基于模块优化强化大肠杆菌合成乳糖-N-新四糖的研究
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(1.中国农业大学 食品科学与营养工程学院, 北京 100083;2.中原食品实验室, 河南 漯河 462300;3.中国农业大学 工学院, 北京 100083;4.蒙牛高科乳制品(北京)有限责任公司, 北京 101100)

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Study on Enhancement of Lacto-N-Neotetraose Synthesis in Escherichia coli Based on Module Optimization
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(1.College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China;2.Food Laboratory of Zhongyuan, Luohe 462300, China;3.College of Engineering, China Agricultural University, Beijing 100083, China;4.Mengniu Gaoke Dairy (Beijing) Co. Ltd., Beijing 101100, China)

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    摘要:

    乳糖-N-新四糖(lacto-N-neotetraose,LNnT)作为人乳寡糖核心组分之一,在婴幼儿成长发育过程中发挥着重要作用。为寻找高效的LNnT生物合成方法,探究模块优化对大肠杆菌合成LNnT产量的影响,以大肠杆菌BL21(DE3)ΔlacZ为出发菌株,根据合成路径中的关键代谢物质将LNnT合成路径划分为:外源酶所在路径的模块A,UDP-半乳糖合成路径的模块B和UDP-N-乙酰氨基葡萄糖合成路径的模块C。利用不同拷贝数质粒初步优化模块A、B和C的表达强度,当大肠杆菌BL21(DE3)ΔlacZ共表达重组质粒pRSF-lgtA-A.act和pET-galE时,LNnT产量最高,达0.87g/L。通过CRISPR/Cas9技术敲除setAugd强化模块A和模块B,获得的重组菌株E20合成LNnT产量达1.16g/L。摇瓶发酵条件优化后,重组菌株E20合成LNnT产量达1.28g/L。在5L发酵罐中,LNnT分批补料发酵产量达15.53g/L,发酵过程最高生产强度为0.43g/(L·h)。模块优化强化大肠杆菌高效合成LNnT有望为人乳寡糖的高效生物合成提供理论基础,进而推进婴幼儿配方食品产业的革新。

    Abstract:

    As one key component of human milk oligosaccharides, lacto-N-neotetraose (LNnT) plays an important role in the growth and development of infants. In order to explore the efficient method for biosynthetic of LNnT and to investigate the influence of module optimization on LNnT synthesis of Escherichia coli, E. coli BL21(DE3)ΔlacZ was used as the initial strain. And the synthetic pathway of LNnT was divided into the following three modules based on the key metabolites in synthetic pathway, module A for the exogenous enzymatic pathway, module B for the synthetic pathway of UDP-galactose, and module C for the synthetic pathway of UDP-N-acetylglucosamine. After preliminarily optimizing the expressions of modules A, B, and C via the plasmids with different copy number, the E. coli BL21(DE3)ΔlacZ harboring the recombinant plasmids pRSF-lgtA-A.act and pET-galE produced the highest LNnT titer of 0.87g/L. Modules A and B were enhanced owing to knocking setA and ugd via CRISPR/Cas9 technique, and the titer of LNnT produced by the recombinant strain E20 was up to 1.16g/L. After optimizing the fermentation conditions of strain E20, the titer of LNnT in shake-flask cultivation was up to 1.28g/L and further was up to 15.53g/L by fed-batch fermentation in a 5L bioreactor. The highest productivity of LNnT was up to 0.43g/(L·h) during fermentation. The enhancement of LNnT synthesis in E. coli with module optimization was expected to provide theoretical basis for efficient biosynthesis of human milk oligosaccharides and to drive innovation in food industry of infant formula.

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刘丹,梁山泉,闫巧娟,杨绍青,李树森,江正强.基于模块优化强化大肠杆菌合成乳糖-N-新四糖的研究[J].食品科学技术学报,2024,42(2):75-83.

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  • 在线发布日期: 2024-04-21
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