Fishing for Catalysis: Experimental Approaches to Narrowing Search Space in Directed Evolution of Enzymes

[Image: see text] Directed evolution has transformed protein engineering offering a path to rapid improvement of protein properties. Yet, in practice it is limited by the hyper-astronomic protein sequence search space, and approaches to identify mutagenic hot spots, i.e., locations where mutations are most likely to have a productive impact, are needed. In this perspective, we categorize and discuss recent progress in the experimental approaches (broadly defined as structural, bioinformatic, and dynamic) to hot spot identification. Recent successes in harnessing protein dynamics and machine learning approaches provide new opportunities for the field and will undoubtedly help directed evolution reach its full potential.