Directed Evolution of Enzymes: How to Improve Nucleic Acid Polymerases for Biocatalysis

Natural selection has created optimal catalysts that exhibit their convincing performance even with a number of sometimes counteracting constraints. Nucleic acid polymerases, for example, provide for the maintenance, transmission, and expression of genetic information and thus play a central role in every living organism. While faithful replication and strict substrate recognition are crucial for long-term survival of species, relaxed fidelity and/or substrate tolerance could be valuable features for biotechnological applications involving the enzymatic synthesis of modified DNA or RNA. Directed evolution that mimics natural evolution on a laboratory time-scale has emerged as a powerful tool for tailoring enzyme functions to specific requirements. In this chapter, we introduce basic techniques necessary for improving enzyme function by directed evolution, and we use the “tuning” of bacteriophage T7 RNA polymerase toward decreased fidelity as an example.