Biomanufacturing of Therapeutically Relevant Chondroitin Sulfate C via Engineered Microbes

Chondroitin sulfate C (CS-C) is a biologically significant glycosaminoglycan in which a precise 6-<i>O</i>-sulfation pattern confers critical structural and signaling functions in connective and neural tissues. Here, we report the first functional expression of human chondroitin 6-<i>O</i>-sulfotransferase-1 (C6ST-1) in <i>Escherichia coli</i>, enabling cell-free biosynthesis of CS-C. By applying structure-guided protein engineering combining transmembrane truncation, His₆ -MBP fusion, and PROSS-directed stabilizing mutations, we generated a soluble and catalytically active variant in <i>E. coli</i> Origami B (DE3) and modified Shuffle T7 Express. Under optimized reaction conditions (MES buffer pH 5.5, 30 °C, Mg²⁺/Ca²⁺/Mn²⁺ with protamine sulfate), the engineered enzyme catalyzed up to 67% sulfation of chondroitin (CS-O) to CS-C, verified via SAX-HPLC. Kinetic and molecular dynamics analyses revealed enhanced substrate affinity and catalytic efficiency for the M9 Δ131 mutant. This study establishes a sustainable, animal-free platform for high-purity CS-C biomanufacturing and provides a generalizable strategy for engineering eukaryotic sulfotransferases for functional expression in bacterial hosts.