TrdL, encoding a flavin-dependent oxidoreductase in the tirandamycin gene cluster, was inactivated to afford a ΔtrdL mutant, the fermentation of which yielded a new intermediate, tirandamycin E (5), and an additional early intermediate, tirandamycin F (6), if XAD-16 resin was introduced. TrdL was overexpressed in E. coli, and the protein was shown to efficiently catalyze the transformations from 5 to tirandamycin A (1) and from 6 to tirandamycin D (4), demonstrating its function as a 10-hydroxy dehydrogenase.