Enabling Glycosyltransferase Evolution:A Facile Substrate-Attachment Strategy for Phage-Display Enzyme Evolution

摘要

Directed enzyme evolution has the potential to generate novel catalysts able to compete with chemical methodologies in the synthesis of complex biomolecules.Directed evolution is often carried out in cells,but for many classes of enzymes,such as glycosyltransferases,it is difficult to couple enzymatic activity to a cell-based selection.M13 phage display is an in vitro methodology that potentially enables the selection of enzymes that do not provide cell-based phenotypes.In order to select phage-bound enzymes based on catalytic activity,the desired substrate needs to be immobilized near the displayed enzyme to allow for affinity capture of the desired product.Schultz and co-workers developed the first catalysis-based display method,in which the substrate molecule was attached next to the displayed enzyme by a coiled-coil interaction.