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Gavin J Williams1, Changsheng Zhang1 and Jon S Thorson1
Natural products, many of which are decorated with essential sugar residues, continue to serve as a key platform for drug development1. Adding or changing sugars attached to such natural products can improve the parent compound's pharmacological properties, specificity at multiple levels2, and/or even the molecular mechanism of action3. Though some natural-product glycosyltransferases (GTs) are sufficiently promiscuous for use in altering these glycosylation patterns, the stringent specificity of others remains a limiting factor in natural-product diversification and highlights a need for general GT engineering and evolution platforms. Herein we report the use of a simple high-throughput screen based on a fluorescent surrogate acceptor substrate to expand the promiscuity of a natural-product GT via directed evolution. Cumulatively, this study presents variant GTs for the glycorandomization of a range of therapeutically important acceptors, including aminocoumarins, flavonoids and macrolides, and a potential template for engineering other natural-product GTs.
As an emerging method to differentially glycosylate natural products, glycorandomization uses the inherent or engineered substrate promiscuity of anomeric kinases (Fig. 1a, E1) and nucleotidyltransferases (E2) for the in vitro synthesis of sugar nucleotide libraries as sugar donors for natural-product GTs4. Although the successful glycorandomization of various natural-product scaffolds (including glycopeptides5, avermectins6 and enediynes7) has been reported, other recent antibiotic glycorandomization attempts have revealed that aminocoumarin and macrolide GTs (NovM and EryBV, respectively) accept only 2 alternative sugar nucleotides out of 25 to 40 potential donors tested8,9. Thus, though permissive GTs open new opportunities for drug discovery, the stringent specificity of other GTs remains a limiting factor in natural-product diversification and highlights a need for general GT engineering and/or evolution platforms. Despite the wealth of GT structural and biochemical information10, attempts to alter GT donor/acceptor specificities via rational engineering have been largely unsuccessful and primarily limited to sequence-guided single-site mutagenesis11. Owing to a lack of high-throughput GT screens and selections, successful reports to alter GT donor/acceptor specificities via directed evolution are equally sparse. Although an in vivo selection for the directed evolution of the sialyltransferase CstII (a unique member of the GT-A superfamily) was recently disclosed12, the directed evolution of any member of the structurally and functionally distinct GT-B superfamily has not been achieved.
(a) General overview of enzymatic glycorandomization. E1 represents a flexible anomeric sugar kinase, E2 represents a flexible sugar-1-phosphate nucleotidyltransferase, GT represents a flexible glycosyltransferase and the gray oval represents a complex natural-product scaffold. (b) The native macrolide glucosyltransferase reaction catalyzed by OleD (upper reaction), and the 4-methylumbelliferone (4) glucosylation reaction used for OleD directed evolution (lower reaction).
