Large-Scale In Vivo Synthesis of the Carbohydrate Moieties of Gangliosides GM1 and GM2 by Metabolically Engineered Escherichia coli

Two metabolically engineered Escherichia coli strains have been constructed to produce the carbohydrate moieties of gangliosides GM2 (GalNAcβ-4(NeuAcα-3)Galβ-4Glc; Gal=galactose, Glc=glucose, Ac=acetyl) and GM1 (Galβ-3GalNAcβ-4(NeuAcα-3)Galβ-4Glc. The GM2 oligosaccharide-producing strain TA02 was devoid of both β-galactosidase and sialic acid aldolase activities and overexpressed the genes for CMP-NeuAc synthase (CMP=cytidine monophosphate), α-2,3-sialyltransferase, UDP-GlcNAc (UDP=uridine diphosphate) C4 epimerase, and β-1,4-GalNAc transferase. When this strain was cultivated on glycerol, exogenously added lactose and sialic acid were shown to be actively internalized into the cytoplasm and converted into GM2 oligosaccharide. The in vivo synthesis of GM1 oligosaccharide was achieved by taking a similar approach but using strain TA05, which additionally overexpressed the gene for β-1,3-galactosyltransferase. In high-cell-density cultures, the production yields for the GM2 and GM1 oligosaccharides were 1.25 g L⁻¹ and 0.89 g L⁻¹, respectively.