into the appropriate heterozygous diploid strain. Transformations (30 μL) were dripped onto Omnitrays, and single colonies were picked. Shuffle plasmid cloning was verified using plasmid primers and a gene internal primer. Following plasmid verification, all strains were sporulated and G418-resistant Ura+ colonies were saved (both MATa and MATα strains were saved). YFEG, your favorite essential gene.
Fig. 2.
Fig. 2.
Screening for best SG strain after integration of the SG construct. Following transformation into the shuffle strains and plating on 5-Foa, 10 candidates from each essential gene were examined by plating on YPGAL vs. YPD. (A) Twelve best-performing strains were chosen for GEV plasmid transformation. Eight isolates from each SG strain were transformed with GEV-containing plasmid and analyzed using a dot assay on dextrose without estradiol. (B) Thirteen good candidates that grew well on SC-Ura without estradiol and did not grow on SC-Ura without estradiol were chosen for further analysis. (C) Liquid cultures were diluted and subjected to optical density measurements every 10 min for 24 h. A growth curve was created, and doubling time was calculated for each strain. The experiment included three independent cultures for each strain to calculate SD. (D) Comparison of our three best SG strains with the previously published ones (20) shows significantly lower growth on YPD medium compared with YPGAL, indicating that this screen identified better-performing SG strains.
Fig. 3.
Fig. 3.
MATa and MATα SG strains compared with WT strain with GEV plasmid. (A) Graphic representation of the relative doubling time of SG strains compared with WT with GEV plasmid. Liquid cultures were diluted and subjected to optical density measurements every 10 min for a 24-h period. Doubling time was calculated as in Fig. 2. (B) Transcriptome profiling of the various SG strains. The graph is organized by gene/promoter pairs. Red dots in the volcano plots represent statistically significantly dysregulated genes (a list of genes affected is provided in SI Appendix, Table S1). The blue-labeled dots represent the SG gene in each sample. The transcriptome profiling shows limited transcriptome changes to the SG strains compared with WT. (C) Metabolomics analysis presented as a heat map of the fold change for each metabolite analyzed. Formic (formic acid 0.1%), tributylamine (TBA) (10 mM, pH 5.0), and NH4 (5 mM, ammonium acetate) represent the solvents used to extract the yeast. MATa SG strains: NAy407 (FAS2), NAy409 (RPB11), and NAy411 (SEC4); MATa control, NAy461; MATα SG strains: NAy408 (FAS2), NAy410 (RPB11), and NAy412 (SEC4); MATα control, NAy462.
Fig. 4.
Fig. 4.
Analysis of SG escapers. (A) We hypothesized that because all of the FAS2 and SEC4 escapers were due to recessive mutations, diploid SG strains will have lower escape rates. Serial dilutions of FAS2 and SEC4 diploid SG strain were compared with the original haploid SG strains and WT strains on the appropriate medium with and without estradiol. This comparison shows that, indeed, the diploids show a decreased escape rate compared with the haploid strains. All strains are shown in biological duplicates to demonstrate reproducibility. (B) Genome sequencing revealed that all escapers carry a mutation in genes encoding subunits of the Rpd3L complex. To reconstitute the escaper phenotype in our SG strains, we deleted ume6 in the FAS2 MATa SG strain. Serial dilutions of the FAS2 SG strain deleted for ume6 and eaf3 and carrying a pRS416-GEV plasmid were plated in appropriate medium with and without estradiol. Cells deleted for ume6 completely recapitulated the escaper phenotype.
Fig. 5.
Fig. 5.
SPAZ promoter. (A) Schematic representation of the SPO13 promoter with the URS1 sequence (purple box), the SPAL5 promoter with five GAL4-binding sites (turquoise boxes), and the SPAZ promoter with the six repeats of the Z4-binding site (orange boxes). (B) SPAZ promoter used to drive our three top SG genes: FAS2, RPB11, and SEC4. Serial dilutions of all strains compared with their shuffle strains containing the ZEV-expressing plasmid were plated on YPD media with or without 1 μM estradiol. (C) Growth of all three SPAZ SG strains was measured compared with BY4741 WT strain in YPD medium containing 30 nM estradiol. (D) Metabolomics analysis presented as a heat map of the fold change from each metabolite analyzed. SG strains used were NAy484 (FAS2), NAy486 (RPB11), and NAy488 (SEC4), and the control was NAy497.
Fig. 6.
Fig. 6.
Engineering the TET promoter. (A) Schematic representation of the TET, SPET, and 3×SPET with the ADH1 transcription terminator sequence (purple box), tetO repeats (blue triangles), and CYC1 TATA region (green arrow); arrowheads indicate the TATA box location and SspI restriction site. For the SPET promoter, a single URS1 sequence was cloned into the SspI site. For 3×SPET, three repeats of the URS1 sequence (with linkers) were cloned into the SspI site. (B) TET (two isolates), SPET (two isolates), and 3×SPET (four isolates) promoters were used to drive the SEC4 gene. Serial dilutions of all strains compared with the shuffle strain were plated on YPD medium with or without 10 μg/mL doxycycline (Dox).
Fig. 7.
Fig. 7.
Decoy molecules: effect on transcription. Graphical representation of the number of transcripts/genes changed in response to each specific candidate decoy compound and concentration. BY4741 WT cells were grown in liquid culture in the presence of each candidate decoy molecule (_1 and _100 represent 1 μM and 100 μM, respectively) and subjected to transcriptome analysis. The letters in brackets refer to the solvent the compound was dissolved in: DMSO (D), ethanol (E), or water (W). DAPG, 2,4-diacetylphloroglucinol; IPTG, isopropyl-β-d-thiogalactopyranoside; NAA, 1-naphthaleneacetic acid; OAH, 3-oxo-octanoyl-l-homoserine; SSA, sodium salicylate.

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