Lankamycin, a 14-membered macrolide antibiotic, contains a 3-hydroxy-2-butyl side chain at C-13. To analyze the function of lkmE, which encodes type-II thioesterase in the lankamycin cluster, we carried out a gene disruption experiment. Disruption of lkmE resulted in a 70% decrease of lankamycin production concomitant with an accumulation of novel lankamycin derivatives (LM-NS01A and LM-NS01B), 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 [3-2 H]3-methyl-2-oxobutyrate into the C-14 position. These results indicate that the biosynthesis of LM-NS01A and LM-NS01B starts from isobutyryl CoA in place of (S)-2-methylbutyryl CoA and LkmE removes the aberrantly loaded starter unit and restores lankamycin production.