Metabolic Engineering of 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 BL21(DE3) strain for the hyperproduction of 2'-FL. Initially, multicopy genomic integration and expression of the lactose permease gene reduced the formation of byproducts. Furthermore, a more efficient Shine-Dalgarno sequence was used to replace the wild-type sequence in the - and - gene clusters, which significantly increased the 2'-FL titer. Based on these results, we overexpressed the sugar efflux transporter SetA and knocked out the 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 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 BL21 (DE3) strains for use in the industrial production of 2'-FL.