Production of chondroitin in metabolically engineered E. coli

Chondroitin sulfates, widely used in the treatment of arthritis, are glycosaminoglycans extracted from food animal tissues. As part of our ongoing efforts to separate the food chain from the drug chain, we are examining the possibility of using metabolic engineering to produce chondroitin sulfate in Escherichia coli. Chondroitin is a valuable precursor in the synthesis of chondroitin sulfate. This study proposes a safer and more feasible approach to metabolically engineer chondroitin production by expressing genes from the pathogenic E. coli K4 strain, which natively produces a capsular polysaccharide that shares the similar structure with chondroitin, into the non-pathogenic E. coli BL21 Star^TM (DE3) strain. The ePathBrick vectors, allowing for multiple gene addition and expression regulatory signal control, are used for metabolic balancing needed to obtain the maximum potential yield. The resulting engineered strain produced chondroitin, as demonstrated by ¹H NMR and disaccharide analysis, relying on chondrotinase treatment followed by liquid chromatography-mass spectrometry. The highest yield from shake flask experiment was 213mg/L and further increased to 2.4g/L in dissolved oxygen-stat fed batch bioreactor.