Directed Evolution by Using Iterative Saturation Mutagenesis Based on Multiresidue Sites

Iterative saturation mutagenesis (ISM) in combination with reduced amino acid alphabets has been shown to be an efficient method for directed evolution. In order to minimize the screening effort, the number of residues in a given randomization site has thus far been restricted to two or three; this prevents oversampling from reaching astronomical numbers when 95 % library coverage is aimed for. In this study, ISM is applied for the first time by using randomization sites composed of five amino acid positions. The use of just two such sites (A and B) results in two different ISM pathways, A→B and B→A. A severely reduced amino acid alphabet (only five members) was employed for the building blocks-a minimal set of structurally representative amino acids. The Baeyer-Villiger monooxygenase PAMO was chosen as the enzyme for this proof-of-principle study. The test system employed tuning of activity and diastereoselectivity in the oxidation of 4-(bromomethylidene)cyclohexanone, which is not accepted by wild-type PAMO. Although only 8-9 % library coverage was ensured (as calculated by traditional statistics), notable activity and 99 % diastereoselectivity were obtained, thus indicating that such an ISM strategy is viable in protein engineering.