食品科学技术学报2024，Vol.42Issue(2)：75-83,9.DOI:10.12301/spxb202300614
基于模块优化强化大肠杆菌合成乳糖-N-新四糖的研究
Study on Enhancement of Lacto-N-Neotetraose Synthesis in Escherichia coli Based on Module Optimization
摘要
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)△lacZ 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)△lacZ harboring the recombinant plasmids pRSF-lgtA-A.act and pET-galE produced the highest LNnT titer of 0.87 g/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 1.16 g/L.After optimizing the fermentation conditions of strain E20,the titer of LNnT in shake-flask cultivation was up to 1.28 g/L and further was up to 15.53 g/L by fed-batch fermentation in a 5 L bioreactor.The highest productivity of LNnT was up to 0.43 g/(Leh)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.关键词
大肠杆菌/乳糖-N-新四糖/模块优化/生物合成/CRISPR/Cas9Key words
Escherichia coli/lacto-N-neotetraose/module optimization/biosynthesis/CRISPR/Cas9分类
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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):75-83,9.基金项目
国家自然科学基金面上项目(32172159).General Program of National Natural Science Foundation of China(32172159). （32172159）
