Summary Directed evolution is a powerful approach for engineering biomolecules and understanding adaptation. However, experimental strategies for directed evolution are notoriously laborintensive and low-throughput, limiting access to demanding functions, multiple functions in parallel, and the study of molecular evolution in replicate. We report OrthoRep, an orthogonal DNA polymerase-plasmid pair in yeast that stably mutates ~100,000-fold faster than the host genome in vivo, exceeding the error threshold of genomic replication that causes singlegeneration extinction. User-defined genes in OrthoRep continuously and rapidly evolve through serial passaging, a highly straightforward and scalable process. Using OrthoRep, we evolved drug-resistant malarial DHFRs in 90 independent replicates. We uncovered a more complex fitness landscape than previously realized, including 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. OrthoRep enables a new paradigm of routine, high-throughput evolution of biomolecular and cellular function.

11Directed evolution is a powerful approach for engineering biomolecules and understanding 12 adaptation 1-3 . However, experimental strategies for directed evolution are notoriously low-13 throughput, limiting access to demanding functions, multiple functions in parallel, and the 14 study of molecular evolution in replicate. Here, we report OrthoRep, a yeast orthogonal 15DNA polymerase-plasmid pair that stably mutates ~100,000-fold faster than the host 16 genome in vivo, exceeding error thresholds of genomic replication that lead to single-17 generation extinction 4 . User-defined genes in OrthoRep continuously and rapidly evolve 18 through serial passaging, a highly scalable process. Using OrthoRep, we evolved drug 19 resistant malarial DHFRs 90 times and uncovered a more complex fitness landscape than 20 previously realized 5-9 . We find rare fitness peaks that resist the maximum soluble 21 concentration of the antimalarial pyrimethamine -these resistant variants support growth 22 at pyrimethamine concentrations >40,000-fold higher than the wild-type enzyme can 23 tolerate -and also find that epistatic interactions direct adaptive trajectories to convergent 24 outcomes. OrthoRep enables a new paradigm of routine, high-throughput evolution of 25 biomolecular and cellular function. 26 27