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

✍ Scribed by Tatiana Antoine; Bernard Priem; Alain Heyraud; Lionel Greffe; Michel Gilbert; Warren W. Wakarchuk; Joseph S. Lam; Eric Samain

• Publisher: John Wiley and Sons
• Year: 2003
• Tongue: English
• Weight: 135 KB
• Volume: 4
• Category: Article
• ISSN: 1439-4227
• DOI: 10.1002/cbic.200200540

✦ Synopsis

Abstract

Two metabolically engineered Escherichia coli strains have been constructed to produce the carbohydrate moieties of gangliosides GM2 (GalNAc__β‐4(NeuAc__α__‐3)Gal__β__‐4Glc; Gal=galactose, Glc=glucose, Ac=acetyl) and GM1 (Gal__β__‐3GalNAc__β__‐4(NeuAc__α__‐3)Gal__β__‐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.