Abstract
Unwanted evolution of designed DNA sequences limits metabolic and genome engineering efforts. Engineered functions that are burdensome to a host cell and slow its replication are rapidly inactivated by mutations, and unplanned mutations with unpredictable effects often accumulate alongside designed changes in large-scale genome editing projects. We developed a directed evolution strategy, Periodic Reselection for Evolutionarily Reliable Variants (PResERV), to discover mutations that prolong the function of a DNA sequence in an engineered organism. Here, we used PResERV to isolate E. coli cells that replicate ColE1 plasmids with higher fidelity. We found mutations in DNA polymerases I and IV and in RNase E that reduce plasmid mutation rates by 6- to 30-fold. Using these antimutator E. coli and applying PResERV to other chassis organisms in the future promises to prevent evolutionary failures and unpredictability to provide a more stable genetic foundation for synthetic biology.