The α-Gal epitope is a carbohydrate structure, Galα-3Galβ-4GlcNAc-R, expressed on glycoconjuguates in many mammals, but not in humans. Species that do not express this epitope have present in their serum large amounts of natural anti-Gal antibodies, which contribute to organ hyperacute rejection during xenotransplantation. We first describe the efficient conversion of lactose into isoglobotriaose (Galα-3Galβ-4Glc) using high cell density cultures of a genetically engineered Escherichia coli strain expressing the bovine gene for α-1,3-galactosyltransferase. Attempts to produce the Galili pentasaccharide (Galα-3Galβ-4GlcNAcβ-3Galβ-4Glc) by additionally expressing the Neisseria meningitis lgtA gene for β-1,3-N-acetylglucosaminyltransferase and the Helicobacter pylori gene for β-1,4-galactosyltransferase were unsuccessful and led to the formation of a series of long chain oligosaccharides formed by the repeated addition of the trisaccharide motif [Galβ-4GlcNAcβ-3Galα-3] onto a lacto-N-neotetraose primer. The replacement of LgtA by a more specific β-1,3-N-acetylglucosaminyltransferase from H. pylori, which was unable to glycosylate α-galactosides, prevented the formation of these unwanted compounds and allowed the successful formation of the Galili pentasaccharide and longer α-Gal epitopes.

Escherichia coli, Oligosaccharides, fermentation, Metabolic engineering, α-Gal epitope, a-Gal epitope

13C NMR data:
Linkage	Residue	C1	C2	C3	C4	C5	C6
4,3,4,3	aDGalp	96.71	?	?	70.39	72.11	62.21
4,3,4	bDGalp	103.94	?	78.52	66.09	?	62.21
4,3,2	Ac	176.10	23.47
4,3	bDGlcpN	104.04	56.44	?	79.75	?	61.24
4	bDGalp	104.14	?	83.27	69.59	?	62.21
	bDGlcp	96.98	75.05	?	79.61	?	61.36


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,3,4,3	aDGalp	5.17	3.89	?	4.05	4.23	?
4,3,4	bDGalp	4.58	3.69	3.82	4.22	?	?
4,3,2	Ac	-	2.06
4,3	bDGlcpN	4.73	3.84	?	3.78	?	?
4	bDGalp	4.47	3.62	3.76	4.19	?	?
	bDGlcp	4.69	3.31	3.68	3.69	?	?


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,3,4,3	aDGalp	96.71/5.17	?/3.89	?/?	70.39/4.05	72.11/4.23	62.21/?
4,3,4	bDGalp	103.94/4.58	?/3.69	78.52/3.82	66.09/4.22	?/?	62.21/?
4,3,2	Ac		23.47/2.06
4,3	bDGlcpN	104.04/4.73	56.44/3.84	?/?	79.75/3.78	?/?	61.24/?
4	bDGalp	104.14/4.47	?/3.62	83.27/3.76	69.59/4.19	?/?	62.21/?
	bDGlcp	96.98/4.69	75.05/3.31	?/3.68	79.61/3.69	?/?	61.36/?

The spectrum also has 10 signals at unknown positions (not plotted).

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

CC(=O)N[C@H]1[C@H](O[C@H]2[C@@H](O)[C@@H](CO)O[C@@H](O[C@@H]3[C@@H](CO)O[C@@H](O)[C@H](O)[C@H]3O)[C@@H]2O)O[C@H](CO)[C@@H](O[C@@H]2O[C@H](CO)[C@H](O)[C@H](O[C@H]3O[C@H](CO)[C@H](O)[C@H](O)[C@H]3O)[C@H]2O)[C@@H]1O

869.773 g/mol (C₃₂H₅₅NO₂₆)