Production of unsulfated chondroitin and associated chondro-oligosaccharides in recombinant Escherichia coli

We designed metabolically engineered non-pathogenic strains of Escherichia coli to produce unsulfated chondroitin with and without chondroitin lyase to produce the chondroitin polymer or its related oligosaccharides. Chondroitin was synthesized using chondroitin synthase KfoC and chondroitin was degraded using Pl35, a chondroitin lyase from Pedobacter heparinus. Pl35 behaved as a true endo-enzyme generating a large panel of oligosaccharides ranging from trimers to 18-mers instead of the di- and tetramers obtained with most chondroitin lyases. Two series of oligosaccharides were characterized, sharing an unsaturated uronic acid (4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid, △UA) residue at their non-reducing end. The major "even-numbered" series was characterized by a terminal reducing N-acetylgalactosaminyl residue. The minor "odd-numbered" series oligosaccharides carried a terminal reducing glucuronic acid residue instead. Cultures were conducted in fed-batch conditions, and led to the production of up to 10 g L^-1 chondroitin or chondroitin oligosaccharides. All products were purified and fully characterized using NMR and mass spectrometry analyses. This is the first report of the microbial production of large chondro-oligosaccharides.