Scalable, Continuous Evolution of Genes at Mutation Rates above Genomic Error Thresholds

Abstract

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.

Potential for Applying Continuous Directed Evolution to Plant Enzymes: An Exploratory Study

“…In contrast, ∆thiG cells carrying the pool of pEvolvR plasmids as well as the TaTHI4 pTarget plasmid showed no growth in cysteine-supplemented medium, although these cells grew well with thiamin supplementation (Figure 5B,C). This result indicates that pEvolvR expression imposes an insupportable burden on cells cultured in minimal medium, unlike the situation in rich medium [27]. Reducing the pEvolvR plasmid copy number by changing the plasmid backbone (from pBR322 to pBBR) [49] (Supplementary Figure S3A,B) or replacing the 0.2% glycerol carbon source with 0.4% or 0.8% glucose did not rectify the problem (Supplementary Figure S3C,D).…”

“…In continuous directed evolution, the function of the target gene is typically coupled to the growth rate of the host microbe by forcing the microbe to rely on the target gene, e.g., by complementing the loss of an essential host activity with a plasmid-borne target gene. Using this method, improved target genes are immediately identifiable with no need for additional screening [27][28][29]. In addition, tracking enzyme function in vivo avoids possible artifacts of in vitro screening assays, which may be poor facsimiles of physiological conditions.…”

“…The yeast OrthoRep system was developed from the p1 and p2 linear cytoplasmic plasmids of Kluyveromyces lactis [27,[31][32][33]. The p1-specific DNA polymerase TP-DNAP1 was transferred from p1 to a nuclear plasmid, leaving a backbone into which a target gene can be introduced ( Figure 2A); p2 remains in its native state, encoding its own DNA polymerase (TP-DNAP2) and the machinery needed to transcribe both p1 and p2 [33].…”

“…For continuous directed evolution, error-prone versions of TP-DNAP1 have been engineered; these polymerases introduce mutations into the p1-borne target gene without increasing the genomic mutation rate (Figure 2A). Error-prone TP-DNAP1s have mutation rates as high as 1 × 10 −5 substitutions per base, which is up to 10 5 times that of the host genome [27,33]. The ~10 5 -fold mutational acceleration, combined with serial passaging, enables rapid, continuous evolution of the target gene entirely in vivo.…”

“…Many herbicides act by inhibiting essential metabolic enzymes, and mutations that alleviate this inhibition confer resistance [58,59]. Continuous directed evolution has already been used to evolve antibiotic resistance in microbial enzymes [27,28]; this approach could be similarly applied to evolve herbicide-resistant crop enzymes. Using genome editing to put the resistant enzyme back in the crop would avoid using transgenes, which are now banned in various countries [60].…”