Sulfated glycosaminoglycans such as chondroitin sulfate (CS) are widely used in biomedical applications and have extensive global markets. CS is traditionally extracted from food animal tissues like bovine trachea, but this is not a sustainable option due to limited availability of source tissues as well as scale-up and quality control issues. To address these challenges and as part of efforts to separate the food chain from the drug chain, we are exploring the use of engineered microorganisms for the biosynthesis of CS. Bacterial production systems offer advantages of lower cost and production time, and eliminate contamination risks associated with more traditional animal extraction production methods. Enzymes involved in the biosynthesis of CS are well known but the cloning and expression of these membrane-bound Golgi enzymes has posed challenges. Toward the development of a biotransformation scheme, where multiple E. coli strains are engineered to generate the components required for in vitro sulfated polysaccharide production, we have expressed chondroitin 6-sulfotransferase, which catalyzes the transfer of sulfate to position-6 of N-acetylgalactosamine residues of chondroitin, by E. coli in active form. Additionally, activity improvements have been made to E. coli-expressed chondroitin-4-sulfotransferase, using engineering strategies adopted to develop more stable mutants. Alongside non-sulfated chondroitin produced by co-expression of E. coli strain K4 genes in the non-pathogenic strain E. coli BL21, two important components of the biotransformation scheme are made available for in vitro CS production. This proof of principle represents an important milestone in the path toward de novo biosynthesis of CS from simple carbon sources by a single microbial host.

Protein engineering and metabolic engineering strategies for animal-free chondroitin sulfate production

2019 Spring National Meeting

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