Project Summary Microbial production of fucosylated human milk oligosaccharides This proposed project aims to establish efficient and specific microbial production processes for human milk oligosaccharides (HMOs). HMOs are potent bioactive compounds that modulate neonatal health and are of interest for development as potential drug treatments for adult diseases. HMOs are a class of over 200 compounds present at 20-23 g/L in colostrum and 12-14 g/L in mature milk. Unlike their common precursor lactose, HMOs are indigestible by human infants and instead improve neonatal health by serving as effective antimicrobials and antivirals, prebiotics, and regulators of inflammatory immune cell-response cascades. These and other potential benefits of HMOs make them attractive targets of study for preventing or treating diseases in both children and adults. β1−3-Linked galactosides Galβ3GlcNAcβOR, which are called Type 1 glycans, are major HMO components found in more than 100 HMOs. Among the 20 HMO core structures that have been identified, 11 contain at least one Type 1 glycan-terminated branch. Lacto-N-tetraose (LNT, Galβ3GlcNAcβ3Lac) is the simplest Type 1 glycan HMO. LNT and its fucosylated derivatives are among the most abundant HMOs. While Type 1 glycan structures are predominant in human milk, they are less abundant (and sometimes completely absent) in the milk of other mammals. Investigating the biological functions of individual Type 1 glycan-containing HMOs and their potential applications as prebiotics and antimicrobials requires access to sufficient quantities of these structurally defined compounds. The potential of these molecules, their limited access from natural sources, and difficulty in large-scale isolation of individual HMOs for studies and applications have motivated the development of novel production methods. Chemical and in vitro enzymatic syntheses of HMOs based on current methods are expected to be costly for industrial-scale synthesis. Whole cell biocatalysts are emerging as alternative self-regulating production platforms that have significant potential to reduce the production cost of HMOs. Short-chain, linear and small monofucosylated HMOs have been produced in whole cell biocatalysts, but structures with higher complexity have not been explored. In this proposed project, we will establish a strategy for producing fucosylated HMOs including lacto-N-fucopentaose II (LNFP II), lacto-N- fucopentaose I (LNFP I) and lacto-N-difucosylhexaose I (LNDFH I) from lactose and L-fucose in live engineered Escherichia coli cells. Notably, we will develop an innovative method to control the order and the site of glycosylation in whole cell systems to lay the groundwork for future microbial production of other complex HMOs.