Although the biosynthesis of rifamycin has been studied for three decades, the biosynthetic formation of the naphthalenic ring remains unclear. In this study, by deletion of all post-PKS modification genes, we identified macrolactam precursors released from rif PKS. Isolated prorifamycins (M3 and M4) have a benzenic chromophore and exist in two sets of macrocyclic atropisomers. The transformation from prorifamycins to benzenoid (5) and naphthalenoid (6) was suggested to be a non-enzymatic process, which is an off-PKS assembly.