Directed evolution of D-2-keto-3-deoxy-6-phosphogluconate aldolase to new variants for the efficient synthesis of D- and L-sugars

Background: Exploitation and improvement of enzymes as catalysts for organi c synthesis is of current interest in biocatalysis. A representative enzyme for investigation is the Escherichia coli D-2-keto-3-deoxy-6-phosphoglucon ate (KDPG) aldolase, which catalyzes the highly specific reversible aldol r eaction using the D-configurated KDPG as substrate. Results: Using in vitro evolution, the aldolase has been converted into ald olases with improved catalytic efficiency, altered substrate specificity an d stereoselectivity. In particular, some evolved aldolases capable of accep ting both D- and L- glyceraldehyde in the non-phosphorylated form as substr ates for reversible aldol reaction have been obtained, providing a new dire ction to the enzymatic synthesis of both D- and L-sugars. Conclusions: This research has demonstrated the effectiveness of using in v itro evolution to rapidly alter the properties of an aldolase to improve it s utility in asymmetric synthesis. The evolved aldolases, differing from th e native enzyme which is highly phosphate- and D-sugar-dependent, catalyze the efficient synthesis of both D- and L-sugars from non-phosphorylated ald ehydes and pyruvate. The principles and strategies described in this study should be applicable to other aldolases to further expand the scope of thei r synthetic utility.