(620p) Microbial Production of Chondrotin Sulfate

Chondroitin sulfate (CS) is an important glycosaminoglycan that predominantly presents in cartilage and surfaces of many cells and extracellular matrices. It has been widely used in treating osteoarthrithis due to its anti-inflammatory and chondro-protective properties. Given the importance of CS in medicine and as a dietary supplement, a traditional animal extraction method that is under potential risk of virus and/or prion contamination should be replaced by standardized bioengineering process. The capsular polysaccharide of pathogenic Escherichia coli K4 strain shares a similar structure to the CS precursor, chondroitin. Most of our effort has been focused on producing chondroitin or K4 polysaccharide. Overexpression of the biosynthesis pathway of K4 polysaccharide in a non-pathogenic production strain E. coli BL21*(DE3) results in ~2.4 g/L chondroitin in DO-STAT fed batch fermenter. Human Golgi chondroitin 4-sulfotransferase (C4ST1) catalyzes the transfer of a sulfo group to the 4-position of chondroitin. Functional expression of a soluble C4ST1 in E. coli BL21*(DE3) remains a challenge due to its sensitivity to the modification of N-glycosylation site. Recently, C4ST1 has been successfully expressed and purified resulting in a high level of conversion of chondroitin to CS type-A in vitro. The metabolic engineering of chondroitin biosynthesis and C4ST1 production into the same organism is underway to produce a bioengineered microbial CS as a promising alternative animal sourced CS.