Reconstitution of a Highly Reducing Type II PKS System Reveals 6π-Electrocyclization Is Required for -Dialkylbenzene Biosynthesis.

Natural products containing an -dialkylbenzene moiety exhibit a wide variety of bioactivities, including antibacterial, antifungal, antitumor, and antiangiogenic activities. However, the biosynthetic scheme of the -dialkylbenzene moiety remains unclear. In this study, we identified the biosynthetic gene cluster (BGC) of compounds and in sp. SANK 60404, which contains a rare -dialkylbenzene moiety, and successfully reconstituted the biosynthesis of using 22 recombinant enzymes in vitro. Our study established a biosynthetic route for the -tolyl group within the -dialkylbenzene moiety, where the triene intermediate loaded onto a unique acyl carrier protein (ACP) is elongated by a specific ketosynthase-chain length factor pair of a type II polyketide synthase system with the aid of a putative isomerase to be termed "electrocyclase" and a thioesterase-like enzyme in the BGC. The C-elongated all-trans diketo-triene intermediate is subsequently isomerized to the 6 configuration by the electrocyclase to allow intramolecular 6π-electrocyclization, followed by coenzyme FAD/FMN-dependent dehydrogenation. Bioinformatics analysis showed that the key genes are all conserved in BGCs of natural products containing an -dialkylbenzene moiety, suggesting that the proposed biosynthetic scheme is a common strategy to form -dialkylbenzenes in nature.