The cylindrocyclophanes are a family of natural products that share a remarkable paracyclophane carbon scaffold. Using genome sequencing and bioinformatic analyses, we have discovered a biosynthetic gene cluster involved in the assembly of cylindrocyclophane F. Through a combination of in vitro enzyme characterization and feeding studies, we have confirmed the connection between this gene cluster and cylindrocyclophane production, elucidated the chemical events involved in initiating and terminating an unusual type I polyketide synthase (PKS) assembly line, and discovered that macrocycle assembly involves functionalization of an unactivated carbon center.
