In Vitro Reconstitution of Cinnamoyl Moiety Reveals Two Distinct Cyclases for Benzene Ring Formation.

Cinnamoyl-containing natural products (CCNPs) are a small class of bacterial metabolites with notable bioactivities. The biosynthesis of cinnamoyl moiety has been proposed to be assembled by an unusual highly reducing (HR) type II polyketide synthases (PKS). However, the biosynthetic route, especially the cyclization step for the benzene ring formation, remains unclear. In this work, we successfully reconstituted the pathway of cinnamoyl moiety in kitacinnamycin biosynthesis through a step-wise approach in vitro and demonstrated that a three-protein complex, Kcn17-Kcn18-Kcn19, can catalyze 6π-electrocyclization followed by dehydrogenation to form the benzene ring. We found that the three-protein homologues were widely distributed among 207 HR type II PKS biosynthetic gene clusters including five known CCNPs. In contrast, in the biosynthesis of youssoufene, a cinnamoyl-containing polyene, we identified that the benzene ring formation was accomplished by a distinct orphan protein. Thus, our work resolved the long-standing mystery in cinnamoyl biosynthesis and revealed two distinct enzymes that can synthesize benzene rings via polyene precursors.