Synthesis of 2'-fucosyllactose using multi-enzyme cascade with cofactor regeneration.

2'-Fucosyllactose (2'-FL) is a major human milk oligosaccharide with nutritional and medicinal benefits, widely used in infant formula and nutraceuticals. Now most studies focus on the synthesis of 2'-FL in vivo starting from glucose and glycerol. Here, we designed the first in vitro multi-enzyme cascade system for the de novo synthesis of 2'-FL using mannose and lactose as substrates. Central to this pathway is an engineered polyphosphate-dependent glucomannokinase (PPGMK), which enables efficient phosphorylation of mannose to G6P and thereby establishes mannose as an effective entry point for the de novo synthetic route. To enhance the production of 2'-FL, the cascade system was optimized, and apart from α-1,2-fucosyltransferase, GDP-D-mannose-4,6-dehydratase (gmd) was determined as a crucial rate-limiting enzyme for the de novo synthesis of 2'-FL. Additionally, an efficient GTP regeneration system was achieved, which converted GDP to GTP using polyphosphate kinase 2, resulting in a 2.94-fold improvement in 2'-FL production. Finally, under optimized conditions incorporating the cofactor regeneration system, 13.18 mM 2'-FL was synthesized through a one-pot enzymatic reaction using 20 mM mannose and lactose as substrates. The engineered system demonstrated exceptional cofactor efficiency, requiring only 2 mM each of the essential cofactor NADPH and GTP representing a 6.6-fold reduction in cofactor consumption. Overall, this study establishes a novel de novo enzymatic route for 2'-FL and provides new insights for both pathway construction and enzyme engineering in oligosaccharide production.