To acquire iron, the N-2-fixing, symbiotic bacterium Rhizobium sp. produce the cyclic trihydroxamate siderophore vicibactin, containing a 30-membered trilactone scaffold. Herein we report the overproduction and purification of the six proteins VbsACGOLS in the bacterial host Escherichia coli and the reconstitution of the biosynthesis of vicibactin from primary metabolites. The flavoprotein VbsO acts as a pathway-initiating L-ornithine M-hydroxylase, followed by VbsA, which transfers (R)-3-hydroxybutyryl- from the CoA thioester to N-6-hydroxyornithine to yield N-6-((R)-3-hydroxybutyryl)-N-6-hydroxy-L-orinithine. VbsL is a PLP-dependent epimerase acting at C-2 of the 10 atom monomer unit. VbsS, a nonribosomal peptide synthetase freestanding module, then activates N-6-((R)-3-hydroxybutyryl)-N-6-hydroxy-D-ornithine as the AMP anhydride on the way to cyclotrimerization to the vicibactin scaffold. The last step, tris-acetylation of the C-2 amino group of desacetyl-D-vicibactin to the mature siderophore vicibactin, is catalyzed distributively by VbsC, using three molecules of acetyl-CoA.