(256c) Animal-Free Chondroitin Sulfate Production through Protein Engineering and Metabolic Engineering Strategies

Sulfated glycosaminoglycans (GAGs) such as chondroitin sulfate are among the top ranked products in industrial biotechnology and are widely used in biomedical applications. Most popularly, chondroitin sulfate is used for osteoarthritis treatment and promoting joint health.Traditionally, this GAG is extracted from animal tissues like bovine trachea. However, despite meeting current worldwide demands, animal extraction is not a sustainable option due to limited availability of source tissues (mostly from food animals), scale-up issues, adverse environmental impacts, and quality control issues.

As part of ongoing efforts to separate the food chain from the drug chain, to circumvent these challenges we are investigating the possibility of using engineered microorganisms for the biosynthesis of chondroitin sulfate. Bacterial production systems offer the advantages of lower cost and production time, as well as eliminating contamination risks associated with the more traditional animal extraction production methods. While the enzymes involved in the biosynthesis of chondroitin sulfates are well known, the cloning end expression of these membrane-bound Golgi enzymes has proved to be challenging.

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 another E. coli-expressed sulfotransferase, chondroitin-4-sulfotransferase, through engineering strategies adopted to develop more stable mutants.

Alongside the production of non-sulfated chondroitin through co-expression of E. coli strain K4 genes kfoA, kfoC, and kfoE in the non-pathogenic strain E. coli BL21, two important components of the biotransformation scheme are made available for in vitro chondroitin sulfate production. This proof of principle represents an important milestone along the path toward de novo biosynthesis of chondroitin sulfate from simple carbon sources like glucose and glycerol in a single microbial host.