N-glycolyl chondroitin synthesis using metabolically engineered E. coli.

Area of Science:

• Biochemistry
• Metabolic Engineering
• Glycoscience

Background:

• N-glycolylneuraminic acid (Neu5Gc) is a sialic acid found in mammals but not humans, incorporated via diet and linked to inflammation and diseases like cancer.
• Humans lack the ability to biosynthesize Neu5Gc due to an evolutionary mutation, leading to challenges in its detection and study.
• N-glycolyl chondroitin (Gc-CN), a metabolite of Neu5Gc, is found in humans and various animals, representing a pathway for Neu5Gc elimination.

Purpose of the Study:

• To establish a bacterial system for the biosynthesis of N-glycolyl chondroitin (Gc-CN).
• To develop a method for producing Gc-CN for further research into its role in human health and disease.
• To investigate the potential of engineered bacteria as a tool for studying sialic acid metabolism and its implications.

Main Methods:

• Metabolically engineered Escherichia coli K4 strain was utilized.
• Bacteria were fed chemically synthesized N-glycolylglucosamine (GlcNGc) supplemented in glucose.
• The produced Gc-CN was enzymatically cleaved into disaccharides using chondroitin lyase ABC and analyzed via LC-MS/MS.

Main Results:

• Engineered E. coli successfully converted GlcNGc to N-glycolylgalactosamine (GalNGc).
• GalNGc was incorporated into the chondroitin biosynthetic pathway, leading to the production of Gc-CN.
• Analysis confirmed the presence of GalNGc within the chondroitin oligosaccharide backbone.

Conclusions:

• Bacterial biosynthesis of Gc-CN is feasible using metabolically engineered E. coli.
• This engineered system provides a novel approach for producing Gc-CN for research purposes.
• The study offers a new avenue for investigating the biological roles of Neu5Gc metabolites and their association with human diseases.