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 (GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc; Gal = galactose, Glc = glucose, Ac = acetyl) and GM1 (Galbeta-3GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc. The GM2 oligosaccharide-producing strain TA02 was devoid of both beta-galactosidase and sialic acid aldolase activities and overexpressed the genes for CMP-NeuAc synthase (CMP = cytidine monophosphate), alpha-2,3-sialyltransferase, UDP-GlcNAc (UDP = uridine diphosphate) C4 epimerase, and beta-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 beta-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.
Antoine
Tatiana
T
Centre de Recherches sur les Macromolécules Végétales 601 rue de la Chimie, BP 53X 38041 Grenoble, cedex 09, France. samain@cermav.cnrs.fr
Priem
Bernard
B
Heyraud
Alain
A
Greffe
Lionel
L
Gilbert
Michel
M
Wakarchuk
Warren W
WW
Lam
Joseph S
JS
Samain
Eric
E
D002236:Carbohydrate Conformation; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D002240:Carbohydrate Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005677:G(M1) Ganglioside / Q000096:biosynthesis* / Q000737:chemistry*; D005678:G(M2) Ganglioside / Q000096:biosynthesis* / Q000737:chemistry*; D005818:Genetic Engineering; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D007785:Lactose / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D000216:N-Acylneuraminate Cytidylyltransferase / Q000235:genetics / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry
D009844:Oligosaccharides; D005678:G(M2) Ganglioside; D005677:G(M1) Ganglioside; D016695:Glycosyltransferases; D000216:N-Acylneuraminate Cytidylyltransferase; D002238:Carbohydrate Epimerases; C023223:UDP-N-acetylglucosamine 4-epimerase; D019158:N-Acetylneuraminic Acid; D007785:Lactose