Nosiheptide is a bicyclic thiopeptide featuring an indole-containing side ring, which is biologically important in maintaining its potent antibacterial activity. By using mutational biosynthesis, the pharmaceutically significant benzothiophene was introduced into the nosiheptide biosynthetic pathway, resulting in the generation of three bioactive nosiheptide analogues with characteristic benzothiophene-containing side rings. Insights were provided into the transformation relationship of these analogues, which effectively improves the yield of S-NOS-1 with favorable activity against Gram-positive pathogens.