Dynamic Regulation Coupled with Metabolic Pathway Optimization Enables High-Efficiency Lacto- <i>N</i> -fucopentaose I Biosynthesis in <i>Escherichia coli</i>

Lacto- N -fucopentaose I (LNFP I), the second most abundant fucosylated human milk oligosaccharide, plays important roles in infant health. In this study, we engineered an efficient Escherichia coli BL21star(DE3) cell factory for LNFP I production. The UDP-GlcNAc precursor pool was increased through glmS A overexpression, and a fructose-1,6-bisphosphate-responsive biosensor was implemented to dynamically regulate pfkA expression, thereby balancing cell growth with LNFP I biosynthesis. Transporter screening identified YhhS from E. coli as an effective exporter that enhanced LNFP I secretion. Strengthening carbon source utilization and optimizing the fermentation process enabled the development of a high-producing strain that achieved 3.50 g/L LNFP I in shake-flask fermentation and 68.10 g/L in a 5 L bioreactor, with an LNT-to-LNFP I conversion rate of 92.10% and a productivity of 0.87 g/L/h.