合成唾液酸乳糖重组大肠杆菌的构建

生物技术通报 ›› 2014, Vol. 0 ›› Issue (10): 201-206.

合成唾液酸乳糖重组大肠杆菌的构建

靳文斌, 张萧萧, 李玉, 路福平

天津科技大学生物工程学院工业发酵微生物教育部重点实验室工业酶国家工程实验室天津市工业微生物重点实验室,天津 300457

收稿日期:2014-05-04 出版日期:2014-10-20 发布日期:2014-10-17
作者简介:靳文斌,男,硕士研究生,研究方向：发酵工程
基金资助:
国家高技术研究发展计划“863计划”资助项目（2012AA021502）,教育部长江学者和创新团队发展计划项目（IRT1166）

The Construction of E.coli Engineering Strain for Sialyllactose Production

Jin Wenbin, Zhang Xiaoxiao, Li Yu, Lu Fuping

Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,National Engineering Laboratory for Industrial Enzymes,Tianjin Key Laboratory of Industrial Microbiology,the College of Biotechnology,Tianjin University of Science & Technology,Tianjin 300457

Received:2014-05-04 Published:2014-10-20 Online:2014-10-17

摘要/Abstract

摘要： 唾液酸寡糖具有抗感染、提高免疫力、促进双歧杆菌增殖等功能,对其微生物合成方法的研究具有重要的理论和应用价值。克隆和表达来源于Campylobacter jejuni的N-乙酰葡萄糖胺异构酶基因（neuC）、乙酰神经氨酸合成酶基因（neuB）、CMP-乙酰神经氨酸合成酶基因（neuA）和来源于Nesseria meningitidis的唾液酸转移酶基因（nst）,利用表达载体pSTV29,在大肠杆菌Escherichia coli JM109内构建合成唾液酸乳糖的代谢途径。在接种量2%、装液量50 mL/250 mL三角瓶、摇床转速180 r/min、温度34℃的条件下发酵30 h,并以10 g/L乳糖为底物,利用HPLC检测到唾液酸乳糖的合成量为2.45 g/L,为人乳唾液酸寡糖及其类似物的异体合成提供了新的思路。

关键词: 唾液酸乳糖, 大肠杆菌, 代谢工程

Abstract: Free sialylated oligosaccharides are known to have anti-infective and immunostimulating properties and also known to promote bifidobacterium proliferation, thus investigating the microbial synthetic route of sialylated oligosaccharides is of great value. Biosynthesis of sialyllactose involves N-acetylglucosamine-6-phosphate-epimerase（neuC）, sialic acid synthase（neuB）, CMP-Neu5Ac synthetase（neuA）and α-2, 3-sialyltransferase（nst）. We engineered a biosynthetic pathway sialyllactose production in E.coli JM109, using the expression vector pSTV29, by coexpressing the α-2, 3-sialyltransferase gene from Neisseria meningitidis with the neuA, neuB and neuC Campylobacter jejuni genes encoding CMP-NeuAc synthetase, sialic acid synthase and N-acetylglucosamine-6-phosphate-epimerase, respectively. Under the optimized fermentation conditions（inoculum volume 2%, 10g/L lactose, fermentation volume 50 mL/250 mL, temperature 34℃, rotation speed 180 r/min, fermentation time 30 h）, sialyllactose of 2.45 g/L was obtained. The above results provide a platform for exploring commercial production of sialylated oligosaccharides and its functional analogues in heterogeneous microbial hosts.

Key words: Sialyllactose, E.coli, Metabolic engineering

靳文斌, 张萧萧, 李玉, 路福平. 合成唾液酸乳糖重组大肠杆菌的构建[J]. 生物技术通报, 2014, 0(10): 201-206.

Jin Wenbin, Zhang Xiaoxiao, Li Yu, Lu Fuping. The Construction of E.coli Engineering Strain for Sialyllactose Production[J]. Biotechnology Bulletin, 2014, 0(10): 201-206.

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