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
Plant evolution has produced enzymes that may not be optimal for maximizing yield and quality in today’s agricultural environments and plant biotechnology applications. By improving enzyme performance, it should be possible to alleviate constraints on yield and quality currently imposed by kinetic properties or enzyme instability. Enzymes can be optimized more quickly than naturally possible by applying directed evolution, which entails mutating a target gene in vitro and screening or selecting the mutated gene products for the desired characteristics. Continuous directed evolution is a more efficient and scalable version that accomplishes the mutagenesis and selection steps simultaneously in vivo via error-prone replication of the target gene and coupling of the host cell’s growth rate to the target gene’s function. However, published continuous systems require custom plasmid assembly, and convenient multipurpose platforms are not available. We discuss two systems suitable for continuous directed evolution of enzymes, OrthoRep in Saccharomyces cerevisiae and EvolvR in Escherichia coli, and our pilot efforts to adapt each system for high-throughput plant enzyme engineering. To test our modified systems, we used the thiamin synthesis enzyme THI4, previously identified as a prime candidate for improvement. Our adapted OrthoRep system shows promise for efficient plant enzyme engineering.
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.
Genetically encoded biosensors are valuable for the optimization of small-molecule biosynthesis pathways, because they transduce the production of small-molecule ligands into a readout compatible with high-throughput screening or selection in vivo. However, engineering biosensors with appropriate response functions and ligand preferences remains challenging. Here, we show that the continuous hypermutation system, OrthoRep, can be effectively applied to evolve biosensors with a high dynamic range, reprogrammed activity toward desired noncognate ligands, and proper operational range for coupling to biosynthetic pathways. In particular, we encoded the allosteric transcriptional factor, BenM, on OrthoRep such that the propagation of host yeast cells resulted in BenM’s rapid and continuous diversification. When these cells were subjected to cycles of culturing and sorting on BenM activity in the presence and absence of its cognate ligand, muconic acid, or the noncognate ligand, adipic acid, we obtained multiple BenM variants that respond to their corresponding ligands. These biosensors outperform previously engineered BenM-based biosensors by achieving a substantially greater dynamic range (up to ∼180-fold induction) and broadened operational range. The expression of select BenM variants in the presence of a muconic acid biosynthetic pathway demonstrated sensitive biosensor activation without saturating response, which should enable pathway and host engineering for higher production of muconic and adipic acids. Given the streamlined manner in which high-performance and versatile biosensors were evolved using OrthoRep, this study provides a template for generating custom biosensors for metabolic pathway engineering and other biotechnology goals.
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