重组大肠杆菌补救途径合成2′-岩藻糖基乳糖的分子克隆和产物合成分析

Construction of engineered Escherichia coli for 2′-fucosyllactose production via salvage pathway

2′-岩藻糖基乳糖作为人乳寡糖中含量最多的一种,对婴幼儿健康生长发育有重要作用,是极具应用价值的母乳概念配方奶粉添加剂。该研究通过比较不同来源的α-1,2-岩藻糖基转移酶,选定幽门螺杆菌来源的α-1,2-岩藻糖基转移酶基因fucT2与脆弱拟杆菌来源的L-岩藻糖激酶基因fkp共表达,构建了2′-FL的补救合成途径。对乳糖转运蛋白LacY以及2′-FL运出蛋白伯氏耶尔森氏菌来源的糖转运蛋白Tpyb或大肠杆菌来源的糖转运蛋白SetA适量过表达,大幅增加了胞外产物浓度。比较BL21(DE3)、C41(DE3)、JM109(DE3)这3种菌株的2′-FL合成能力,得到了基于JM109(DE3)的2′-FL最终合成菌株J2-V8。通过发酵条件优化,在摇瓶中48 h得到2′-FL的胞外质量浓度为2.67 g/L,较初始菌株提高了5倍,转化率为0.82 mol/mol岩藻糖,以上结果为2′-FL的生物制备技术开发提供了借鉴。

2′-Fucosyllactose(2′-FL)is one of the most abundant oligosaccharides found in human milk,which delivering many health benefits to the neonate.It is now available in some commercial infant formulas with great market potentials.In this study,salvage pathway was constructed by comparingα-1,2-fucosyltransferases from different sources,and choosing the co-expression of fucT2 from Helicobacter pylori and fkp.Then by over-expressing the substrate transporter LacY and product transporter Tpyb or SetA,the extracellular concentration of 2′-FL was greatly increased.Comparing the production capacity of BL21(DE3),C41(DE3),and JM109(DE3),the 2′-FL synthetic strain J2-V8 was obtained.Finally,through the optimization of fermentation conditions,the extracellular concentration of 2′-FL was 2.67 g/L in shaker for 48 h,which was 5 times higher than that of the initial strain,and the conversion rate was 0.82 mol/mol L-fucose,which provided a reference for the biological production technology of 2′-FL.