To get what we aim for - Progress in diversity generation methods

Abstract

Protein re-engineering by directed evolution has become a standard approach for tailoring enzymes in many fields of science and industry. Advances in screening formats and screening systems are fueling progress and enabling novel directed evolution strategies, despite the fact that the quality of mutant libraries can still be improved significantly. Diversity generation strategies in directed enzyme evolution comprise three options: (a) focused mutagenesis (selected residues are randomized); (b) random mutagenesis (mutations are randomly introduced over the whole gene); and (c) gene recombination (stretches of genes are mixed to chimeras in a random or rational manner). Either format has both advantages and limitations depending on the targeted enzyme and property. The quality of diverse mutant libraries plays a key role in finding improved mutants. In this review, we summarize methodological advancements and novel concepts (since 2009) in diversity generation for all three formats. Advancements are discussed with respect to the state of the art in diversity generation and high-throughput screening capabilities, as well as robustness and simplicity in use. Furthermore, limitations and remaining challenges are emphasized 'to get what we aim for' through 'optimal diversity' generation. Methodological advancements and novel concepts of the three diversity generation strategies (I. Focused mutagenesis, II. Random mutagenesis, and III. Gene recombination) in directed enzyme evolution are summarized. Advancements are discussed in respect to the state of the art in diversity generation and high throughput screening capabilities as well as robustness and simplicity in use. © 2013 FEBS.