Chondroitin sulfate A (CSA) is an essential component in articular cartilage repair.CSA is synthesized by the hydroxyl sulfonation of N-acetylgalactosamine (GalNAc) at position 4 in chondroitin catalyzed by chondroitin 4-O-sulfotransferase-1 (C4ST-1).However, the low enzyme activity of C4ST-1 limits its catalytic efficiency, thereby hindering the industrial production of CSA.For this reason, this study aimed to enhance C4ST-1 enzyme activity by combining recombinant strain construction and medium optimization.Initially, based on the screening and determination of Komagataella phaffii GS115 as the chassis cell, the combination of OST1-α secretion signal peptide and SUMO Pro 3 solubility-enhancing tag was further optimized for the secretion and expression of C4ST-1, and the highest enzyme activity of C4ST-1 was 1,889.2 U/L after shake flask fermentation.Given that previous studies found inorganic salts to inhibit C4ST-1 enzyme activity and the high cost of the existing medium, this study optimized the medium components and found that omitting the expensive component, yeast nitrogen base without amino acids (YNB), resulted in a 68.4% increase in C4ST-1 enzyme activity compared to the original medium.Furthermore, the screening and optimization of carbon sources, other nitrogen sources, and biotin further increased the C4ST-1 enzyme activity.Finally, fed-batch fermentation in a 5 L fermenter for 72 hours achieved a maximum enzyme activity of 5 040.7 U/L.This study not only lays the foundation for the large-scale production of CSA, but will also provide a reference for the fermentation and production of sulfotransferases required for the synthesis of other glycosaminoglycans (e.g., heparin, dermatan sulfate).
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