Methods

Akin to natural evolution, enzymatic function can be modulated in the laboratory by creating genetic diversity and selecting variants with desirable traits. This process, called directed evolution, usually requires isolation of single genetic variants in order to assay the desired functions (phenotype) while retaining knowledge about the genetic changes (genotype) responsible for the improvements. Although directed evolution is a simple and robust technique, it is bedeviled by the astronomical number of possible amino acid changes, most of which are neutral or deleterious. Thus, obtaining a suitable mutant typically requires extensive screening. 

Higher-throughput screening methods enable sampling of regions of sequence space that would otherwise be inaccessible, dramatically accelerating enzyme optimization. As such, they have the potential to illuminate the emergence of catalytic function during evolution. The ultrahigh throughput of fluorescence-activated cell sorting (FACS) and fluorescence-activated droplet sorting (FADS) have proved advantageous for the directed evolution of enzymes.
Complementary to screening, genetic selection can be used for directed evolution. Here, the sample size is only limited by the transformation efficiency of the expression strain, making it a very productive method for cases where enzyme function is directly influencing the organism’s ability to proliferate. By fine tuning selection stringency, such systems act as an in vivo testing ground for enzymatic function and evolution.