Abstract We extensively analyzed the giant linear plasmid pSLA2-L in Streptomyces rochei 7434AN4, a producer of two structurally unrelated polyketide antibiotics, lankacidin and lankamycin. It was found that amine oxidase LkcE oxidizes an acyclic amine to an imine, which is in turn converted to the 17-membered carbocyclic lankacidin. Heterologous expression and translational fusion experiments indicated the modular-iterative mixed polyketide biosynthesis of lankacidin. Concerning to lankamycin biosynthesis, starter unit biosynthesis and the post-PKS modification pathway were elucidated by feeding and gene inactivation experiments. It was shown that pSLA2-L contains many regulatory genes, which constitute the signaling molecule/receptor system for antibiotic production and morphological differentiation in this strain. Two signaling molecules, SRB1 and SRB2, that induce production of lankacidin and lankamycin were further isolated and their structures were elucidated. Each contains a 2,3-disubstituted butenolide skeleton, and the stereochemistry at C-1′ position is crucial for inducing activity.