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. 2020 Aug 17;10(1):144.
doi: 10.1186/s13568-020-01084-6.

N-glycolyl chondroitin synthesis using metabolically engineered E. coli

Adeola E Awofiranye  1 , Sultan N Baytas  2   3 , Ke Xia  2 , Abinaya Badri  2 , Wenqin He  2 , Ajit Varki  4 , Mattheos Koffas  5   6 , Robert J Linhardt  7   8   9
Affiliations

N-glycolyl chondroitin synthesis using metabolically engineered E. coli

Adeola E Awofiranye et al. AMB Express. .

Abstract

N-glycolyl chondroitin (Gc-CN) is a metabolite of N-glycolylneuraminic acid (Neu5Gc), a sialic acid that is commonly found in mammals, but not humans. Humans can incorporate exogenous Neu5Gc into their tissues from eating red meat. Neu5Gc cannot be biosynthesized by humans due to an evolutionary mutation and has been implicated in causing inflammation causing human diseases, such as cancer. The study Neu5Gc is important in evolutionary biology and the development of potential cancer biomarkers. Unfortunately, there are several limitations to detecting Neu5Gc. The elimination of Neu5Gc involves a degradative pathway leading to the incorporation of N-glycolyl groups into glycosaminoglycans (GAGs), such as Gc-CN. Gc-CN has been found in humans and in animals including mice, lamb and chimpanzees. Here, we present the biosynthesis of Gc-CN in bacteria 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.

Keywords: Biotransformation; Metabolite; N-glycolyl chondroitin; N-glycolyl glucosamine; Sialic acid.

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Conflict of interest statement

The authors declare that have no conflict of interest.

Figures

Fig. 1
Fig. 1
The Cmah enzyme catalyzes the conversion of CMP-Neu5Ac to CMP-Neu5Gc through the addition of an oxygen atom
Fig. 2
Fig. 2
Neu5Gc is converted to UDP-GlcNGc and ultimately UDP-GalNGc. UDP-GalNGc can then be incorporated into Gc-CN and Gc-CS
Fig. 3
Fig. 3
Synthetic route for GlcNGc synthesis
Fig. 4
Fig. 4
LC–MS/MS MRM result for chondroitin control and N-glycolyl chondroitin sample. a Chondroitin control has a clear peak (MRM transition pair 572/396) for CN but no peak for Gc-CN (588/412). b N-glycolyl chondroitin sample both have clear peaks (572/396) for chondroitin and Gc-CN (588/412)
Fig. 5
Fig. 5
Metabolic pathway for N-glycolyl chondroitin synthesis in E. coli K4
See this image and copyright information in PMC

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