糖蛋白,其中多糖通常附着在蛋白质上,是一类重要的生物分子,几十年来在临床治疗中广泛用作治疗剂。尿路致病性大肠杆菌(UPEC)O21已被确定为诱导尿路感染的血清群,随着全球妇女和幼儿人数的增加。因此,迫切需要建立针对UPEC感染的保护性疫苗.在这里,我们设计了非致病性大肠杆菌MG1655以实现稳健,针对UPECO21的O21O-抗原多糖基糖蛋白的经济有效的从头生物合成。具体来说,该糖工程大肠杆菌MG1655被操作用于高效葡萄糖-甘油共利用以及基因簇安装和O-糖基化机器组装。UDP-糖前体的关键途径也得到了加强,以迫使更多的碳通量流向糖基供体,将糖蛋白滴度提高了5.6倍。进一步优化培养条件可产生高达35.34mg/L的糖蛋白。糖肽MS证实了糖蛋白的精确生物合成。该糖蛋白引发抗原特异性IgG免疫应答并显著减少肾脏和膀胱定植。这种基于细菌细胞的糖平台和优化策略可以为其他增值糖蛋白的生物合成提供指导。
Glycoproteins, in which polysaccharides are usually attached to proteins, are an important class of biomolecules that are widely used as therapeutic agents in clinical treatments for decades. Uropathogenic Escherichia coli (UPEC) O21 has been identified as a serogroup that induces urinary tract infections, with a global increasing number among women and young children. Therefore, there is an urgent need to establish protective vaccines against UPEC infection. Herein, we engineered non-pathogenic E. coli MG1655 to achieve robust, cost-effective de novo biosynthesis of O21 O-antigen polysaccharide-based glycoprotein against UPEC O21. Specifically, this glycoengineered E. coli MG1655 was manipulated for high-efficient glucose-glycerol co-utilization and for the gene cluster installation and O-glycosylation machinery assembly. The key pathways of UDP-sugar precursors were also strengthened to enforce more carbon flux towards the glycosyl donors, which enhanced the glycoprotein titer by 5.6-fold. Further optimization of culture conditions yielded glycoproteins of up to 35.34 mg/L. Glycopeptide MS confirmed the preciset biosynthesis of glycoprotein. This glycoprotein elicited antigen-specific IgG immune responses and significantly reduced kidney and bladder colonization. This bacterial cell-based glyco-platform and optimized strategies can provide a guideline for the biosynthesis of other value-added glycoproteins.