(502f) Elucidating the Evolvability of Ancestral Proteins Using a Continuous Stirred Tank Bioreactor

Evolution is persistent. In nature, multiple forces act on the evolutionary trajectory of each species. A tug-of-war takes place over the fitness landscape of a protein during the course of evolution with neutral drift pushing to explore new space and purifying selection blocking off other regions. Modern species may showcase useful diversity offered by neutral drift. However, since a tremendous variety of selective pressures have driven purifying selection over the course of evolution, ancient proteins may benefit to an even greater extent and offer a much less constrained fitness landscape. At present, how these forces balance is largely unknown. Quantifying this balance for essential enzymes would provide valuable information for predicting the evolvability of proteins based on ancestry. In this presentation, we evaluate the hypothesis that ancient proteins are ideally suited for adopting new functionality and improved fitness. This perspective is motivated by the fact that ancestral proteins have inherently been exposed to fewer selective pressures, relative to their modern homologs. To test this, we have developed a continuous evolution system where the catalytic speed of an essential enzyme (adenylate kinase, Adk) directly influences the growth rate of host E. coli cells (i.e. cells expressing highly functional variants of Adk grow more quickly than cells expressing less functional Adk variants). Paired with this system, combinatorial libraries were designed based on a modern thermophile and a resurrected ancestor of Adk. To assess the evolvability of the modern and ancestral Adk, the two diversified populations were grown within a continuous stirred tank reactor under harsh low-temperature conditions. Over time, the populations became dominated by cells expressing Adk variants with the highest fitness. High-throughput sequencing at multiple time points throughout the continuous selection process elucidated the evolutionary trajectory of both the modern and ancient proteins. By comparing these mutational trajectories, we were able to draw insights regarding the mechanisms by which new function evolves. The knowledge gained from these experiments enable the community to identify more efficient starting points for drug discovery campaigns and has the direct potential to quicken the pace of developing new protein-based therapeutics.