Wholly enzymatic programmable sulfation in nonanimal heparin synthesis

Heparin is a critically important clinical anticoagulant. The biosynthesis of heparin using Escherichia coli-based multienzyme cascades represents a promising alternative to animal-derived production. However, efficient deployment of heparin-synthesizing enzymes faces significant challenges, particularly in achieving recombinant expression of functionally active heparin N-deacetylase/N-sulfotransferase (NDST) enzymes in bacterial systems. In this article, we implement a strategy termed model animal-guided sequence-structure-activity to discover functional NDST orthologs compatible with prokaryotic expression. Coupled with computationally assisted focused rational iterative site-specific mutagenesis, we engineered the high-performance variant NDST-M8, which exhibited a 10.65-fold increase in activity and a 3.84-fold improvement in stability relative to the truncated variant AgNDST-M0. This methodology enabled an E. coli multienzyme cascade that synthesizes bioactive heparin from heparosan backbones, with tunable N-sulfation levels (30-90%) that precisely modulate anticoagulant activity. Our work resolves critical bottlenecks in enzymatic heparin production, establishing a scalable, nonanimal platform for the industrial manufacturing of activity-graded heparin therapeutics.