(576a) Production of Chondroitin Sulfate with Escherichia coli K4

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, kfoA, kfoC 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. coli BL21. 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.