Large scale in vivo synthesis of globotriose and globotetraose by high cell density culture of metabolically engineered Escherichia coli

Large amounts of globotriose (Gal alpha-4Gal beta-4Glc) are shown to be produced by the high cell density Culture of an Escherichia coli strain over-expressing the Neisseria meningitidis lgtC gene for alpha-1,4-Gal transferase. The strain which was devoid of both alpha and beta glactosidase activity was fed with glycerol as the energy and carbon source and with lactose as precursor for globotriose synthesis. After complete exhaustion of lactose. globotriose Could serve as an alternative acceptor for LgtC and the formation of a series of polyglactosylated compounds was observed. The system was extended to the synthesis of globotetraose (GalNAc beta-3Gal alpha-4Gal beta-4Glc) by overexpressing two additional genes lgtD from Haemophilus influenzae Rd which encodes a beta-1,3-GalNAc transferase and wbpP from Pseudomonas aeruginosa which encodes a UDP-GalNAc C4 epimerase, Globotetraose could also be produced from exogenous globotriose which was shown to be actively taken Lip by the cells. (c) 2004 Elsevier SAS. All rights reserved.