Metabolic Engineering of Escherichia coli BL21(DE3) for 2′-Fucosyllactose Synthesis in a Higher Productivity

2′-Fucosyllactose (2′-FL) is the most abundant human milk oligosaccharides (HMOs). 2′-FL exhibits great benefits for infant health, such as preventing infantile diarrhea and promoting the growth of intestinal probiotics. The microbial cell factory technique has shown promise for the massive production of 2′-FL. Here, we aimed to construct a recombinant E. coli BL21­(DE3) strain for the hyperproduction of 2′-FL. Initially, multicopy genomic integration and expression of the lactose permease gene lacY reduced the formation of byproducts. Furthermore, a more efficient Shine–Dalgarno sequence was used to replace the wild-type sequence in the manC-manB and gmd-wcaG gene clusters, which significantly increased the 2′-FL titer. Based on these results, we overexpressed the sugar efflux transporter SetA and knocked out the pgi gene. This further improved 2′-FL synthesis when glycerol was used as the sole carbon source. Finally, a new α-1,2-fucosyltransferase was identified in Neisseria sp., which exhibited a higher capacity for 2′-FL production. Fed-batch fermentation produced 141.27 g/L 2′-FL in 45 h with a productivity of 3.14 g/L × h. This productivity rate achieved the highest recorded 2′-FL levels, indicating the potential of engineered E. coli BL21 (DE3) strains for use in the industrial production of 2′-FL.

2′-岩藻糖基乳糖（2′-Fucosyllactose, 2′-FL）是丰度最高的人乳寡糖（human milk oligosaccharides, HMOs）。2′-FL对婴幼儿健康具有诸多益处，例如可预防婴儿腹泻、促进肠道益生菌增殖。微生物细胞工厂技术已展现出用于2′-FL大规模生产的潜力。本研究旨在构建一株重组大肠杆菌BL21(DE3)菌株，以实现2′-FL的高产合成。初始实验中，通过多拷贝基因组整合并表达乳糖通透酶基因lacY，降低了副产物的生成。进一步，我们使用更高效的夏因-达尔加诺序列（Shine–Dalgarno sequence）替换manC-manB与gmd-wcaG基因簇中的野生型序列，显著提升了2′-FL的发酵效价。基于上述结果，我们过表达糖外排转运蛋白SetA并敲除pgi基因，当以甘油作为唯一碳源时，该策略进一步优化了2′-FL的合成途径。最终，我们在奈瑟菌属（Neisseria sp.）中鉴定到一种新型α-1,2-岩藻糖基转移酶，其具备更高的2′-FL合成能力。通过补料分批发酵，工程菌株在45小时内可合成141.27 g/L的2′-FL，生产强度达3.14 g/(L·h)。该生产强度达到目前已报道的2′-FL最高水平，表明经工程改造的大肠杆菌BL21(DE3)菌株具备用于2′-FL工业化生产的潜力。