The validamycin biosynthetic gene cluster in Streptomyces hygroscopicus var. limoneus contains vldI, the gene encoding a glucoamylase (1,4-alpha-D-glucan glucohydrolase). The knock-out of vldI (vldI::neo) reduced the yield of validamycin-A, thus indicating that VldI contributes to validamycin-A productivity by supplying glucose with the hydrolysis of 1,4-alpha-D-glucan(s). Promoter-probe assays employing xylE fusions indicated that the transcription of vldI correlates to those of other biosynthetic genes, which are organized with two divergently arranged vldABC and vldDEFGH sets. These results reveal that the contiguous region covering nine genes of vldABCDEFGHI represents the core of the validamycin biosynthetic cluster. After confirming that genes vldABCDEFGH confer validamycin production ability to Streptomyces lividans, genes vldBCHI were eliminated from the expression construct and the remaining genes, vldADEFG, were tested for the ability to confer validamycin-A production to S. lividans. Ion-exchange chromatographic purification and a subsequent HPLC analysis confirmed that S. lividans/vldADEFG yielded a 75 microg/l of validamycin-A, showing that the validamycin pathway involves a single NDP-sugar glycosyltransferase reaction. It was also demonstrated that VldE is capable of coupling validoxylamine-A and UDP-glucose to generate validamycin-A. The proposal that VldADEFG catalyze the biosynthesis of validamycin-A from sedo-heptulose 7-phosphate and UDP-glucose and include a N-bridge-forming catalyst will serve as a guideline for future biochemical studies and a platform to explore this m-C7N cyclitol pathway for biotechnological applications.