Strategies for Applying Gene Mutagenesis Methods

This chapter considers enzymes as catalysts in organic chemistry and biotechnology in which the following parameters are manipulated: stereo- and regioselectivity, activity, and substrate scope. Whenever more than one enzyme parameter needs to be improved, two strategic options are possible: engineering the parameters separately in two sequential projects or optimizing both parameters simultaneously in a single project. It has been recommended that for practical reasons it is useful to start directed evolution from a robust enzyme, since protein stability promotes evolvability. Traditionally, NNK codon degeneracy encoding all 20 canonical amino acids has been used in saturation mutagenesis, especially when targeting stereoselectiity. Two representative examples of the traditional approach based on NNK codon degeneracy are analyzed. In a study concerning the directed evolution of a stereoselective transaminase, iterative saturation mutagenesis (ISM) played a crucial role, but DNA shuffling and site-specific mutagenesis was also employed.