One-pot synthesis of lacto-N-tetraose using a glycosynthase engineered from GH136 lacto-N-biosidase LnbX

Lacto-N-tetraose (LNT: Galβ1-3GlcNAcβ1-3Galβ1-4Glc) is the most abundant core structure of human milk oligosaccharides (HMOs) and plays important roles in establishing healthy microbiota in the infant gut. Several enzymatic strategies have been developed for the synthesis of LNT. In the conventional linear approach, LNT is synthesized by the sequential addition of N-acetylglucosamine (GlcNAc) and galactose (Gal) to lactose (Lac) through glycosidase-catalyzed transglycosylation or glycosyltransferase reactions. By contrast, the convergent approach developed more recently involves enzymatic coupling of preformed lacto-N-biose I (LNB, Galβ1-3GlcNAc) derivatives to Lac, with relatively high yields. Here, we propose a new convergent one-pot, two-enzyme strategy for LNT synthesis using GH112 galacto-N-biose/LNB phosphorylase (GLNBP) and a glycosynthase mutant of GH136 lacto-N-biosidase (LnbX). In the first reaction, α-LNB-fluoride (LNB-F) was produced from galactose-1-phosphate (Gal-1P) and α-GlcNAc-fluoride by GLNBP. In the second reaction, LNB-F is coupled to Lac using a glycosynthase variant LnbX D418S to yield LNT. Increasing the Lac concentration to 200 mM improved the apparent yield to 49%, based on the initial amount of Gal-1P. Structural analysis of the LnbX D418S-LNB complex revealed that the hydroxy group of Ser418 is positioned to facilitate the departure of the fluoride group during glycosyl transfer, providing mechanistic insight into the observed glycosynthase activity.