Lankamycin (2), produced by Streptomyces rochei 7434AN4, is a 14-membered macrolide antibiotic attached with two deoxysugars, L-arcanose and D-chalcose. To reveal the order of glycosylation steps in lankamycin biosynthesis, we carried out gene disruption of two glycosyltransferase genes, Ikm1 and IkmL. The /km/ mutant produced 3-O-L-arcanosyl lankanolide (3), while the IkmL mutant accumulated 8-deoxylankanolide (4). These results indicated that LkmL transfers L-arcanose to the C-3 hydroxyl of 4, while Lkml does D-chalcose to the C-5 hydroxyl of 3. Taking together with previous results of gene disruption of two P450 hydroxylases, we propose the biosynthetic pathway of lankamycin including two hydroxylation and two glycosylation steps. Compound 2 contains a 3-hydroxy-2-butyl side chain at C-13. To analyze the function of IkmE which encodes type-II thioesterase in the lankamycin cluster, we carried out a gene disruption experiment. Disruption of IkmE resulted in a 70% decrease of lankamycin production concomitant with an accumulation of novel lankamycin derivatives (LM-NSO1A and LM-NSO1B), in which the C-13 side chain is replaced by a 1-carboxyethyl group. The biosynthetic origin of 1-carboxyethyl group was confirmed by incorporation of deuterium in [3-2H]3-methyl-2-oxobutyrate (10) into the C-14 position. These results indicate that the biosynthesis of LM-NSO1A and LM-NSO1B starts from isobutyryl CoA in place of (S)-2-methylbutyryl CoA and LkmE removes the aberrantly loaded starter unit and restores lankamycin production.