A library of structurally homogeneous humanN-glycans synthesized from microbial oligosaccharide precursors

Abstract

1Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the 2 production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and 3 reference standards. Here, we describe a combined biological/enzymatic method termed 4 bioenzymatic synthesis that is capable of efficiently converting microbially-derived precursor 5 oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material 6 obtained by chemical synthesis or direct isolation from natural sources, which can be time 7consuming and costly to generate, bioenzymatic synthesis involves precursors derived from 8 renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked 9 oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these 10 biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly 11 simplified purification scheme followed by structural remodeling using commercially available 12 and recombinantly produced glycosyltransferases including key N-13 acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of 14 glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of 15 hybrid and complex-type N-glycans including asymmetric multi-antennary structures were 16 generated all without the need of a specialized skillset. Collectively, our results reveal 17 bioenzymatic synthesis to be a user-friendly methodology for rapidly supplying homogeneous 18 oligosaccharide structures that can be used to understand the human glycome and probe the 19 biological roles of glycans in health and disease.