Assessing Directed Evolution Methods for the Generation of Biosynthetic Enzymes with Potential in Drug Biosynthesis

Abstract

To address the synthesis of increasingly structurally diverse small-molecule drugs, methods for the generation of efficient and selective biological catalysts are becoming increasingly important. 'Directed evolution' is an umbrella term referring to a variety of methods for improving or altering the function of enzymes using a nature-inspired twofold strategy of mutagenesis followed by selection. This article provides an objective assessment of the effectiveness of directed evolution campaigns in generating enzymes with improved catalytic parameters for new substrates from the last decade, excluding studies that aimed to select for only improved physical properties and those that lack kinetic characterization. An analysis of the trends of methodologies and their success rates from 81 qualifying examples in the literature reveals the average fold improvement for k (cat) (or V (max)), K (m) and k (cat)/K (m) to be 366-, 12- and 2548-fold, respectively, whereas the median fold improvements are 5.4, 3 and 15.6. Further analysis by enzyme class, library-generation methodology and screening methodology explores relationships between successful campaigns and the methodologies employed.