Escherichia coli immobilized fermentation for continuous 3-fucosyllactose production via manipulating biofilm formation.

3-Fucosyllactose (3-FL), a representative human milk oligosaccharide, is witnessing rapidly increasing demand. Establishing an immobilized fermentation system using microbial cell factories presents a promising strategy for continuous 3-FL production. In this study, the effects of nine biofilm-related genes on biofilm-forming capacity and 3-FL synthesis in Escherichia coli were systematically evaluated. Among them, pslA from Pseudomonas aeruginosa driven by a constitutive promoter demonstrated the best performance. Subsequently, the strategy of "prioritizing biofilm formation, following product synthesis" was implemented to develop an E. coli biofilm-based immobilized fermentation based on cotton fiber. In repeated-batch fermentation, BZJP05-JpslA exhibited higher cell density, improved cellular viability, and a 38.4% increase in average 3-FL yield. Furthermore, transcriptional level analysis revealed that pslA overexpression promoted glycerol metabolism, TCA cycle, fluxes toward 3-FL biosynthesis pathway, and expression of key genes involved in stress response. These findings provide an effective strategy and mechanism insights for optimizing E. coli cell-factory platforms.