Engineered Biosynthesis of Glycoproteins with Eukaryotic N-Glycans In Escherichia Coli

Thursday, October 20, 2011: 4:55 PM
L100 J (Minneapolis Convention Center)
Juan D. Valderrama-Rincon1, Adam C. Fisher2, Judith Merritt2, Yao-Yun Fan3, Craig Reading2, Krishan Chhiba4, Markus Aebi3 and Matthew P. DeLisa1, (1)Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, (2)Glycobia Inc., Ithaca, NY, (3)Department of Biology, Institute of Microbiology, ETH Zürich, Zürich, Switzerland, (4)Department of Neurobiology and Behavior, Cornell University, Ithaca, NY

Asparagine-linked (N-linked) glycosylation pathways have recently been discovered in bacteria. However, bacterial N-glycans are structurally distinct from their eukaryotic counterparts, rendering bacteria marginally useful for therapeutic applications. Here we report the engineering of a eukaryotic protein glycosylation pathway in the Gram-negative bacterium Escherichia coli. This involved the heterologous expression of four eukaryotic proteins, including yeast uridine diphosphate-N-acetylglucosamine transferases (Alg13, Alg14) and mannosyltransferases (Alg1, Alg2), and a bacterial oligosaccharyltransferase (PglB). The resulting synthetic pathway enabled a laboratory strain of E. coli to produce secretory glycoproteins bearing asparagine-linked eukaryotic trimannosyl core N-glycans (mannose3-N-acetylglucosamine2). This development opens the door for the production of therapeutic glycoproteins in E. coli and the biosynthesis of novel N-glycan structures.

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