Steering directed protein evolution: strategies to manage combinatorial complexity of mutant libraries

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Abstract
How to explore protein sequence space efficiently and how to generate high‐quality mutant libraries that allow to identify improved variants with current screening technologies are key questions for any directed protein evolution experiment. High‐quality mutant libraries can be generated through improved random mutagenesis methodologies and by restricting diversity generation through computational methods to residues which have high success probabilities. Advances in mutant library design and computational tools to focus diversity generation are summarized in this minireview and discussed from an experimentalist point of view in the context of directed protein evolution.