Abstract
The widespread application of recombinant DNA and synthetic biology approaches for microbial metabolic engineering pursuits has motivated the development of biocontainment strategies, targeting safe and secure deployment of genetically modified microorganisms (GMMs). However, the design rules and mechanistic drivers governing biocontainment efficacy, as well as impacts of biocontainment upon microbial fitness, remain to be comprehensively evaluated, hindering predictive design and application of these strategies. We have developed a platform for high-resolution analysis of a transactivated kill switch in laboratory and industrial strains of Saccharomyces cerevisiae to assess modes of biocontainment escape and establish design rules for development of kill switch systems in diverse microbes. A camphor-regulated, RelE toxin system was systematically deployed to assess the impacts of differential kill switch copy number and ploidy in laboratory vs industrial strains. CRISPR-mediated integration of the biocontainment system at various loci revealed rapid escape events driven, in part, by mutations to both the Cam-transactivator (cam-TA) and RelE toxin. Genetic engineering enabled recapitulation of escape phenotypes, confirming mechanisms of escape and establishing structure-function relationships in the cam-TA system. Interestingly, genomic resequencing of escape mutants also revealed a series of off-target mutations, implicating additional modes of kill switch escape. Multi-copy integration of the kill switch system mitigated these effects by orders of magnitude, without compromising the biosynthetic capacity of the microbes, but proved insufficient to establish sustained biocontainment. The resultant data define a series of key design rules for next-generation biocontainment strategies and add to a growing foundational knowledge base targeting establishment of secure biosystems designs.IMPORTANCEThe development of biocontainment mechanisms is essential for safe deployment of microbes in industrial processes and to minimize escape into the natural environment. To achieve secure biosystems designs, a deeper understanding of the mechanistic drivers governing biocontainment efficacy and associated impacts of biocontainment upon microbial fitness are needed. This study uncovers successful biocontainment strategies for kill switch deployment in addition to mechanistic information conferring kill switch escape. Additionally, differential effects were observed in laboratory vs industrial yeasts, implicating auxotrophies and heterothallism as additional drivers of biocontainment. These learnings can inform iterative designs, with the goal of improving the efficacy of biocontainment while maintaining fitness in engineered microbes.