Machine-Directed Evolution of an Imine Reductase for Activity and Stereoselectivity
J Gilbert Eric, Elina Siirola, Charles M. Moore, Arkadij Kummer, Markus Stoeckli, Michael Faller, Caroline Bouquet, Fabian Eggimann, Mathieu Ligibel, Dan Huynh, Geoffrey J. Cutler, Luca Siegrist, Richard A. Lewis, Anne-Christine Acker, Ernst Freund, Elke Koch, Markus Vogel, Holger Schlingensiepen, Edward J. Oakeley, Radka Šnajdrová
Abstract
Biocatalysis is an effective tool to access chiral molecules that are otherwise hard to synthesize or purify. Time-efficient processes are needed to develop enzymes that adequately perform the desired chemistry. We evaluated machine-directed evolution as an enzyme engineering strategy using a moderately stereoselective imine reductase as the model system. We compared machine-directed evolution approaches to deep mutational scanning (DMS) and error-prone PCR. Within one cycle, it was found that machine-directed evolution yielded a library of high-activity mutants with a dramatically shifted activity distribution compared to that of traditional directed evolution. Structure-guided analysis revealed that linear additivity might provide a simple explanation for the effectiveness of machine-directed evolution. The most active and selective enzyme mutant, which was identified through DMS and error-prone PCR, was used for the gram-scale synthesis of the H4 receptor antagonist ZPL389 with full conversion, > 99% ee (R), and a 72% yield.