A Population-Based Experimental Model for Protein Evolution: Effects of Mutation Rate and Selection Stringency on Evolutionary Outcomes

Abstract

Protein evolution is a critical component of organismal evolution and a valuable method for the generation of useful molecules in the laboratory. Few studies, however, have experimentally characterized how fundamental parameters influence protein evolution outcomes over long evolutionary trajectories or multiple replicates. In this work, we applied phage-assisted continuous evolution (PACE) as an experimental platform to study evolving protein populations over hundreds of rounds of evolution. We varied evolutionary conditions as T7 RNA polymerase evolved to recognize the T3 promoter DNA sequence and characterized how specific combinations of both mutation rate and selection stringency reproducibly result in different evolutionary outcomes. We observed significant and dramatic increases in the activity of the evolved RNA polymerase variants on the desired target promoter after 96 hours of selection, confirming positive selection occurred under all conditions. We used high-throughput sequencing to quantitatively define convergent genetic solutions, including mutational “signatures” and non-signature mutations that map to specific regions of protein sequence. These findings illuminate key determinants of evolutionary outcomes, inform the design of future protein evolution experiments, and demonstrate the value of PACE as a method to study protein evolution.

Low Selection Pressure Aids the Evolution of Cooperative Ribozyme Mutations in Cells

“…Here the evolving molecule, usually T7 RNA polymerase, is repeatedly selected for high activity inside E. coli cells, allowing for the completion of hundreds of evolution rounds within a few days. With this technique, populations that were evolved under low selection stringency followed by high selection stringency reproducibly arrived at different sets of mutations than when evolution was done at high stringency alone, similar to the results of our study (69). Additionally, low selection stringency generated larger genetic diversity, which appeared to be the case in our study as well.…”

Evolution of a split RNA polymerase as a versatile biosensor platform

“…Such a protein engineering problem presents substantial challenges, which can in principle be overcome by molecular evolution. Therefore, we chose to deploy PACE, a rapid evolution system 35 , which has been used to evolve RNAP promoter specificity, protein-DNA interactions, protease activities, and protein-protein interactions 3,30,35–40 . Briefly, PACE involves providing an evolving gene of interest to M13 bacteriophage, linking the life cycle of the phage to a desired activity of interest to be evolved in the target gene, and then propagating the virus until the activity evolves.…”

“…Mixed selection pressures, indicated by listing multiple posAP/negAP sets for a given timepoint, were utilized as appropriate to enhance the likelihood of successful evolution. 24,30,40 …”

Experimental interrogation of the path dependence and stochasticity of protein evolution using phage-assisted continuous evolution

“…We recently demonstrated that PACE can experimentally explore the effects of mutation rate and selection stringency on evolutionary outcomes (23). In this work, we used PACE to experimentally address the following questions: (i) If initially identical enzyme populations are subjected to distinct selection pressures before converging toward a common evolutionary goal (Fig.…”