Directed Evolution of Protein Catalysts

Abstract

Directed evolution is a powerful technique for generating tailor-made enzymes for a wide range of biocatalytic applications. Following the principles of natural evolution, iterative cycles of mutagenesis and screening or selection are applied to modify protein properties, enhance catalytic activities, or develop completely new protein catalysts for non-natural chemical transformations. This review briefly surveys the experimental methods used to generate genetic diversity and screen or select for improved enzyme variants. Emphasis is placed on a key challenge, namely how to generate novel catalytic activities that expand the scope of natural reactions. Two particularly effective strategies, exploiting catalytic promiscuity and rational design, are illustrated by representative examples of successfully evolved enzymes. Opportunities for extending these approaches to more complex biocatalytic systems are also considered.

Biocatalytic Strategies towards [4+2] Cycloadditions

“…Enzyme engineers are working hard to expand the toolkit of organic chemists with efficient, tailor made cyclases. Great potential seems to reside in artificial metalloenzymes, which could possibly reach the efficiencies of typical, natural enzymes if they were subjected to extensive genetic optimization . A growing number of natural and designed [4+2] cyclases will hopefully become available in the years to come and finally equip one of the most important reactions in synthetic chemistry with versatile biocatalysts.…”

Upgrading aminoacyl-tRNA synthetases for genetic code expansion

“…High-quality libraries of diverse aaRS variants are paramount to obtaining highly active and ncAA-specific aaRSs (Figure 1c). Oligo-directed libraries depend on the availability of high-resolution crystal structures of aaRSs [14], as well as docking studies to identify active site residues that may contact the ncAA [15,16]. In practice, the size of site saturated libraries that can be subjected to comprehensive analysis with standard lab resources is limited to 10 9 , which corresponds to 6 residues randomized using redundant NNK primers [16].…”

“…Oligo-directed libraries depend on the availability of high-resolution crystal structures of aaRSs [14], as well as docking studies to identify active site residues that may contact the ncAA [15,16]. In practice, the size of site saturated libraries that can be subjected to comprehensive analysis with standard lab resources is limited to 10 9 , which corresponds to 6 residues randomized using redundant NNK primers [16]. Proficient o-aaRS variants are identified by selection or screening techniques, typically using reporter genes that confer antibiotic resistance or encode fluorescent proteins (Figure 1c).…”

Navigating within thiamine diphosphate‐dependent decarboxylases: Sequences, structures, functional positions, and binding sites

“…Over the last three decades, enzyme properties have been tailored both through evolutionary approaches as well as by applying computer‐aided rational strategies for protein design and selection. As computational methods improve, the synergy between these approaches is likely to intensify to achieve faster and robust systematic approaches for navigating sequence space …”

“…As computational methods improve, the synergy between these approaches is likely to intensify to achieve faster and robust systematic approaches for navigating sequence space. 3 Although structures and sequences of enzymes are expansively made available through databases, extracting complex information in a systematic way remains a difficult task. Ideally, the rational analysis of the active site of an enzyme should provide quantitative information on the relationship between structural features and the ability of the protein to stabilize the transition state of a given reaction.…”