Comparing the evolvability of an ancestrally reconstructed and modern adenylate kinase

Abstract Directed evolution transformed protein engineering by providing a customizable framework for generating enzymes with improved catalytic performance across diverse functions. Yet modern enzymes often stall during directed evolution because populations become trapped on local fitness peaks. Researchers have suggested that ancestral enzymes offer better starting points because they are typically more thermostable. Here we propose and experimentally test an alternative explanation that does not depend on ancestral thermostability. We posit that ancestrally reconstructed sequences are unusually evolvable because they are inferred from the evolutionary lineages that survived to produce extant proteins. Less evolvable ancestors, and the trajectories emanating from them, disappeared by extinction and therefore do not contribute to reconstructed ancestors. Using thermophilic ancestral and modern adenylate kinases, we performed independent single-round selection experiments for activity in vivo and in vitro. In both settings, the ancestral enzyme tolerates a larger number of mutations, yielding more viable variants with greater genetic diversity than its modern descendants. Because mutational robustness promotes evolvability, these results support an intrinsic evolvability of reconstructed ancestral sequences that makes them superior launch points for directed evolution.