RT Journal Article SR Electronic T1 A library of structurally homogeneous human N-glycans synthesized from microbial oligosaccharide precursors JF bioRxiv FD Cold Spring Harbor Laboratory SP 118216 DO 10.1101/118216 A1 Brian S. Hamilton A1 Joshua D. Wilson A1 Marina A. Shumakovich A1 Adam C. Fisher A1 James C. Brooks A1 Alyssa Pontes A1 Radnaa Naran A1 Christian Heiss A1 Chao Gao A1 Robert Kardish A1 Jamie Heimburg-Molinaro A1 Parastoo Azadi A1 Richard D. Cummings A1 Judith H. Merritt A1 Matthew P. DeLisa YR 2017 UL http://biorxiv.org/content/early/2017/03/18/118216.abstract AB Synthesis of homogenous glycans in quantitative yields represents a major bottleneck to the production of molecular tools for glycoscience, such as glycan microarrays, affinity resins, and reference standards. Here, we describe a combined biological/enzymatic method termed bioenzymatic synthesis that is capable of efficiently converting microbially-derived precursor oligosaccharides into structurally uniform human-type N-glycans. Unlike starting material obtained by chemical synthesis or direct isolation from natural sources, which can be time consuming and costly to generate, bioenzymatic synthesis involves precursors derived from renewable sources including wild-type Saccharomyces cerevisiae glycoproteins and lipid-linked oligosaccharides from glycoengineered Escherichia coli. Following deglycosylation of these biosynthetic precursors, the resulting microbial oligosaccharides are subjected to a greatly simplified purification scheme followed by structural remodeling using commercially available and recombinantly produced glycosyltransferases including key N- acetylglucosaminyltransferases (e.g., GnTI, GnTII, and GnTIV) involved in early remodeling of glycans in the mammalian glycosylation pathway. Using this approach, preparative quantities of hybrid and complex-type N-glycans including asymmetric multi-antennary structures were generated all without the need of a specialized skillset. Collectively, our results reveal bioenzymatic synthesis to be a user-friendly methodology for rapidly supplying homogeneous oligosaccharide structures that can be used to understand the human glycome and probe the biological roles of glycans in health and disease.