(513f) Continuous Evolution of Engineered Synthetic Auxotrophs for Industrial Application

The development of safeguards to prevent undesired proliferation of genetically modified organisms is vital as the ease and scale of genetic engineering increases. The recently demonstrated biocontainment strategy of synthetic auxotrophy has conferred Escherichia coli strains with escape rates below a detection limit of 10^-12 escapees per cell in the absence of the non-standard amino acid biphenylalanine. However, the long-term effectiveness of synthetic auxotrophy, which is critical for its adoption in settings ranging from industrial bioreactors to environmental remediation, is unknown. Here, we report continuous evolution of several synthetic auxotrophic strains while adapting them to decreasing biphenylalanine concentrations. After 100 days of evolution, strains derived from one synthetic auxotroph exhibit no observable escape and are capable of normal growth rates at 100-fold lower biphenylalanine concentration than their initial ancestor. We use next-generation sequencing (NGS) and multiplex automatable genome engineering (MAGE) to determine and reconstruct selective mutations in the ancestral strain for investigation of their contribution to increased fitness at low biphenylalanine concentration. Overall, our findings reveal that synthetic auxotrophy is an effective biocontainment strategy for durations relevant to potential industrial or therapeutic uses.