High-Titer <i>De Novo</i> Biosynthesis of the Predominant Human Milk Oligosaccharide 2′-Fucosyllactose from Sucrose in <i>Escherichia coli</i>

Abstract

Human milk oligosaccharides (HMOs) are unique components of human breast milk. Their large-scale production by fermentation allows infant formulas to be fortified with HMOs, but current fermentation processes require lactose as a starting material, increasing the costs, bioburden, and environmental impact of manufacturing. Here we report the development of an <i>Escherichia coli</i> strain that produces 2'-fucosyllactose (2'-FL), the most abundant HMO, <i>de novo</i> using sucrose as the sole carbon source. Strain engineering required the expression of a novel glucose-accepting galactosyltransferase, overexpression of the <i>de novo</i> UDP-d-galactose and GDP-l-fucose pathways, the engineering of an intracellular pool of free glucose, and overexpression of a suitable α(1,2)-fucosyltransferase. The export of 2'-FL was facilitated using a sugar efflux transporter. The final production strain achieved 2'-FL yields exceeding 60 g/L after fermentation for 84 h. This efficient strategy facilitates the lactose-independent production of HMOs by fermentation, which will improve product quality and reduce the costs of manufacturing.