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To overproduce erythromycin C, B or D and evaluate the effect of disruption of tailoring genes eryK and eryG in an industrial erythromycin producer.The tailoring genes eryG and eryK were inactivated individually or simultaneously by targeted gene disruption in an industrial strain Saccharopolyspora erythraea HL3168 E3, resulting in the overproduction of erythromycin C (2·48 g l−1), B (1·70 g l−1) or D (2·15 g l−1) in the mutant strain QL-G, QL-K or QL-KG, respectively. Analysis of the erythromycin congeners throughout the fermentation indicated that, at the end of fermentation, comparatively large amount of erythromycin D (0·67 g l−1) was accumulated in QL-G, whereas only small amount of erythromycin D (0·10 g l−1) was produced in QL-K.Inactivation of tailoring genes eryG and eryK in the high producer did not affect the biosynthesis of erythromycin. However, erythromycin D could be more efficiently methylated by EryG than be hydroxylated by EryK.Development of the mutant strains provides a method for the economical large-scale production of potent lead compounds. The information about the accumulation and conversion of erythromycins in the industrial strains may contribute to further improving erythromycin production.