An engineered eukaryotic protein glycosylation pathway in Escherichia coli

Abstract

We performed bottom-up engineering of a synthetic pathway in Escherichia coli for the production of eukaryotic trimannosyl chitobiose glycans and the transfer of these glycans to specific asparagine residues in target proteins. The glycan biosynthesis was enabled by four eukaryotic glycosyltransferases, including the yeast uridine diphosphate-N-acetylglucosamine transferases Alg13 and Alg14 and the mannosyltransferases Alg1 and Alg2. By including the bacterial oligosaccharyltransferase PglB from Campylobacter jejuni, we successfully transferred glycans to eukaryotic proteins.

Figure 1. Engineering eukaryotic glycan biosynthesis in E. coli

Schematic of the synthetic pathway for synthesis of a trimannosyl core glycan and transfer to acceptor sites in target proteins. Enzyme names in black are native to E. coli; enzyme names in red are heterologous. See text for details. Glycan in brackets to the right of open arrows depicts terminally sialylated structure common in human glycoproteins, but outside the scope of this study.

Figure 2. Characterization of LLOs produced by glycoengineered E. coli

(a) Flow cytometric analysis of E. coli MC4100 gmd::kan or MC4100 gmd::kan ΔwaaL cells carrying plasmids as indicated. Cells were labeled with ConA-AlexaFluor prior to flow cytometry. Median cell fluorescence (M) values are given for each histogram. (b) MALDI-MS profile of permethylated glycans released from LLOs by acid hydrolysis. LLOs were extracted from E. coli MC4100 gmd::kan carrying plasmid pYCG. The major signal at m/z 1171 corresponds to [M+Na]⁺ of Hex₃HexNAc₂.

Figure 3. Transfer of eukaryotic glycans to target proteins in E. coli

(a) Western blot analysis of scFv13-R4^4x-DQNAT affinity purified from E. coli MC4100 gmd::kan ΔwaaL cells carrying pYCG-PglB_Cj or pYCG-PglB_Cjmut as indicated. Proteins isolated from cells expressing wild-type PglB_Cj were further treated with PNGase F for removal of N-linked glycans. Polyhistidine tags on the proteins were detected using anti-His antibodies while mannose glycans on the proteins were detected using ConA. See Supplementary Fig. 8 for full, uncut gel image. (b) MALDI-MS profile of permethylated glycopeptides generated by digestion of scFv13-R4^1x-DQNAT with Pronase E. The major signal at m/z 1282 corresponds to the permethylation product of Hex₃HexNAc₂-N, where the asparagine residue underwent β-elimination during the permethylation procedure (see inset). (c) MALDI-MS profile of permethylated glycans released from scFv13-R4^4x-DQNAT by PNGase F treatment. The major signal at m/z 1171 corresponds to [M+Na]⁺ of Hex₃HexNAc₂.