硫酸软骨素E(CS-E)是一种重要的硫酸化糖胺聚糖,具有多种生物学功能和治疗潜力。这项研究标志着一个重要的里程碑,通过在大肠杆菌中首次成功地微生物生产4-硫酸软骨素6-O-磺基转移酶(GalNAc4S-6ST),使重组CS-E生物合成。最初,我们鉴定了能够将硫酸软骨素A(CS-A)转化为CS-E的磺基转移酶,但是这些酶在大肠杆菌中表达时是无功能的。此外,在蛋白质数据库中,这种特定的磺基转移酶没有实验衍生的三维结构。为了克服这一挑战,我们使用AlphaFold2开发了GalNAc4S-6ST的3D模型,并采用PROSS稳定性设计来鉴定具有不同N末端截短的增强酶溶解度和稳定性的突变.这些突变的实验验证导致了几种功能酶的鉴定。在测试酶表达的各种大肠杆菌菌株中,折纸B(DE3)是最有效的宿主。这促进了CS-A到CS-E的酶促转化,达到50%以上的转化率,并标志着无动物CS-E的首次成功生物合成这些发现代表了使用具有成本效益的碳源大规模合成CS-E的重大进展,为鲨鱼等濒危动物的传统采购提供了可持续的替代方案。关键点:•在简单的原核生物中实现GalNAc4S-6ST的功能性表达。•首次完成无动物硫酸软骨素E的生物合成。
Chondroitin sulfate E (CS-E) is a vital sulfated glycosaminoglycan with diverse biological functions and therapeutic potential. This study marks a significant milestone by achieving the first successful microbial production of chondroitin 4-sulfate 6-O-
sulfotransferase (GalNAc4S-6ST) in Escherichia coli, enabling recombinant CS-E biosynthesis. Initially, we identified sulfotransferases capable of converting chondroitin sulfate A (CS-A) to CS-E, but these enzymes were non-functional when expressed in E. coli. Moreover, there is no experimentally derived three-dimensional structure available for this specific
sulfotransferase in the protein databases. To overcome this challenge, we developed a 3D model of GalNAc4S-6ST using AlphaFold2 and employed PROSS stability design to identify mutations that enhance enzyme solubility and stability with different N-terminal truncations. Experimental validation of these mutations led to the identification of several functional enzymes. Among various E. coli strains tested for enzyme expression, Origami B (DE3) emerged as the most effective host. This facilitated the enzymatic conversion of CS-A to CS-E, achieving a conversion rate of over 50%, and marking the first successful biosynthesis of animal-free CS-E. These findings represent a significant advancement towards the large-scale synthesis of CS-E using cost-effective carbon sources, offering a sustainable alternative to traditional sourcing from endangered animals like sharks. KEY POINTS: • Functional expression of GalNAc4S-6ST in a simple prokaryote was accomplished. • First-time biosynthesis of animal-free chondroitin sulfate E was accomplished.