Redesigning chondroitin 6-O-sulfotransferase for efficient whole-cell biosynthesis of chondroitin sulfate C

Osteoarthritis remains a global health problem and chondroitin sulfate C (CSC) is effective to treat arthritis. The biosynthesis of CSC offers a sustainable and promising alternative to conventional extraction from animal tissues. However, the scarcity of chondroitin-6-O-sulfotransferases and their low catalytic performance have severely hindered the large-scale biosynthesis of CSC. To overcome these limitations, a novel chondroitin-6-O-sulfotransferase from Rattus norvegicus (RnCHST3) was identified via gene fishing, which demonstrates regioselectivity in transferring a sulfate group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the 6-position of chondroitin. Subsequent optimization of expression conditions and truncation of the N-terminal region enhanced the sulfation rate from 4.6% to 11.5%. For the first time, the regioselective mechanism of RnCHST3 was elucidated through molecular dynamics (MD) simulations and site-directed mutagenesis. A mechanism-guided rational design led to the development of mutant M5, which increased the sulfation rate to 46.2%. Furthermore, a novel "physical sieve"-guided PROSS design was developed to improve kinetic thermostability, resulting in mutant M7. This variant exhibited an extended half-life from 9.4 h to 12.3 h and achieved a sulfation rate to 74.5%. Finally, a whole-cell catalytic system was constructed for CSC production, reaching a sulfation rate of 86.4% and a titer of 12.9 g·L^-1-the highest level reported to date. This study lays a solid foundation for the industrial biosynthesis of CSC.