(599bt) Metabolic Engineering and Applied Enzymology for the Preparation of Nutraceutical Chondroitin Sulfate

He, W., Rensselaer Polytechnic Institute
Koffas, M. A. G., Rensselaer Polytechnic Institute
Linhardt, R. J., Rensselaer Polytechnic Institute

Chondroitin sulfate is an important homopolymeric glycosaminoglycan consisting of repeating disaccharide units of N-acetyl-D-galactosamine and glucuronic acid with sulfation at various positions. It has been widely used for treating knee osteoarthrithis due to its anti-inflammatory and chondro-protective properties. The current method for manufacturing chondroitin sulfate relies solely on animal sourced tissues, which may be unreliable due to the risk of virus and/or prion contamination.  Given the importance of its use in medicine and as a dietary supplement, alternative production methods are necessary in order to provide stable and safe supply of chondroitin sulfate. The capsular polysaccharide of the pathogenic Escherichia coli K4 strain shares a similar structure to the chondroitin sulfate except for a β-linked fructose branching residue on the chondroitin backbone. The three genes, kfoAkfoC and kfoF, which encode the major enzymes for the biosynthesis of the backbone of this chondroitin-like capsular polysaccharide in K4, were cloned into ePathbrick system vectors allowing a tunable gene copy number and promoter strength in a single plasmid, and then transformed into the non-pathogenic production strain E. coliBL21. A significant level of polysaccharide production was observed with an average yield of ~2.2 g/L chondroitin in shake flask. Functional expression of sulfotransferases has further allowed the production of a chondroitin sulfate derivative for the first time in E.coli.