Efficient biosynthesis of difucosyllatose(DFL) by engineered Escherichia coli

Abstract

Human milk oligosaccharides (HMOs) are essential bioactive components that perform various physiological functions, such as shaping the gut microbiota, inhibiting the adhesion of pathogenic bacteria, and modulating the immune system. Among them, difucosyllactose (DFL) exhibits superior bioactivity due to its unique structure containing two fucose residues, making it a promising candidate for use as a functional food ingredient and pharmaceutical material. In this study, a metabolic engineering strategy was developed to enable DFL production using Escherichia coli. To enhance the accumulation of the donor molecule GDP-L-fucose, the genes waaF and wcaJ, which are involved in competing biosynthetic pathways, were deleted. In addition, to prevent the degradation of the acceptor molecule lactose, the lacZ gene was also deleted. To synthesize the precursor 2'-fucosyllactose (2'-FL), two α-1,2-fucosyltransferases —WbgL and FucT2—were introduced, and their expression was systematically evaluated under various constitutive promoter conditions to identify the optimal configuration. Subsequently, a dual fucosylation pathway was constructed by introducing Hp3/4FT, an α-1,3/4-fucosyltransferase that works in conjunction with WbgL to facilitate DFL biosynthesis while minimizing the formation of the byproduct 3-FL. To assess the potential for enhanced enzyme expression and catalytic performance, both wild-type and truncated variants of Hp3/4FT were compared in terms of DFL productivity. Initial fermentation using the engineered strain exhibited low DFL titers. To address this limitation, the biosynthesis of 2'-FL was enhanced by replacing the promoter, and fermentation conditions were further optimized. Additionally, a mutated variant of Hp3/4FT was introduced to improve enzyme expression and overall DFL production.