Fig. 2

Discovery of a natural and synthetic pathway for rhizopine synthesis. a Gas chromatography-mass spectrometry (GC-MS) total ion chromatograms (TICs) (left) and extracted ion chromatograms (m/z 245 = 3-O-MSI-TMS) (EIC [245]) (right) of metabolites from nodules formed by wild-type S. meliloti L5-30 compared to mosB:pK19 and mosE:pK19 mutants. Chemically synthesised (±)-3-O-MSI (±)-2 and purchased (+)-1d-ononitol (+)-1d-4 as standards are shown below. b GC-MS chromatograms of metabolites from nodules formed by wild-type S. meliloti Rm1021 maintaining an empty vector (EV) and expressing mosDEF. Chemically synthesised (±)-3-O-methyl-scyllo-inosose (±)-6 and purchased (+)-1d-ononitol (+)-1d-4 as standards are shown below. c GC-MS chromatograms (TIC) of MosB in vitro assay in the absence of protein or with MosB added. Chemically synthesised (±)-3-O-methyl-scyllo-inosose (±)-6 and (±)-3-O-MSI (±)-2 as standards are shown below. d Proposed natural pathway of 3-O-MSI biosynthesis in S. meliloti L5-30. e Linked in vitro assay of inositol dehydrogenase IolG and MosB. GC-MS TICs of assay mix in the absence of added protein, with IolG added, with MosB added and with IolG and MosB added together. Standards represent a mix of SIA 1 and myo-inositol 3. f GC-MS TICs of extracts prepared from tobacco leaves agro-infiltrated with either empty vector (control) or IdhA or MosB or IdhA-MosB together. Standards represent a mix of SIA 1 and myo-inositol 3. g Proposed synthetic pathway of SIA 1 synthesis. Metabolites were identified by comparison with authentic standards. Highlighted peaks indicate scyllo-inosamine 1 (orange), scyllo-inosose 7 (green), myo-inositol 3 (dark blue), (+)-1d-ononitol 4 (light blue), 1d-3-O-methyl-scyllo-inosose 6 (purple) and (−)-1l-3-O-methyl-scyllo-inosamine 2 (3-O-MSI) (red). All chromatograms are representative of experiments repeated at least three independent times. Source data underlying  a–c, e and f are provided as a Source Data file