Engineering all human milk oligosaccharide fucosidic linkages in planta

Human milk oligosaccharides (HMOs) are a component of breast milk which improve infant health outcomes by modulating immune responses, inhibiting pathogen adhesion, and promoting healthy gut microbiota. Although their effects are well-documented, few HMOs have been clinically or biochemically studied because only a handful of the simplest HMOs out of >200 species are currently possible to chemically synthesize or produce in microbes via metabolic engineering in microbial hosts. In contrast, plants are naturally adept at biosynthesizing a wide range of complex polysaccharides, and they have recently been established as a heterologous system for HMO production. However, much of the structural diversity of HMOs has not yet been reproduced in a plant, particularly fucosylations. Four fucosidic linkages can be found in fucosylated HMOs, but only Fucα1-2Gal has been biosynthesized in planta. Here, we screened bacterial fucosyltransferases using the model plant Nicotiana benthamiana to produce 3-fucosyllactose (Fucα1-3Glc), lacto-N-fucopentaose II (Fucα1-4GlcNAc), and lacto-N-fucopentaose III (Fucα1-3GlcNAc), demonstrating that all four fucosidic linkages can be made in planta. Just twelve glycosidic linkages make up all the HMOs found in breast milk, and our findings increase the number of linkages made in planta from seven to ten, greatly expanding the variety of HMOs accessible in a plant.