ABSTRACT
Enzymes are naturally occurring biocatalysts operating in living cells. During biological evolution for billions of years, they have been optimized to catalyze a given reaction with high activity and substrate specificity. Nowadays, several weeks are sufficient to mimic natural evolution in a test tube by using “directed” or in vitro evolution, during which enzyme variants with desired properties are identified in large libraries of mutated genes. In principle, this technique does not require knowledge of the enzymes’ structure, its catalytic mechanism, or biosynthesis, making it a powerful novel tool for enzyme optimization (1-5). The directed evolution of a given biocatalyst is a multistep process including the (a) identification of a candidate enzyme, which preferably should have a catalytic activity toward the substrate of interest; (b) cloning of the respective enzyme gene; (c) generation of a large number of mutant genes; (d) expression of these genes to generate large libraries of enzyme variants; (e) identification of better-performing biocatalysts in the libraries by high-throughput screening or selection; and, finally, (f) produc-tion of the best-performing biocatalyst at a large scale. Therefore, the construction of a potent overexpression system for a gene of interest constitutes a major part of devising an efficient directed evolution strategy.
