Overexpression of a homogeneous oligosaccharide with 13C labeling by genetically engineered yeast strain

This report describes a novel method for overexpression of 13C-labeled oligosaccharides using genetically engineered Saccharomyces cerevisiae cells, in which a homogeneous high-mannose-type oligosaccharide accumulates because of deletions of genes encoding three enzymes involved in the processing pathway of asparagine-linked oligosaccharides in the Golgi complex. Using uniformly 13C-labeled glucose as the sole carbon source in the culture medium of these engineered yeast cells, high yields of the isotopically labeled Man8GlcNAc2 oligosaccharide could be successfully harvested from glycoprotein extracts of the cells. Furthermore, 13C labeling at selected positions of the sugar residues in the oligosaccharide could be achieved using a site-specific 13C-enriched glucose as the metabolic precursor, facilitating NMR spectral assignments. The 13C-labeling method presented provides the technical basis for NMR analyses of structures, dynamics, and interactions of larger, branched oligosaccharides.