(596f) Microbial Production of Chondroitin Sulfates: An Eco-Friendly Alternative to Animal-Based Extraction

Chondroitin sulfate is a crucial component of connective tissues and is widely used in the treatment of osteoarthritis. However, commercial extraction of chondroitin sulfates from animal tissues results in undefined mixtures of various types of chondroitin sulfates, potentially affecting their biological functions. This method is also susceptible to cross-species viral or prion infection. The microbial production of chondroitin sulfotransferases has garnered attraction as a promising method for the efficient and sustainable production of chondroitin sulfates to overcome these difficulties. In this study, we report the successful production of chondroitin-6-sulfate (CS-C) and chondroitin-4,6-disulfate (CS-E) by the accumulation of sulfo-donor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) and expression of chondroitin 6-sulfotransferase (C6ST) and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) enzymes, respectively, in E. coli. We employed protein engineering strategies such as PROSS and performed docking studies to identify potential mutations of the enzymes which would enhance their activities. We also optimized fermentation conditions for high-level production of the enzymes. A high level of sulfation was achieved in CS-C and CS-E by metabolic engineering, indicating their potential for industrial-scale production. Our findings suggest a significant advancement towards the development of animal-free and eco-friendly bioprocesses for the production of chondroitin sulfate which is highly valued by the pharmaceutical industry.