Production of Complex Human Glycoproteins in Yeast

Abstract

We report the humanization of the glycosylation pathway in the yeast Pichia pastoris to secrete a human glycoprotein with uniform complex N-glycosylation. The process involved eliminating endogenous yeast glycosylation pathways, while properly localizing five active eukaryotic proteins, including mannosidases I and II, N-acetylglucosaminyl transferases I and II, and uridine 5′-diphosphate (UDP)-N-acetylglucosamine transporter. Targeted localization of the enzymes enabled the generation of a synthetic in vivo glycosylation pathway, which produced the complex human N-glycan N-acetylglucosamine₂-mannose₃-N-acetylglucosamine₂ (GlcNAc₂Man₃GlcNAc₂). The ability to generate human glycoproteins with homogeneous N-glycan structures in a fungal host is a step toward producing therapeutic glycoproteins and could become a tool for elucidating the structure-function relation of glycoproteins.