Design and Engineering of an Artificial Bifunctional N ‐Deacetylase/ N ‐Sulfotransferase for the Biosynthesis of N ‐Sulfated Heparosan

ABSTRACT Heparin is a widely used anticoagulant, while its traditional animal‐derived production faces significant challenges in safety and scalability. The active expression of the initiating‐step bifunctional enzyme N ‐deacetylase/ N ‐sulfotransferase (NDST) in bacteria remains a key bottleneck in heparin biosynthesis. Here, we firstly mined and characterized diverse polysaccharide N ‐deacetylases (NDases) in Escherichia coli and revealed several NDases that are active toward the deacetylation of heparosan. An artificial ND Dr ‐RL‐ST was designed and constructed by fusing an active NDase with a heterogenous NST domain. The ND Dr –MEc /ST variant with a 7.48‐fold improvement in catalytic efficiency was engineered by combining dynamic cross‐correlation analysis, sequence consensus analysis, and mutation‐effect prediction with sequential linker optimization and rational mutagenesis, resulting in enhanced substrate binding, active‐site stabilization, and improved inter‐domain coordination. ND Dr –MEc /ST efficiently converted heparosan to N ‐sulfated polysaccharide with a sulfation degree of 82.8%. The results highlighted the critical role of N ‐sulfotransferase interaction with ND Dr domain in stabilizing the catalytic conformation essential for bifunctional enzyme activity. Moreover, this work also provided valuable insights for the rational design of artificial multifunctional enzymes for polysaccharide modification.