Enzyme engineering by targeted libraries

This review outlines the strategies we apply for directed enzyme evolution using targeted libraries, namely, libraries that diversify specific residues with predefined mutational compositions. The theoretical grounds underlining the design of such libraries are described, including the mutational load, the ratio of beneficial versus deleterious mutations, and screening capacity. We point out the advantage of using mutational spiking strategies for "hedging the bets," exploring a large number of potentially beneficial mutations, and tuning the library's mutational load. Also highlighted are the merits of low-throughput screens that measure multiple parameters at high accuracy, and of using the desired substrate and reaction conditions rather than surrogates. We subsequently describe library construction strategies (rational and analytical) based on structure and sequence analyses, including ancestral libraries, which are particularly suitable for low-throughput screens. We also discuss the critical role of including compensatory, stabilizing mutations during library construction. Finally, the design efficiency and the optimal mutational loads of libraries are assessed by comparing targeted mutational libraries versus libraries of random mutations.