An engineered eukaryotic protein glycosylation pathway in Escherichia coli

Abstract

We performed bottom-up engineering of a synthetic pathway in E. coli for the production of eukaryotic trimannosyl chitobiose glycans and the transfer of these glycans to specific asparagine residues in target proteins. 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 C. jejuni, glycans were successfully transferred to eukaryotic proteins.

Structural Biology of Glycoproteins

“…The glycan is then transferred onto an N-glycosylation site using the oligosaccharyltransferase PglB from Campylobacter jejuni. Valderrama-Rincon et al tested production of three eukaryotic glycoproteins; the Fc domain of human IgG1, bovine RNaseA and the placental variant of human growth hormone, and detected expression of glycosylated proteins [7]. Although currently only ~1 % of the expressed proteins were found to be glycosylated, this technology represents a huge potential for the cost-effective production of glycoproteins with a defined glycosylation pattern.…”

“…Therefore, production of glycoproteins is most often performed using eukaryotic cells, although the aglycosylated protein can be expressed as inclusion bodies in E. coli and refolded and also produced using cell-free systems. Recent advances in glycosylation pathway engineering have resulted in both E. coli [7] and cell-free systems [8] which are capable of introducing Nglycosylation onto a protein. These methods are discussed in the relevant sections below.…”

“…Recently an engineered eukaryotic protein glycosylation pathway has been inserted into E. coli resulting in cells capable of producing glycoproteins with Man3GlcNAc2 sugars attached [7]. Four glycosyltransfereases from Saccharomyces cerevisae, the uridine diphosphate-Nacetylglucosamine transferases Alg13 and Alg14 and the mannosyltransferases Alg1 and Alg2, are used to generate the glycan.…”

Application of Quality by Design Paradigm to the Manufacture of Protein Therapeutics

“…The challenge was to introduce a pathway which would lead to an AsnGlcNAc linkage, then couple this with the mammalian glycosylation pathway which would generate an acceptable sugar moiety, but this was achieved using metabolic engineering ( [123] [124] [125]. The E. coli was then further engineered to synthesis a mannose3-Nacetylglucosamine2 (Man3GlcNAc2) glycan chain, a common core structure shared in all eukaryotes [126]. However, many challenges still remain.…”

Les capteurs biomimétiques en recherche biomédicale

“…C'est pour cette raison que certains laboratoires tentent de développer des hôtes plus adaptés. Par exemple, pour E. coli, des souches telles que C41, C43 ou Origami résistent mieux à la toxicité des protéines membranaires [6] ou sont capables de réaliser une partie de la glycosylation (E. coli MC4100) [7]. D'autres laboratoires ont développé des étiquettes peptidiques favorisant l'agrégation ou l'insertion des protéines dans les membranes, telles que Zera ou Mistic [8,9].…”