Efficient Production of 2′-Fucosyllactose from l‑Fucose <i>via</i> Self-Assembling Multienzyme Complexes in Engineered <i>Escherichia coli</i>

2′-Fucosyllactose (2′-FL) has been widely used as a nutritional additive in infant formula due to its multifarious nutraceutical and pharmaceutical functions in neonate health. As such, it is essential to develop an efficient and extensive microbial fermentation platform to cater to the needs of the 2′-FL market. In this study, a spatial synthetic biology strategy was employed to promote 2′-FL biosynthesis in recombinant Escherichia coli. First, the salvage pathway for 2′-FL production from l-fucose and lactose was constructed by introducing a bifunctional enzyme l-fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) derived from Bacteroides fragilis and an α-1,2-fucosyltransferase (FutC) derived from Helicobacter pylori into engineered E. coli BL21­(DE3). Next, the endogenous genes involved in the degradation and shunting of the substrate and key intermediate were inactivated to improve the availability of precursors for 2′-FL biosynthesis. Moreover, to further improve the yield and titer of 2′-FL, a short peptide pair (RIAD-RIDD) was used to form self-assembling multienzyme complexes in vivo. The spatial localization of peptides and stoichiometry of enzyme assemblies were subsequently optimized to further improve 2′-FL production. Finally, cofactor regeneration was also considered to alleviate the potential cofactor deficiency and redox flux imbalance in the biocatalysis process. Fed-batch fermentation of the final WLS20 strain accumulated 30.5 g/L extracellular 2′-FL with the yield and productivity of 0.661 mol/mol fucose and 0.48 g/L/h, respectively. This research has demonstrated that the application of spatial synthetic biology and metabolic engineering strategies can dramatically enlarge the titer and yield of 2′-FL biosynthesis in engineered E. coli