Metabolic engineering strategies of de novo pathway for enhancing 2'-fucosyllactose synthesis in Escherichia coli

Abstract

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 α1,2-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.