通过在工程大肠杆菌中同时转化葡萄糖和甘油可持续生产基于多糖的糖蛋白,Green Chemistry

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通过在工程大肠杆菌中同时转化葡萄糖和甘油可持续生产基于多糖的糖蛋白
Green Chemistry ( IF 9.3 ) Pub Date : 2023-05-18 , DOI: 10.1039/d3gc01279d
Yuhui Wang _{1,

2} , Xiaohan Wang _{1,

2} , Guozhen Ma _{1,

2} , Lijie Xie _{1,

2} , Dan Liu _{1,

2} , Yanling Wang _{1,

2} , Xinyu Zhao _{1,

2} , Yingying Su _{1,

2} , Andrei V. Perepelov ₃ , Peng Ding _{1,

2} , Xiao Zhang _{1,

2} , Bo Xu _{1,

2} , Bin Liu _{1,

2} , Di Huang _{1,

2}

Affiliation

尿路感染 (UTI) 是一种主要的传染病，通常由尿路致病性大肠杆菌(UPEC) 引起，其中普遍存在的血清群主要是抗生素耐药性 O4。因此，需要针对 UPEC 感染的保护性疫苗，尤其是血清群 O4。基于多糖的糖蛋白是开发抗菌疫苗的有前途的候选者；然而，由于需要复杂的连接策略和化学试剂，目前的化学和化学酶促合成成本高昂且对环境不友好。开发高效的微生物细胞工厂，使用廉价的可再生原料并避免使用有毒试剂，将减少有毒废物的产生和环境问题。O-糖基化大肠杆菌底盘的开发是为了安装 O 抗原基因簇，通过共同利用具有高协同代谢效率的葡萄糖/甘油混合物来生物合成基于多糖的糖蛋白。为了实现这一目标，从大肠杆菌MG1655 中删除了 32 个与竞争途径相关的基因，并引入了 28 个与糖蛋白生物合成相关的基因，从而成功生产了目标重组糖蛋白。随后，通过调节相应酶的表达来优化细胞内糖衍生物前体 dTDP- L -Rha 和 UDP- L -FucNAc的可用性。发酵条件的进一步优化导致糖蛋白滴度为 27.71 mg L ⁻¹. MS 和 EMR 证实了目标糖蛋白的精确生物合成和高效糖基化。最后，用糖蛋白接种疫苗可刺激小鼠产生强烈的免疫反应，这显着降低了 UPEC 攻击后膀胱和肾脏中的细菌负荷。本研究为开发特定的高价值治疗性糖蛋白的可持续且环保的制造平台提供了指导。

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Sustainable production of a polysaccharide-based glycoprotein by simultaneous conversion of glucose and glycerol in engineered Escherichia coli

Urinary tract infection (UTI) is a major infectious disease commonly induced by uropathogenic Escherichia coli (UPEC), of which a prevalent serogroup is the mostly antibiotic-resistant O4. Therefore, protective vaccines against UPEC infection, especially serogroup O4, are needed. Polysaccharide-based glycoproteins are promising candidates for development of anti-bacterial vaccines; however, current chemical and chemoenzymatic syntheses are costly and environmentally unfriendly, because of the complex ligation strategies and chemical reagents required. Development of efficient microbial cell factories, using cheap, renewable feedstocks and avoiding the use of toxic reagents would reduce toxic waste generation and environmental concerns. An O-glycosylation Escherichia coli chassis was developed to install the O-antigen gene cluster to biosynthesize a polysaccharide-based glycoprotein, by co-utilization of a glucose/glycerol mixture with high synergistic metabolic efficiency. Towards this goal, 32 genes related to competing pathways were deleted from E. coli MG1655 and 28 genes relevant to the biosynthesis of the glycoprotein were introduced, resulting in the successful production of the target recombinant glycoprotein. Subsequently, the availability of the intracellular sugar derivative precursors dTDP-L-Rha and UDP-L-FucNAc was optimized by tuning the expression of the corresponding enzymes. Further optimization of fermentation conditions resulted in a glycoprotein titre of 27.71 mg L⁻¹. MS and EMR confirmed the precise biosynthesis of the target glycoprotein and highly efficient glycosylation. Finally, vaccination with the glycoprotein stimulated a strong immune response in mice, which markedly reduced the bacterial burden in the bladder and kidneys after UPEC challenge. This study provides guidance for the development of a sustainable and environmentally friendly manufacturing platform for specific high-value therapeutic glycoproteins.

更新日期：2023-05-18

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