Transferring a biosynthetic cycle into a productive Escherichia coli strain: large-scale synthesis of galactosides.

Discoveries concerning the important roles of glycoconjugates in a variety of biological processes have led to an increased demand for efficient synthetic methodologies adaptable for their large-scale production.1 Compared to chemical synthesis, an enzymatic approach utilizing glycosyltransferases has been proven more efficient in the production of complex carbohydrates.2 Despite the increasing availability of various glycosyltransferases, the high cost and inadequate availability of sugar nucleotides required by these enzymes have severely limited their applications.3 Since sugar nucleotides only serve as cofactors in the overall glycosylation in biological system, the best way is mimicking nature to recycle them in situ.4 Among the existing uridine 5′-diphosphoglucose (UDP-Glc) and uridine 5′-diphosphogalactose (UDP-Gal) recycling schemes, the most elegant one is based on a sucrose synthase (SusA, EC 2.4.1.13).5 The synthesis and cleavage of sucrose catalyzed by sucrose synthase (sucrose + UDP S UDP-Glc + fructose) is the only readily reversible transglycosylation reaction involving a sugar nucleotide6 (Scheme 1). On the basis of this in vitro biosynthetic cycle, our objective has been to create a bacterial strain transformed with a single plasmid which contains the three enzymes along the biosynthetic cycle. The bacterial cells will then be used as a galactoside-producing factory, in which UDP is recycled back to UDP-Gal, providing the donor for galactosyltransferases for largescale production of custom carbohydrates.