Metabolic engineering of Escherichia coli for the production of 2'-fucosyllactose and 3-fucosyllactose through modular pathway enhancement
Metabolic engineering of Escherichia coli for the production of 2'-fucosyllactose and 3-fucosyllactose through modular pathway enhancement
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DOI：
10.1016/j.ymben.2017.03.001
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发表时间：
2017-05-01
影响因子：
8.4
通讯作者：
Feng, Lu
中科院分区：
文献类型：
--
作者：
Huang, Di;Yang, Kexin;Feng, Lu
岩藻糖基乳糖，包括2 '-岩藻糖基乳糖（2'-FL）和3-岩藻糖基乳糖（3-FL），是人乳中的重要寡糖，由于其生物学功能，如促进双歧杆菌生长、抑制病原体感染和改善免疫应答，通常用作婴儿配方食品中的营养添加剂。在本研究中，我们开发了一种合成生物学方法来促进工程大肠杆菌中2 '-FL和3-FL的高效生物合成。为了提高2 '-FL和3-FL的产量，以即插即用的方式应用了多种模块优化策略。首先，比较各种外源性α 1，2-岩藻糖基转移酶和α 1，3-岩藻糖基转移酶候选物，以及一系列E. coli宿主菌中，以BL 21（DE 3）为宿主菌的幽门螺杆菌的futC和futA产生了最高滴度的2 '-FL和3-FL。随后，通过灭活竞争性（或抑制性）途径和增强受体（或供体）的可用性来优化乳糖受体底物的可用性和GDP-L-岩藻糖供体底物的细胞内库，以实现过量生产。此外，还对细胞内氧化还原再生途径进行了改造，以进一步提高2 '-FL和3-FL的产量。最后，对各种培养条件进行了优化，以使2'-FL和3-FL生物合成菌株的性能达到最佳。2 '-FL和3-FL的最终浓度分别为9.12和12.43 g/L。这项工作提供了一个平台，使模块化的建设，优化和表征，以促进FL生产细胞工厂的发展。
Fucosyllactoses, including 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL), are important oligosaccharides in human milk that are commonly used as nutritional additives in infant formula due to their biological functions, such as the promotion of bifidobacteria growth, inhibition of pathogen infection, and improvement of immune response. In this study, we developed a synthetic biology approach to promote the efficient biosynthesis of 2'-FL and 3-FL in engineered Escherichia coli. To boost the production of 2'-FL and 3-FL, multiple modular optimization strategies were applied in a plug-and-play manner. First, comparisons of various exogenous alpha 1,2-fucosyltransferase and alpha 1,3-fucosyltransferase candidates, as well as a series of E. coli host strains, demonstrated that futC and futA from Helicobacter pylori using BL21(DE3) as the host strain yielded the highest titers of 2'-FL and 3-FL. Subsequently, both the availability of the lactose acceptor substrate and the intracellular pool of the GDP-L-fucose donor substrate were optimized by inactivating competitive (or repressive) pathways and strengthening acceptor (or donor) availability to achieve overproduction. Moreover, the intracellular redox regeneration pathways were engineered to further enhance the production of 2'-FL and 3-FL. Finally, various culture conditions were optimized to achieve the best performance of 2'-FL and 3-FL biosynthesizing strains. The final concentrations of 2'-FL and 3-FL were 9.12 and 12.43 g/L, respectively. This work provides a platform that enables modular construction, optimization and characterization to facilitate the development of FL-producing cell factories.