2′-Fucosyllactose (2′-FL), a major oligosaccharide of human breast milk, and is currently supplemented into infant formula. For the overproduction of 2′-FL via fucosylation of lactose, conventional approaches have focused on the episomal overexpression of de novo or salvage GDP-L-fucose biosynthetic pathway and α-1,2-fucosyltransferase (FucT2) through T7 RNA polymerase expression system in engineered E. coli. However, these approaches have drawbacks of metabolic burden, plasmid instability, and inclusion body formation. In this study, a deletion mutant of waaF coding for ADP-heptose:LPS heptosyltransferase II was employed for 2′-FL production. As the waaF deletion induces accumulation of colanic acid, additional deletion of wcaJ coding for UDP-glucose-1-phosphate transferase in the waaF deletion mutant resulted in enhanced accumulation of GDP-L-fucose. Besides, 2′-FL yields and titers were drastically improved when T7 promoter was replaced with Trc promoter for α-1,2 fucosyltransferase expressions in the waaF and wcaJ deleted strain. As a result, when FucT2 was expressed under Trc promoter in the E. coli JM109(DE3) ΔwaaFΔwcaJ, 14.7 g/L of 2′-FL was produced with a productivity of 0.31 g/L/h in a fed-batch fermentation. We envision that the deletion-based metabolic design and decreased promoter strength for fucosyltransferase expression can resolve the drawbacks of T7 RNA polymerase-based expression design for 2′-FL production in E. coli.

2'-岩藻糖基乳糖 (2'-FL) 是人类母乳的主要低聚糖，目前已添加到婴儿配方奶粉中。对于通过乳糖的岩藻糖基化过度生产 2'-FL，传统方法集中在从头的附加型过表达或通过 T7 RNA 聚合酶表达系统挽救GDP- L-岩藻糖生物合成途径和 α-1,2-岩藻糖基转移酶 (FucT2 )在工程大肠杆菌中。然而，这些方法具有代谢负担、质粒不稳定性和包涵体形成的缺点。在本研究中，使用编码 ADP-庚糖:LPS 庚糖基转移酶 II的waaF缺失突变体来生产 2'-FL。作为waaF缺失诱导可拉酸的积累，waaF缺失突变体中编码UDP-葡萄糖-1-磷酸转移酶的wcaJ的额外缺失导致GDP- L-岩藻糖的积累增强。此外，当waaF和wcaJ缺失菌株中的 α-1,2 岩藻糖基转移酶表达用 Trc 启动子替换 T7 启动子时，2'-FL 产量和滴度显着提高。结果，当 FucT2 在大肠杆菌JM109(DE3) 的 Trc 启动子下表达时，Δ waaF Δ wcaJ，在补料分批发酵中以 0.31 g/L/h 的生产率生产 14.7 g/L 的 2'-FL。我们设想基于缺失的代谢设计和降低的岩藻糖基转移酶表达启动子强度可以解决基于 T7 RNA 聚合酶的表达设计在大肠杆菌中产生 2'-FL 的缺点。