State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, China.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
Biotechnol Appl Biochem. 2023 Jun;70(3):1332-1345. doi: 10.1002/bab.2443. Epub 2023 Jan 25.
Monophosphoryl lipid A (MPL), mainly isolated from Salmonella minnesota R595, has been used as adjuvant in several vaccines. In this study, an Escherichia coli strain that can efficiently produce the MPL has been constructed. The gene clusters related to the biosynthesis of O-antigen, core oligosaccharide, enterobacterial common antigen, and colanic acid were sequentially removed to save the carbon source and to increase the activity of PagP in E. coli MG1655. Then, the genes pldA, mlaA, and mlaC related to the phospholipid transport system were further deleted, resulting in the strain MW012. Finally, the genes lpxE from Francisella novicida and pagP and pagL from Salmonella were overexpressed in MW012 to modify the structure of lipid A, resulting in the strain MW012/pWEPL. Lipid A species were isolated from MW012/pWEPL and analyzed by thin-layer chromatography and liquid chromatography-mass spectrometry. The results showed that mainly two MPL species were produced in E. coli MW012/pWEPL, one is hexa-acylated, and the other is penta-acylated. More importantly, the proportion of the hexa-acylated MPL, which is the most effective component of lipid A vaccine adjuvant, reached 75%. E. coli MW012/pWEPL constructed in this study provided a good alternative for the production of lipid A vaccine adjuvant MPL.
单磷酰脂质 A（MPL）主要从沙门氏菌 minnesota R595 中分离出来，已被用作几种疫苗的佐剂。在这项研究中，构建了一种能够高效生产 MPL 的大肠杆菌菌株。依次去除与 O-抗原、核心寡糖、肠杆菌共同抗原和柯立酸生物合成相关的基因簇，以节省碳源并增加大肠杆菌 MG1655 中 PagP 的活性。然后，进一步删除与磷脂转运系统相关的基因 pldA、mlaA 和 mlaC，得到菌株 MW012。最后，在 MW012 中过表达来自弗朗西斯氏菌 novicida 的 lpxE 基因以及来自沙门氏菌的 pagP 和 pagL 基因，以修饰脂质 A 的结构，得到菌株 MW012/pWEPL。从 MW012/pWEPL 中分离出脂质 A 种类，并通过薄层层析和液相色谱-质谱进行分析。结果表明，大肠杆菌 MW012/pWEPL 主要产生两种 MPL 种类，一种是六酰化的，另一种是五酰化的。更重要的是，最有效的脂质 A 疫苗佐剂成分六酰化 MPL 的比例达到 75%。本研究构建的大肠杆菌 MW012/pWEPL 为生产脂质 A 疫苗佐剂 MPL 提供了一个很好的替代方案。
