Biosynthesis of trialkyl-substituted aromatic polyketide NFAT-133 involves unusual P450 monooxygenase-mediating aromatization and a putative metallo-beta-lactamase fold hydrolase.

The bacterial trialkyl-substituted aromatic polyketides are structurally featured with the unusual aromatic core in the middle of polyketide chain such as TM-123 (), veramycin A (), NFAT-133 () and benwamycin I (), which were discovered from species and demonstrated with antidiabetic and immunosuppressant activities. Though the biosynthetic pathway of - was reported as a type I polyketide synthase (PKS), the PKS assembly line was interpreted inconsistently, and it remains a mystery how the compound was generated. Herein, the PKS assembly logic of - was revised by site-mutagenetic analysis of the PKS dehydratase domains. Based on gene deletion and complementation, the putative P450 monooxygenase E and metallo-beta-lactamase (MBL) fold hydrolase F were verified as essential genes for the biosynthesis of -. The absence of E led to abolishment of - and accumulation of new products (-). Structural elucidation reveals - as the non-aromatic analogs of , suggesting the NftE-catalyzed aromatic core formation. Deletion of F resulted in disappearance of and with the compounds and unaffected. As a rare MBL-fold hydrolase from type I PKSs, NftF potentially generates the compound through two strategies: catalyze premature chain-offloading as a -acting thioesterase or hydrolyze the lactone-bond of compound as an esterase.