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- O -sulfation pattern confers critical structural and signaling functions in connective and neural tissues. Here, we report the first functional expression of human chondroitin 6- O -sulfotransferase-1 (C6ST-1) in Escherichia coli, enabling cell-free biosynthesis of CS–C. By applying structure-guided protein engineering combining transmembrane truncation, His 6 -MBP fusion, and PROSS-directed stabilizing mutations, we generated a soluble and catalytically active variant in E. coli Origami B (DE3) and modified Shuffle T7 Express. Under optimized reaction conditions (MES buffer pH 5.5, 30 °C, Mg 2+ /Ca 2+ /Mn 2+ 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.