Directed evolution of stereoselective enzymes based on geneticselection as opposed to screening systems

Abstract

tDirected evolution of stereoselective enzymes provides a means to generate useful biocatalysts for asym-metric transformations in organic chemistry and biotechnology. Almost all of the numerous examplesreported in the literature utilize high-throughput screening systems based on suitable analytical tech-niques. Since the screening step is the bottleneck of the overall procedure, researchers have consideredthe use of genetic selection systems as an alternative to screening. In principle, selection would be themost elegant and efficient approach because it is based on growth advantage of host cells harboring ste-reoselective mutants, but devising such selection systems is very challenging. They must be designed sothat the host organism profits from the presence of an enantioselective variant. Progress in this intrigu-ing research area is summarized in this review, which also includes some examples of display systemsdesigned for enantioselectivity as assayed by fluorescence-activated cell sorting (FACS). Although thecombination of display systems and FACS is a powerful approach, we also envision innovative ideascombining metabolic engineering and genetic selection systems with protein directed evolution for thedevelopment of highly selective and efficient biocatalysts.