Directed evolution is a powerful approach for engineering new biomolecular and cellular functions [1–3]. In contrast to rational design approaches, directed evolution exploits diversity and evolution to shape the behavior of biological matter by applying the Darwinian cycle of mutation, selection, and amplification of genes and genomes. By doing so, the field of directed evolution has generated important insights into the evolutionary process [4–6] as well as useful RNAs, proteins, and systems with wide-ranging applications across biotechnology and medicine [7–11]. To mimic the evolutionary process, classical directed evolution approaches carry out cycles of ex vivo diversification on genes of interest (GOIs), transformation of the resulting gene libraries into cells, and selection of the desired function (Figure 1.1). Each iteration of this cycle is defined as a round of evolution, and as selection stringency increases over rounds, either automatically through competition or manually through changing conditions (or both), this process can lead GOIs closer and closer to the desired function. This overall process makes practical sense for a number of reasons, especially for the goal of protein engineering (i.e. GOI encodes a protein). First, ex vivo diversification is appropriate, because test tube molecular biology techniques such as DNA shuffling, site-directed saturation mutagenesis, and error-prone (ep) polymerase chain reaction (PCR) [2] are capable of generating exceptionally high and precise levels of sequence diversity for any GOI. Second, transforming diversified libraries of the GOI into cells is appropriate, because each GOI variant needs to be translated into a protein in order to express its function, and cells, especially model microbes, are naturally robust hosts for protein expression. Third, carrying out selection inside cells is appropriate, because (i) cells automatically maintain the genotype–phenotype connection between the GOI and expressed protein that is necessary for amplification of desired variants,

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, multiparameter 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.

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, the components of which (e.g., promoters) have previously limited the strength with which OrthoRep-encoded genes can be expressed. Here, we report a collection of synthetic and 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.

The yeast cytoplasmically localized pGKL1/TP-DNAP1 plasmid/DNA polymerase pair forms an orthogonal DNA replication system whose mutation rate can be drastically increased without influencing genomic replication, thereby supporting in vivo continuous evolution. Here, we report that the pGKL2/TP-DNAP2 plasmid/DNA polymerase pair forms a second orthogonal replication system. We show that custom genes can be encoded and expressed from pGKL2, that error-prone TP-DNAP2s can be engineered, and that pGKL2 replication by TP-DNAP2 is both orthogonal to genomic replication in Saccharomyces cerevisiae and mutually orthogonal with pGKL1 replication by TP-DNAP1. This demonstration of two mutually orthogonal DNA replication systems with tunable error rates and properties should enable new applications in cell-based continuous evolution, genetic recording, and synthetic biology at large.

Directed evolution is a powerful approach for engineering biomolecules and understanding adaptation. However, experimental strategies for directed evolution are notoriously labor intensive 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 single-generation 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 dihydrofolate reductases (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.

It's only unnatural: Reengineered genetic codes can achieve biocontainment by specifying different meanings for protein sequences in modified versus natural organisms; the synthetic bacterium discussed here is dependent on unnatural amino acids because its reengineered genetic code specifies unnatural amino acids in essential genes.

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.