School of Pharmaceutical Sciences, Shandong University, Jinan, China.
National Glycoengineering Research Center, Shandong University, Jinan, China.
Nat Commun. 2024 May 4;15(1):3755. doi: 10.1038/s41467-024-48193-5.
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.
肝素是一种重要的抗凝药物，微生物来源的肝素生物合成是动物来源肝素生产的潜在替代方法。然而，有效利用肝素合成酶面临挑战，特别是在微生物重组表达活性硫酸乙酰肝素 N-脱乙酰基/N-磺基转移酶时。在这里，我们将单糖 N-三氟乙酰氨基葡萄糖引入大肠杆菌 K5 中，以促进硫酸化修饰。采用 Protein Repair One-Stop Service-Focused Rational Iterative Site-specific Mutagenesis (PROSS-FRISM) 平台提高磺基转移酶的效率，得到了工程化的 NST-M8 酶，与野生型 N-磺基转移酶相比，稳定性提高了 11.32 倍，活性提高了 2.53 倍。该方法可应用于各种磺基转移酶的工程化。多酶级联反应使生物工程化的肝素聚糖能够生产出具有活性的肝素，其抗 FXa（246.09 IU/mg）和抗 FIIa（48.62 IU/mg）活性。本研究为克服肝素合成和修饰中的挑战提供了思路，为未来采用基于细胞体系的半合成策略开发无动物来源的肝素铺平了道路。
