{Reference Type}: Journal Article
{Title}: Biosynthetic production of anticoagulant heparin polysaccharides through metabolic and sulfotransferases engineering strategies.
{Author}: Deng JQ;Li Y;Wang YJ;Cao YL;Xin SY;Li XY;Xi RM;Wang FS;Sheng JZ;
{Journal}: Nat Commun
{Volume}: 15
{Issue}: 1
{Year}: 2024 May 4
{Factor}: 17.694
{DOI}: 10.1038/s41467-024-48193-5
{Abstract}: Heparin is an important anticoagulant drug, and microbial heparin biosynthesis is a potential alternative to animal-derived heparin production. However, effectively using heparin synthesis enzymes faces challenges, especially with microbial recombinant expression of active heparan sulfate N-deacetylase/N-sulfotransferase. Here, we introduce the monosaccharide N-trifluoroacetylglucosamine into Escherichia coli K5 to facilitate sulfation modification. The Protein Repair One-Stop Service-Focused Rational Iterative Site-specific Mutagenesis (PROSS-FRISM) platform is used to enhance sulfotransferase efficiency, resulting in the engineered NST-M8 enzyme with significantly improved stability (11.32-fold) and activity (2.53-fold) compared to the wild-type N-sulfotransferase. This approach can be applied to engineering various sulfotransferases. The multienzyme cascade reaction enables the production of active heparin from bioengineered heparosan, demonstrating anti-FXa (246.09 IU/mg) and anti-FIIa (48.62 IU/mg) activities. This study offers insights into overcoming challenges in heparin synthesis and modification, paving the way for the future development of animal-free heparins using a cellular system-based semisynthetic strategy.