LAUSR

As one of the most abundant components in human milk oligosaccharides, 2′‐fucosyllactose (2′‐FL) possesses versatile beneficial health effects. Although most studies focused on overexpressing or fine‐tuning the expression of pathway enzymes and achieved a striking increase of 2′‐FL production, directly facilitating the metabolic flux toward the key intermediate GDP‐l‐fucose seems to be ignored. Here, multienzyme complexes consisting of sequential pathway enzymes were constructed by using specific peptide interaction motifs in recombinant Escherichia coli to achieve a higher titer of 2′‐FL. Specifically, we first fine‐tuned the expression level of pathway enzymes and balanced the metabolic flux toward 2′‐FL synthesis. Then, two key enzymes (GDP‐mannose 4,6‐dehydratase and GDP‐ l‐fucose synthase) were self‐assembled into enzyme complexes in vivo via a short peptide interaction pair RIAD–RIDD (RI anchoring disruptor–RI dimer D/D domains), resulting in noticeable improvement of 2′‐FL production. Next, to further strengthen the metabolic flux toward 2′‐FL, three pathway enzymes were further aggregated into multienzyme assemblies by using another orthogonal protein interaction motif (Spycatcher–SpyTag or PDZ–PDZlig). Intracellular multienzyme assemblies remarkably enlarged the flux toward 2′‐FL biosynthesis and showed a 2.1‐fold increase of 2′‐FL production compared with a strain expressing free‐floating and unassembled enzymes. The optimally engineered strain EZJ23 accumulated 4.8 g/L 2′‐FL in shake flask fermentation and was capable of producing 25.1 g/L 2′‐FL by fed‐batch cultivation. This work provides novel approaches for further improvement and large‐scale production of 2′‐FL and demonstrates the effectiveness of spatial assembly of pathway enzymes to improve the production of valuable products in the engineered host strain.