Gene Mutagenesis Methods in Directed Evolution and Rational Enzyme Design

The traditional approaches used in directed evolution, including mutator strains, error-prone polymerase chain reaction, gene insertion and deletion, saturation mutagenesis, DNA shuffling and related recombinant gene mutagenesis, circular mutation and domain swapping, as well as solid-phase gene synthesis techniques, are presented critically in this chapter. Computational tools and approaches, as well as machine learning algorithms established in rational enzyme design, are also described. As outlined in Section 3.18 of this chapter, the techniques in directed evolution and rational design no longer develop on separate tracks but have merged recently with the construction of powerful techniques such as Focused Rational Iterative Site-specific Mutagenesis (FRISM).