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 ( 1), veramycin A ( 2), NFAT-133 ( 3) and benwamycin I ( 4), which were discovered from Streptomyces species and demonstrated with antidiabetic and immunosuppressant activities. Though the biosynthetic pathway of 1- 3 was reported as a type I polyketide synthase (PKS), the PKS assembly line was interpreted inconsistently, and it remains a mystery how the compound 3 was generated. Herein, the PKS assembly logic of 1- 4 was revised by site-mutagenetic analysis of the PKS dehydratase domains. Based on gene deletion and complementation, the putative P450 monooxygenase nftE 1 and metallo-beta-lactamase (MBL) fold hydrolase nftF 1 were verified as essential genes for the biosynthesis of 1- 4. The absence of nftE 1 led to abolishment of 1- 4 and accumulation of new products ( 5- 8). Structural elucidation reveals 5- 8 as the non-aromatic analogs of 1, suggesting the NftE 1-catalyzed aromatic core formation. Deletion of nftF 1 resulted in disappearance of 3 and 4 with the compounds 1 and 2 unaffected. As a rare MBL-fold hydrolase from type I PKSs, NftF 1 potentially generates the compound 3 through two strategies: catalyze premature chain-offloading as a trans-acting thioesterase or hydrolyze the lactone-bond of compound 1 as an esterase.