Clarifying the Difference between Iterative Saturation Mutagenesis as a Rational Guide in Directed Evolution and...

A recent directed-evolution study by Schwaneberg and co-workers comparing the widely used iterative saturation mutagenesis (ISM) with the OmniChange version of saturation mutagenesis (SM) prompts us to point out some flaws in the conclusions presented therein. Most importantly, ISM is a semirational strategy in directed evolution that is independent of the particular type of SM that the experimenter may choose; this means that OmniChange should not be compared with ISM. When aiming to improve enzyme selectivity or activity by the ISM strategy, the state-of-the-art calls for SM at randomization sites lining the enzyme binding pocket as part of the combinatorial active-site saturation test (CAST). Our recent studies focusing on the refinement of CAST/ISM have shown that this approach works best when using multiresidue randomization sites as opposed to single-residue sites owing to the possibility of cooperative mutational effects. This advance was not considered by Schwaneberg and co-workers, thus leading to questionable conclusions when pitching CAST/ISM against OmniChange.