%A Zhu,Jing %A Ruan,Yao %A Fu,Xin %A Zhang,Lichao %A Ge,Gaoshun %A Wall,J. Gerard %A Zou,Teng %A Zheng,Yang %A Ding,Ning %A Hu,Xuejun %D 2020 %J Frontiers in Bioengineering and Biotechnology %C %F %G English %K sialylation,sialic acid,Escherichia coli,N-Glycoprotein,α-2,6-sialyltransfease %Q %R 10.3389/fbioe.2020.00313 %W %L %M %P %7 %8 2020-April-15 %9 Brief Research Report %# %! terminally sialylated N-glycoproteins in Escherichia coli %* %< %T An Engineered Pathway for Production of Terminally Sialylated N-glycoproteins in the Periplasm of Escherichia coli %U https://www.frontiersin.org/articles/10.3389/fbioe.2020.00313 %V 8 %0 JOURNAL ARTICLE %@ 2296-4185 %X Terminally sialylated N-glycoproteins are of great interest in therapeutic applications. Due to the inability of prokaryotes to carry out this post-translational modification, they are currently predominantly produced in eukaryotic host cells. In this study, we report a synthetic pathway to produce a terminally sialylated N-glycoprotein in the periplasm of Escherichia coli, mimicking the sialylated moiety (Neu5Ac-α-2,6-Gal-β-1,4-GlcNAc-) of human glycans. A sialylated pentasaccharide, Neu5Ac-α-2,6-Gal-β-1,4-GlcNAc-β-1,3-Gal-β-1,3-GlcNAc-, was synthesized through the activity of co-expressed glycosyltransferases LsgCDEF from Haemophilus influenzae, Campylobacter jejuni NeuBCA enzymes, and Photobacterium leiognathi α-2,6-sialyltransferase in an engineered E. coli strain which produces CMP-Neu5Ac. C. jejuni oligosaccharyltransferase PglB was used to transfer the terminally sialylated glycan onto a glyco-recognition sequence in the tenth type III cell adhesion module of human fibronectin. Sialylation of the target protein was confirmed by lectin blotting and mass spectrometry. This proof-of-concept study demonstrates the successful production of terminally sialylated, homogeneous N-glycoproteins with α-2,6-linkages in the periplasm of E. coli and will facilitate the construction of E. coli strains capable of producing terminally sialylated N-glycoproteins in high yield.