Terminal differentiation strategy for evolutionary stability. Integrase-mediated differentiation circuits improve evolutionary stability of burdensome and toxic functions in E. coli

Advances in synthetic biology, bioengineering, and computation allow us to rapidly and reliably program cells with increasingly complex and useful functions. However, because the functions we engineer cells to perform are typically burdensome to cell growth, they can be rapidly lost due to the processes of mutation and natural selection. To improve the evolutionary stability of engineered functions in a general manner, we develop a genetic differentiation circuit in Escherichia coli using unidirectional integrase-recombination, and apply it to a strategy of terminal differentiation. With this strategy differentiated cells uniquely express burdensome functions driven by the orthogonal T7 RNA polymerase, but their capacity to proliferate is limited in order to prevent the propagation of advantageous loss-of-function mutations that inevitably occur. Here we demonstrate computationally and experimentally that terminal differentiation increases duration and yield of high-burden expression, its evolutionary stability can be improved with strategic redundancy, and it can be applied to even toxic functions. Overall, this study provides an effective generalizable strategy for protecting burdensome engineered functions from evolutionary degradation.