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 fromStreptomycesspecies and demonstrated with antidiabetic and immunosuppressant activities. Though the biosynthetic pathway of1−3was reported as a type I polyketide synthase (PKS), the PKS assembly line was interpreted inconsistently, and it remains a mystery how the compound3was generated. Herein, the PKS assembly logic of1−4was revised by site-mutagenetic analysis of the PKS dehydratase domains. Based on gene deletion and complementation, the putative P450 monooxygenasenftE1and metallo-beta-lactamase (MBL) fold hydrolasenftF1were verified as essential genes for the biosynthesis of1−4. The absence ofnftE1led to abolishment of1−4and accumulation of new products (5−8). Structural elucidation reveals5−8as the non-aromatic analogs of1, suggesting the NftE1-catalyzed aromatic core formation. Deletion ofnftF1resulted in disappearance of3and4with the compounds1and2unaffected. As a rare MBL-fold hydrolase from type I PKSs, NftF1potentially generates the compound3through two strategies: catalyze premature chain-offloading as atrans-acting thioesterase or hydrolyze the lactone-bond of compound1as an esterase.