Scalable continuous evolution of genes at mutation rates above genomic error thresholds

Abstract

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

Automated continuous evolution of proteinsin vivo

“…Additionally, the monotonically increasing pyrimethamine concentrations we observed for most replicates (Fig. 2b) are consistent with step-wise fixation of beneficial mutations expected for the evolution of PfDHFR resistance under strong selection 2,16 . Finally, ACE demonstrated a substantial increase in speed over our previous evolution campaign performed by manual passaging; with ACE, culture growth rates in 5/6 vials stabilized at the maximum pyrimethamine concentration after ~550 hours, which is over 200 hours faster than for the manual evolution campaign done with serial passaging 2 .…”

“…Yeast transformations were done with roughly 100 ng -1 µg of plasmid or donor DNA via the Gietz high-efficiency transformation method 18 . For integration of genes onto the orthogonal plasmid (pGKL1), cassettes were linearized with ScaI and subsequently transformed as described previously 2,7 . Standard preparations of YPD and drop-out synthetic media were obtained from US Biological.…”

“…( ) would then be used to determine the ratios of media to add during each dilution step by controlling the pump runtime. For example, if an ( ) = 0.25 was determined with a pump runtime of 5 seconds, the pump for the base media would run for [1 − ( )] * 5 seconds = 3.75 seconds while the pump for the full selection media would run for ( ) * 5 seconds = 1.25 seconds.The integral error (∫ ( )2 3…”

Biosystems design by directed evolution

“…(Figure b) . OrthoRep was recently used to evolve drug‐resistant malarial dihydrofolate reductases and uncovered more complex fitness landscapes than previously identified through normal adaptive evolution …”

A high-throughput platform for feedback-controlled directed evolution

“…Continuous directed evolution enables rapid population diversification and selection by coupling a single desired protein function to fitness, and has been used to engineer biological systems that are not tractable with rational design alone. [1][2][3][4][5] The most well-established continuous evolution method, Phage-Assisted Continuous Evolution (PACE), 6 has been successfully applied to a diverse set of protein engineering challenges, including the evolutions of proteases with altered substrate specificity, 7 of proteins with improved soluble expression, 8 and of CRISPR-Cas proteins with broadened PAM recognition. 9,10 The power of this technique comes from the ability to perform many rounds of directed evolution in a single day, making it ideal for challenging evolution goals that require numerous sequential mutations.…”