Metabolic engineering strategies of de novo pathway for enhancing 2′‐fucosyllactose synthesis in Escherichia coli

Summary 2′‐Fucosyllactose (2′‐FL), one of the most abundant human milk oligosaccharides (HMOs), is used as a promising infant formula ingredient owing to its multiple health benefits for newborns. However, limited availability and high‐cost preparation have restricted its extensive use and intensive research on its potential functions. In this work, a powerful Escherichia coli cell factory was developed to ulteriorly increase 2′‐FL production. Initially, a modular pathway engineering was strengthened to balance the synthesis pathway through different plasmid combinations with a resulting maximum 2′‐FL titre of 1.45 g l −1 . To further facilitate the metabolic flux from GDP‐ l ‐fucose towards 2′‐FL, the CRISPR‐Cas9 system was utilized to inactivate the genes including lacZ and wcaJ , increasing the titre by 6.59‐fold. Notably, the co‐introduction of NADPH and GTP regeneration pathways was confirmed to be more conducive to 2′‐FL formation, achieving a 2′‐FL titre of 2.24 g l −1 . Moreover, comparisons of various exogenous α 1,2‐fucosyltransferase candidates revealed that futC from Helicobacter pylori generated the highest titre of 2′‐FL. Finally, the viability of scaled‐up production of 2′‐FL was evidenced in a 3 l bioreactor with a maximum titre of 22.3 g l −1 2′‐FL and a yield of 0.53 mole 2′‐FL mole −1 lactose.