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Directed evolution of stereo- and regioselective enzymes constitutes a prolific source of catalysts for asymmetric transformations in organic chemistry. In this endeavor (iterative) saturation mutagenesis at sites lining the binding pocket of enzymes has emerged as the method of choice, but uncertainties regarding the question of how to group many residues into randomization sites and how to choose optimal upward pathways persist. Two new approaches promise to beat the numbers problem effectively. One utilizes a single amino acid as building block for the randomization of a 10-residue site, the other also employs only one but possibly different amino acid at each position of a 9-residue site. The small but smart libraries provide highly enantioselective epoxide hydrolase or lipase mutants, respectively.

Directed evolution has emerged as a general way to engineer essentially any catalytic property of enzymes, but due to the bottleneck imposed by the necessity to screen large libraries of mutants, it is often time-consuming. In order to make this type of protein engineering faster and more efficient than in the past, improved methods for probing protein sequence space need to be developed. This review focuses on recent advances which help to solve the traditional numbers problem in laboratory evolution, as in the directed evolution of enantioselective enzymes. Our contribution in this endeavour is iterative saturation mutagenesis (ISM), which can be used to enhance the enantioselectivity and/or the thermostability of enzymes. The option to use reduced amino acid alphabets as defined by the appropriate codon degeneracies supplements in a crucial way the toolbox in this knowledge-guided approach to laboratory evolution.

Directed evolution based on saturation mutagenesis at sites lining the binding pocket is a commonly practiced strategy for enhancing or inverting the stereoselectivity of enzymes for use in organic chemistry or biotechnology. However, as the number of residues in a randomization site increases to five or more, the screening effort for 95 % library coverage increases astronomically until it is no longer feasible. We propose the use of a single amino acid for saturation mutagenesis at superlarge randomization sites comprising 10 or more residues. When used to reshape the binding pocket of limonene epoxide hydrolase, this strategy, which drastically reduces the search space and thus the screening effort, resulted in R,R- and S,S-selective mutants for the hydrolytic desymmetrization of cyclohexene oxide and other epoxides. X-ray crystal structures and docking studies of the mutants unveiled the source of stereoselectivity and shed light on the mechanistic intricacies of this enzyme.

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.

Saturation mutagenesis probes define sections of the vast protein sequence space. However, even if randomization is limited this way, the combinatorial numbers problem is severe. Because diversity is created at the codon level, codon redundancy is a crucial factor determining the necessary effort for library screening. Additionally, due to the probabilistic nature of the sampling process, oversampling is required to ensure library completeness as well as a high probability to encounter all unique variants. Our trick employs a special mixture of three primers, creating a degeneracy of 22 unique codons coding for the 20 canonical amino acids. Therefore, codon redundancy and subsequent screening effort is significantly reduced, and a balanced distribution of codon per amino acid is achieved, as demonstrated exemplarily for a library of cyclohexanone monooxygenase. We show that this strategy is suitable for any saturation mutagenesis methodology to generate less-redundant libraries.

Enantioselective mutants of the thermally robust phenyl acetone monooxygenase (PAMO) as catalysts in Baeyer-Villiger reactions have been evolved by utilizing saturation mutagenesis in which drastically reduced amino acid alphabets are employed at homologous enzyme positions.