N-Glycosylation is a ubiquitous protein post-translational modification mechanism in eukaryotes. In this work, a synthetic pathway containing glycosyltransferases from Saccharomyces cerevisiae was introduced to Escherichia coli to synthesize lipid-linked mannosyl-chitobiose (Man-GlcNAc(2)) and trimannosyl-chitobiose (Man(3)-GlcNAc(2)). Transfer of Man(3)-GlcNAc(2) onto a model periplasmic protein occurred in the engineered E. con cell using oligosaccharyltransferase PglB. from Campylobacter jejuni. Mass spectrometric analysis of the fluorescently labeled N-glycan indicated a glycan signal composed of 2 HexNAc and 3 Hex residues. The reversed-phase HPLC analysis suggested that the Hex residues were alpha 1,3-, alpha 1,6- and beta 1,4-linked mannoses. These results indicated that the constructed system synthesizes a Man(3)-GlcNAc(2), identical to that observed in an early eukaryotic dolichol pathway. Finally, glycopeptide mass spectrometry confirmed the transfer of the assembled glycan moiety onto an engineered glycosylation motif of recombinant maltose binding protein. Surprisingly, the Man(3)-GlcNAc(2) structure but not Man-GlcNAc(2) was transferred onto maltose binding protein. This work showed that PglB protein might be able to accommodate the transfer of the further engineered glycan with greater complexity. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.