Chondroitin sulfate C (CS-C) is a biologically important glycosaminoglycan garnering increased interest for its neuroprotective effects, particularly in low molecular weight form that mitigates amyloid-β toxicity in Alzheimer’s disease models . However, current supplies of CS-C rely on animal-derived sources results in heterogeneous mixtures and poses risks related to pathogen contamination and product variability. To address these challenges, we developed a microbial platform for the biosynthesis of structurally defined chondroitin-6-sulfate (CS-C) in Escherichia coli.The platform centers on heterologous expression of a mammalian chondroitin 6-O-sulfotransferase (C6ST) that installs sulfate at the 6-position of N-acetylgalactosamine, thereby converting unsulfated chondroitin into CS-C.Because C6ST is an integral Golgi glycoprotein with poor solubility and multiple disulfide bonds, initial expression in bacteria yielded a misfolded, low-activity enzyme . We overcame these challenges by applying structure-guided rational design and computational sequence optimization to improve C6ST folding, stability, and catalytic efficiency, and by engineering the host strain (e.g., an oxidative cytoplasmic environment) to facilitate proper disulfide bond formation for functional enzyme expression . With these interventions, the engineered E. coli strain successfully synthesized CS-C, as confirmed by chromatographic and structural analyses of the product. This work demonstrates the first one-step microbial synthesis of CS-C, paving the way for scalable, animal-free production of therapeutic glycosaminoglycans and supporting further development of CS-C-based interventions for Alzheimer’s disease.
