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.

An extranuclear replication system, consisting of an orthogonal DNA plasmid-DNA polymerase pair, was developed in Saccharomyces cerevisiae. Engineered error-prone DNA polymerases showed complete mutational targeting in vivo: per-base mutation rates on the plasmid were increased substantially and remained stable with no increase in genomic rates. Orthogonal replication serves as a platform for in vivo continuous evolution and as a system whose replicative properties can be manipulated independently of the host's.

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

We present automated continuous evolution (ACE), a platform for the hands-free directed evolution of biomolecules. ACE pairs OrthoRep, a genetic system for continuous targeted mutagenesis of user-selected genes in vivo, with eVOLVER, a scalable and automated continuous culture device for precise, multi-parameter regulation of growth conditions. By implementing real-time feedback-controlled tuning of selection stringency with eVOLVER, genes of interest encoded on OrthoRep autonomously traversed multimutation adaptive pathways to reach desired functions, including drug resistance and improved enzyme activity. The durability, scalability, and speed of biomolecular evolution with ACE should be broadly applicable to protein engineering as well as prospective studies on how selection parameters and schedules shape adaptation.Continuous evolution has emerged as a powerful paradigm for the evolution of proteins and enzymes 1-3 towards challenging functions 4,5 . In contrast to classical directed evolution approaches that rely on stepwise rounds of ex vivo mutagenesis, transformation into cells, and selection 6 , continuous evolution systems achieve rapid diversification and functional selection autonomously, often through in vivo targeted mutagenesis systems (Fig. 1a). The result is a mode of directed evolution that requires only the basic culturing of cells, in theory, enabling extensive speed, scale, and depth in evolutionary search 3 . In practice, however, developing a continuous evolution method that realizes all three properties has been challenging.Recently, our groups made two independent advances that can pair to achieve continuous evolution at significant speed, scale, and depth. These advances are OrthoRep and eVOLVER. First, OrthoRep. OrthoRep is an engineered genetic system for continuous in vivo targeted mutagenesis of genes of interest (GOIs) 2,7 . OrthoRep uses a highly error-prone, orthogonal DNA polymerase-plasmid pair in yeast that replicates GOIs at a mutation rate of 10 -5 substitutions per base (spb) without increasing the genomic mutation rate of 10 -10 spb (Fig. 1a). This ~100,000-fold increase in the mutation rate of GOIs drives their accelerated evolution (speed). Because the OrthoRep system functions entirely in vivo and culturing yeast is straightforward, independent GOI evolution experiments can be carried out in high-throughput (scale). In addition, long multimutation pathways can be traversed using OrthoRep, owing to the durability of mutagenesis over many generations (depth). However, to practically realize depth in evolutionary search, in vivo