The Dawn of Directed Evolution
Tuning the Activity of an Enzyme for Unusual Environments: Sequential Random Mutagenesis of Subtilisin E for Catalysis in Dimethylformamide.
Proc. Natl. Acad. Sci. USA 1993;
14 December 2018 (online)
In 1993, Chen and Arnold reported a revolutionary approach to protein design. This strategy, later referred to as ‘directed evolution’, relies on the accumulation of positive mutations over generations of enzymes through sequential rounds of random mutagenesis. The authors applied this strategy to serine protease subtilisin E to form mutated enzyme PC3 after three rounds of mutagenesis. The variant contains ten amino acid substitutions clustered near the active site and substrate binding pocket and it proved substantially more reactive than the wild-type subtilisin, hydrolyzing a peptide substrate 256 times faster in a 60% aqueous DMF solution.
In an era dominated by metal-catalyzed reactions and years before the organocatalysis revolution, the Arnold group showed the first practical application of directed evolution, a protein-engineering method that would bring enzymatic reactions to the frontline of modern catalysis. In this seminal report, Chen and Arnold outline the key steps of the process, which they and many others have since refined and further developed. For example, Reetz et al. evolved enzymes for asymmetric catalysis. Most recently the Arnold group has used this approach to develop enzymes capable of constructing highly strained carbocycles (Science 2018, 360, 71) and oxidizing olefins (Science 2017, 358, 215), as well as activating benzylic C–H bonds toward amination (Nat. Chem. 2017, 9, 629). In the 25th year since this seminal report, we recognize its vast impact and look forward to further applications.