Diversity generation is the first step for the Directed Evolution of enzymes and other proteins. In this section, we will find out why artificially generating diversity is essential to understand some features of enzymes (i.e. organic solvent resistance and pH resistance) that are not accessible by rational design. We will explore the vastness of an average enzyme amino acid sequence space (which is larger than actual “space”).

After a short introduction on how to mutate a gene, we will learn how to accelerate natural evolution millionfold through iterative rounds of artificial mutagenesis methods such as errorprone PCR (epPCR). We will get to know the limitations of this method and the reasons for it. We will also introduce the SeSaM method, which allows us to explore the previously unknown regions of protein amino acid sequence space.

Finally, we will discover the KnowVolution strategy which allows us to both generate improved enzyme variants and to understand the molecular basis of their improvements.