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	bDGalp	104.18	?	73.77	69.82	?	62.25
4,3,2	Ac	176.13	23.41
4,3	bDGlcpN	103.92	56.43	73.43	79.49	?	61.14
4	bDGalp	104.13	?	83.27	69.61	?	62.18
	bDGlcp	96.98	?	?	79.59	?	61.35


1H NMR data:
Linkage	Residue	H1	H2	H3	H4	H5	H6
4,3,4	bDGalp	4.51	?	?	?	?	?
4,3,2	Ac	-	2.06
4,3	bDGlcpN	4.73	?	?	?	?	?
4	bDGalp	4.47	?	?	4.18	?	?
	bDGlcp	4.69	3.31	?	?	?	?


1H/13C HSQC data:
Linkage	Residue	C1/H1	C2/H2	C3/H3	C4/H4	C5/H5	C6/H6
4,3,4	bDGalp	104.18/4.51	?/?	73.77/?	69.82/?	?/?	62.25/?
4,3,2	Ac		23.41/2.06
4,3	bDGlcpN	103.92/4.73	56.43/?	73.43/?	79.49/?	?/?	61.14/?
4	bDGalp	104.13/4.47	?/?	83.27/?	69.61/4.18	?/?	62.18/?
	bDGlcp	96.98/4.69	?/3.31	?/?	79.59/?	?/?	61.35/?

The spectrum also has 8 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]2O)[C@@H]1O

707.632 g/mol (C₂₆H₄₅NO₂₁)