Efficient Expression of an Engineered Heparan Sulfate 2-<i>O</i>-Sulfotransferase with Improved Catalytic Properties

Heparin, as a type of highly sulfated polysaccharide, is crucial for various physiological and pathophysiological functions. Heparan sulfate 2-<i>O</i>-sulfotransferase (2<i>O</i>ST) is responsible for the second sequential sulfation modification in heparin biosynthesis. However, challenges such as low expression and poor enzyme activity performance limit its application. To this end, a combination of strategies was employed to improve expression level and catalytic performance. First, SUMO fusion tag was used to enable the active expression of <i>Gallus gallus</i>-derived 2<i>O</i>ST (Ga2<i>O</i>ST), followed by enhancing its expression level through N-terminal synonymous codon optimization. Under the principle of considering both catalytic activity and stability, the combinatorial mutant SUMO-Ga2<i>O</i>ST A98K/Y145F was successfully constructed, resulting in a 2.32-fold increase in catalytic activity and a 7.80-fold extension of its half-life. Eventually, the enzyme activity was improved to 5720 U/mL with a 14.79-fold increase in a 5 L fermenter, which, to the best of our knowledge, is the highest reported to date. The engineered mutant SUMO-Ga2<i>O</i>ST A98K/Y145F with markedly enhanced active expression and catalytic performance could provide a solid foundation for heparin biomanufacturing.