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

AbstractWe 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 multi-mutation 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.

We recently developed an orthogonal DNA replication (OrthoRep) system capable of driving the rapid continuous evolution of genes in vivo. However, OrthoRep uses a special transcription system whose components (e.g. promoters) have previously limited the strength with which OrthoRep-encoded genes can be expressed. Here, we report a collection of synthetic or evolved OrthoRep expression parts that allow OrthoRep-encoded genes to span expression levels matching those of endogenous Saccharomyces cerevisiae genes. Specifically, we found that various promoter mutations as well as a genetically-encoded poly(A) tail enable us to tune the expression level of OrthoRep-encoded genes over a large range and up to levels 43-fold higher than were previously attained, reaching at least ~40% of the strength of the genomic TDH3 promoter. We further show that expression level gains using our new parts are stable over passaging and consistent across multiple genes and OrthoRep systems of different mutation rates. This new set of expression parts further expands OrthoRep’s applicability to the continuous in vivo evolution of proteins and pathways.

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