Multistrategy Optimization for High-Yield 3‑Fucosyllactose Production in Escherichia coli BL21 Star (DE3)

3-Fucosyllactose (3-FL), an essential component of human milk oligosaccharides, is crucial for infant health. However, the extraction of 3-FL from milk presents a significant challenge. In this study, E. coli BL21 Star (DE3) was engineered for 3-FL biosynthesis, and the gene combinations and metabolic pathways were optimized to obtain a high-yield 3-FL production strain. First, the 3-FL production module was integrated into E. coli. Second, an efficient alpha-1,3-fucosyltransferase was screened. Then, different integration sites were used to optimize the precursor synthesis gene cluster and the transferase genes and to screen for transporter proteins and glycerol utilization genes that could enhance 3-FL production. The 3FL05–1 strain yielded a 3-FL titer of 11.26 g/L in shake flasks. Further amplification in a 5 L bioreactor resulted in a 3-FL titer of 60.24 g/L, which represents the highest reported titer to date.