Directed evolution of enzyme catalysts.

Directed enzyme evolution has emerged in the past few years as a powerful alternative to rational approaches for engineering biocatalysts. Prerequisites for successful directed evolution are functional expression in a suitable microbial host, a rapid screen for the desired feature(s) and a well-thought-out working strategy for navigating protein landscapes. The rapidly growing body of literature on enzyme evolution in vitro includes techniques for creating and searching combinatorial enzyme libraries, as well as several successful examples of different evolutionary strategies being used.

Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences.

Sets of genes improved by directed evolution can be recombined in vitro to produce further improvements in protein function. Recombination is particularly useful when improved sequences are available; costs of generating such sequences, however, must be weighed against the costs of further evolution by sequential random mutagenesis. Four genes encoding para-nitrobenzyl (pNB) esterase variants exhibiting enhanced activity were recombined in two cycles of high-fidelity DNA shuffling and screening.