Engineering stringent genetic biocontainment of yeast with a protein stability switch

Abstract

AbstractSynthetic biology holds immense promise to tackle key problems we are facing, for instance in resource use, environmental health, and human health care. However, comprehensive safety measures are needed to deploy genetically engineered microorganisms in open-environment applications. Here, we describe a genetic biocontainment system based on conditional stability of essential proteins. We used a yeast-adapted destabilizing domain degron, which can be stabilized by estradiol addition (ERdd). Leveraging the yeast GFP collection and lab automation platforms, we ERdd-tagged 775 essential genes and screened for strains with estradiol dependent growth. Three genes,SPC110, DIS3andRRP46, were found to be particularly suitable targets. Respective strains showed no growth defect in the presence of estradiol and strong growth inhibition in its absence.SPC110-ERddoffered the most stringent containment, with an escape frequency of 7.0×10-8, and full growth restoration at 100 nM estradiol. By systematically analyzing the containment escapees, we identified the non-essential C-terminal region ofSPC110as target for escape mutations. Its removal decreased the escape frequency with a single ERdd tag further to 4.3×10-9. CombiningSPC110-ERddwith a second ERdd tag on eitherDIS3orRRP46resulted in escape frequencies below the detection limit of the used assay (<2×10-10). Being based on conditional protein stability, this approach is mechanistically orthogonal to previously reported intrinsic biocontainment systems. It thus can be readily combined with other systems, for instance ones based on transcriptional or translational control of essential gene expression, to achieve multiplexed, extremely stringent control over the survival of engineered organisms.SignificanceSynthetic biology holds enormous potential to tackle key issues humanity is facing and can for instance revolutionize agriculture, bioremediation or health care. In each case, the unchecked spread of engineered organisms in natural environments must be prevented. This is particularly problematic with use cases of engineered microbes in open environments. Intrinsic, genetically encoded biocontainment systems, which control cell survival based on environmental cues, can solve this issue. We have developed such a genetic biocontainment system acting on the stability of essential proteins, leveraging a switchable degron. Through a large-scale screening for suitable essential target genes, we were able to create yeast strains that are strictly dependent on estradiol. Supplied with this small molecule, the engineered cells maintain high fitness and grow as robustly as the unmodified strains.