Expression Optimization and Semirational Engineering of Ga6OST1 for Improved Catalytic Performance in Heparin Biosynthesis.

Bioengineered heparin represents a safe and sustainable alternative to animal-derived anticoagulants. However, inefficient 6-O-sulfation catalyzed by heparan sulfate 6-O-sulfotransferase remains a bottleneck hindering the synthesis of heparin. Here, we establish a high-efficiency expression platform for Gallus gallus heparan sulfate 6-O-sulfotransferase 1 (Ga6OST1) in Komagataella phaffii. First, a systematic secretion-engineering strategy established an optimized production chassis with markedly elevated extracellular Ga6OST1 activity. Building on this platform, a semirational design strategy integrating structural modeling, evolutionary analysis, and hotspot prediction identified a triple mutant (MBP-Ga6OST1-A77R/V107L/V248T) with markedly improved properties. This variant exhibited a 2.4-fold increase in specific activity, a 2.3-fold enhancement in catalytic turnover, and a 5.3-fold extension in half-life at 37 °C. Structural analysis and molecular dynamics simulations revealed that cooperative reinforcement of hydrophobic packing and electrostatic networks underlies the observed gains in stability and catalytic durability. Under preparative catalytic conditions, the engineered enzyme displayed enhanced performance toward distinct sulfated heparin precursors, enabling the production of a final product with a 6-O-sulfation degree of 75.7%, comparable to that of porcine intestinal heparin. This work establishes an efficient platform for the production of structurally defined, highly 6-O-sulfated heparin and related derivatives.