Applications of Directed Evolution

With directed evolution we can engineer enzyme properties rapidly and with a high probability of success. Many enzymes that have been improved by directed evolution are listed in Tab. 4-3. This powerful biocatalyst engineering strategy creates new opportunities in organic synthesis new and improved bioconversion processes can be developed and novel compounds that are otherwise inaccessible by classical chemistry can be synthesized. In addition, the molecules created by directed evolution offer an excellent opportunity for improving our still poor understanding of sequence-structure-function relationships. 

The specific applications of directed evolution that are described below focus on properties that are important for efficient enzyme production as well as on those that are of special interest for applications in organic synthesis, including enzyme 

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The application of directed evolution approaches for the change or extension of the specificity of a restriction enzyme is hampered by the fact that an in vivo selection assay is not available and that examination of endonuclease activity in vitro usually requires purification of the enzymes to avoid background activity by other nucleases prevalent in all cells. This means that an altered or extended specificity can only be observed with sufficiently purified protein preparations, thereby unfortunately separating genotype and phenotype. As neither a reliable in vivo test nor the secretion of restriction endo-... 

Selected improved variants were later recombined using a method similar to DNA shuffling. Three or four rounds of mutation allowed the selection of a mutant enzyme with 15-fold higher activity, and 5°C increase in stability. Substrate selectivity was unchanged with respect to the wild-type enzyme from FusariumP This example is one of the many possible applications of directed evolution for the generation of new bioassay reagents with new or improved characteristics. 

David L. Ollis completed a B.Sc. (Hons) at the University of New South Wales before moving across town to complete a Ph.D. in the Department of Chemistry in Sydney University. He then worked in Tom Steitz s laboratory in Yale University before taking a faculty position in the Department of Biochemistry, Molecular Biology, and Cell Biology in Northwestern University, Chicago. He returned to Australia in 1992 to take up a position within the Research School of Chemistry of the Australian National University. His research interests include the application of directed evolution and crystallography in protein structure-function studies. 

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