Cellular selection systems for continuous directed evolution using OrthoRep

In the past decade, newly developed continuous evolution systems have allowed biomolecules with demanding functions to be engineered with greatly reduced time and labor requirements. In this work, we expanded the range of proteins that can be evolved by our laboratory’s continuous evolution system, OrthoRep, and studied nuances regarding its replication system. By leveraging the scalability of OrthoRep, we evolved drug-resistant malarial dihydrofolate reductases (DHFRs) in 90 independent replicates and uncovered a more complex fitness landscape than previously realized. This included common adaptive trajectories constrained by epistasis, rare outcomes that avoid a frequent early adaptive mutation, and a suboptimal fitness peak that occasionally traps evolving populations. Next, we developed a selection for protein-protein interactions by linking the split DHFR protein-complementation assay (PCA) with OrthoRep. The dynamic range of selection was expanded to improve binders from low (Kd ~ 10 uM) to high (Kd ~ 1 nM) affinity. Using this system, we matured the affinity of leucine zipper pairs. Lastly, we showed that the p1 and p2 linear plasmids comprising OrthoRep are replicated through mutually orthogonal mechanisms. This finding sets the ground for future applications in dual-channel recording, as well as evolution of multiple proteins at custom mutation rates