N-glycolyl chondroitin synthesis using metabolically engineered E. coli

Abstract

N -glycolylneuraminic acid (Neu5Gc) is a sialic acid missing in humans but commonly found in mammals and is the proposed cause of “xenosialitis” in humans, a “xenoautoimmune” inflammatory reaction believed to result from humans consuming Neu5Gc from red meat that human cells incorporate into some cell types, but is also recognized as a foreign antigen. The production of antibodies against Neu5Gc-containing glycans causes inflammation that is hypothesized to be an aggravating factor to multiple diseases associated with red meat consumption, including cancers arising from epithelial cells lining hollow organs and atherosclerosis involving vascular endothelial cells. The small amounts of Neu5Gc involved is glycosidically-linked at the reducing end of glycans but is difficult to quantitate because of its location on endothelial and epithelial cells. A more stable recently discovered metabolite of Neu5Gc, N-glycolyl chondroitin (Gc-CN), is present in the serum of humans who eat red meat and is more easily isolated and analyzed. Here, we present the biosynthesis of Gc-CN by feeding chemically synthesized N-glycolylglucosamine to Escherichia coli. A metabolically engineered strain of E. coli K4, fed with glucose supplemented with GlcNGc, converted it to N-glycolylgalactosamine (GalNGc) that could then be utilized as a substrate in the chondroitin biosynthetic pathway. The final product, Gc-CN was converted to disaccharides using chondroitin lyase ABC and analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring detection. This analysis showed the incorporation of GalNGc into the backbone of the chondroitin oligosaccharide.