Fig. 1: Development of an artificial biosynthetic pathway for chemically tagged UDP-GalNAc/GlcNAc analogues.

a Strategy of metabolic oligosaccharide engineering. A chemically modified GalNAc analogue that is not accepted by the GalNAc salvage pathway should be processed by an artificial biosynthetic pathway. B. longum NahK and mut-AGX1 biosynthesise UDP-GalNAc analogues and, by epimerisation, UDP-GlcNAc analogues. Incorporation into glycoconjugates can be traced by CuAAC. b In vitro evaluation of GalNAc-1-phosphate analogue synthesis by human GALK2 or B. longum NahK (left) and UDP-GalNAc analogue synthesis by WT- or mut-AGX1 (right). Data were recorded in Liquid Chromatography-Mass Spectrometry (LC-MS) assays and processed by integrated ion counts against a calibration curve or a single run of authentic product. Data are from two independent experiments and depicted as individual data points and means (left) or from three independent experiments and depicted as means + standard deviation (SD, right) overlaid to the individual data points. c Biosynthesis of UDP-GalNAc/GlcNAc analogues in cells stably expressing both NahK and mut-AGX1 or either component, as assessed by High Performance Anion Exchange Chromatography (HPAEC). A hygromycin resistance gene allows for stable transfection. Data are representative of one out of two independent experiments collected on two different days. Mock: pSBbi-GH empty plasmid. Source data are provided as a Source Data file.