神经节苷脂氟化寡糖在大肠杆菌中的生物合成

doi:10.13560/j.cnki.biotech.bull.1985.2019-0229

摘要/Abstract

摘要：

神经节苷脂是一类存在于所有脊椎动物细胞中的糖脂分子,具有重要的生理功能和药用活性。合成生物学为构建神经节苷脂类的人工高效合成体系提供了契机,而其神经节苷脂氟化寡糖中间体的合成是至关重要的步骤。旨在实现神经节苷脂氟化寡糖在大肠杆菌中的人工生物合成途径：通过在大肠杆菌JM107中引入来源于脑膜炎奈瑟氏菌（Neisseria meningitidis）的CMP-NeuAc合成酶（neuA）,空肠弯曲杆菌（Campylobacter jejuni）来源的α 2,3-NeuAc转移酶（cst）、β 1,4-GalNAc转移酶（cgtA）、β 1,3-Gal转移酶（cgtB）和UDP-GlcNAc异构酶（gne）,成功构建了以氟化乳糖和唾液酸为底物的3种神经节苷脂GM3、GM2、GM1氟化寡糖在大肠杆菌中的生物合成途径;开发了高灵敏度的氟化寡糖柱前衍生-HPLC定量检测方法;并通过对工程菌株进行透酶过表达和启动子优化,在摇瓶发酵中GM3、GM2及GM1氟化寡糖的产量分别达到81.5 mg/L、18.1 mg/L及12.3 mg/L,为进一步的代谢工程改造奠定了良好的基础。

关键词: 大肠杆菌, 神经节苷脂, 合成生物学, 氟化寡糖, 生物合成

Abstract:

Gangliosides are glycosphingolipid molecules that present in all vertebrate cells and show important physiological functions and pharmaceutical activities. Synthetic biology provides an opportunity to construct biological synthetic system for the efficient production of gangliosides,in which the synthesis of oligosaccharide fluoride intermediates is a critical step. Herein,the genes of CMP-NeuAc synthase（neuA）from Neisseria meningitidis,α-2,3-sialyltransferase（cst）,β-1,4-GalNAc transferase（cgtA）,β-1,3-galactosyltransferase（cgtB）and UDP-GlcNAc C4 epimerase（gne）from Campylobacter jejuni were introduced into Escherichia coli JM107. Then the biosynthesis pathways of GM3,GM2,and GM1 oligosaccharide fluoride were successfully constructed in E. coli by using lactose fluoride and sialic acid as substrates. A highly sensitive detection method was developed based on pre-column-derivatization-HPLC. The engineered strains were optimized by permeases overexpression and promoter replacement,and the yields of GM3,GM2,and GM1 oligosaccharide fluoride reached 81.5 mg/L,18.1 mg/L,and 12.3 mg/L respectively in shaking flasks fermented,which laid a promising foundation for further transformation by metabolic engineering.

Key words: Escherichia coli, gangliosides, synthetic biology, oligosaccharide fluoride, biosynthesis

刘新平, 谭玉萌, 张雪, 冯雁, 杨广宇. 神经节苷脂氟化寡糖在大肠杆菌中的生物合成[J]. 生物技术通报, 2019, 35(8): 162-169.

LIU Xin-ping, TAN Yu-meng, ZHANG Xue, FENG Yan, YANG Guang-yu. Biosynthesis of Ganglioside Oligosaccharide Fluoride in Escherichia coli[J]. Biotechnology Bulletin, 2019, 35(8): 162-169.

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