N-glycolyl chondroitin synthesis using metabolically engineered E. coli

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.

Heterologous production of chondroitin

“…In the work of Awofiranye et al. [196] , N ‑glycolyl chondroitin has been produced from chemically synthesized precursors ( N -glycolylglucosamine replacing GlcNAc in the pathway) in E. coli K4 lacking kfoE gene.…”

Chondroitin Sulfate and Its Derivatives: A Review of Microbial and Other Production Methods

“…This GAG is found in many animals [76] but has just recently been synthesized in bacteria. Gc-CN was synthesized in E. coli K4 with a deleted kfoE gene (K4_∆kfoE) by feeding the bacteria with chemically synthesized N-glycolylglucosamine [72]. This modified GAG is being explored as a potential cancer biomarker and also has some evolutionary applications.…”