Sulfated glycosaminoglycans (GAGs) like heparan sulfates and chondroitin sulfates constitute an essential component of the extracellular matrix in higher eukaryotes. Hence, they serve as important pharmaceuticals. Of these, chondroitin sulfates (CS) are majorly used in the treatment of osteoarthritis. CS is also used to improve liver function, lower blood sugar and inhibit tumor metastasis. It is also used in drug delivery, food thickeners and preservatives. CS is commercially extracted from porcine, bovine, chicken, fish and shark tissues. There is a growing interest in animal-free CS production world-wide. Among such efforts, we have engineered for the first time, single strains of Escherichia coli that have the capability to produce CS in a single scalable fermentation. CS production involves biosynthesis and sulfation of chondroitin polymer. We previously engineered E. coli K4 to produce chondroitin by deleting the fructosyltransferase gene. However, expressing chondroitin sulfotransferase in this strain does not enable CS synthesis. In this work, we show that in addition to sulfotransferase expression, accumulation of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is crucial for CS synthesis. We employ different genetic constructs for accumulation of PAPS and expression of sulfotransferase to produce CS in E. coli. We also study the effect of different PAPS levels and polysaccharide transport efficiencies on sulfation and yield of CS from our engineered strains. This is the first report of animal-free one-pot, one-strain microbial CS synthesis. We propose that our animal-free product will also enable pharmaceutical and cell-culture applications, thus opening avenues to understand the importance of specific CS types in biological processes.

One-step microbial synthesis of chondroitin sulfates using engineered Escherichia coli

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.