The linear tetracyclic TAN-1612 (1) and BMS-192548 (2) were isolated from different filamentous fungal strains and have been examined as potential neuropeptide Y and neurokinin-1 receptor antagonists, respectively. Although the biosynthesis of fungal aromatic polyketides has attracted much interest in recent years, the biosynthetic mechanism for such naphthacenedione-containing products has not been established. Using a targeted genome mining approach, we first located the ada gene cluster responsible for the biosynthesis of 1 in Aspergillus niger ATCC 1015. The connection between 1 and the ada pathway was verified through overexpression of the Zn(2)Cys(6)-type pathway-specific transcriptional regulator AdaR and subsequent gene expression analysis. The enzymes encoded in the ada gene duster share high sequence similarities to the known apt pathway linked to the biosynthesis of anthraquinone asperthecin 3. Subsequent comparative investigation of these two highly homologous gene clusters by heterologous pathway reconstitution in Saccharomyces cerevisiae revealed a novel alpha-hydroxylation-dependent Claisen cyclization cascade, which involves a flavin-dependent monooxygenase that hydroxylates the alpha-carbon of an acyl carrier protein-bound polyketide and a bifunctional metallo-beta-lactamase-type thioesterase (M beta L-TE). The bifunctional M beta L-TE catalyzes the fourth ring cyclization to afford the naphthacenedione scaffold upon alpha-hydroxylation, whereas it performs hydrolytic release of an anthracenone product in the absence of a-hydroxylation. Through in vitro biochemical characterizations and metal analyses, we verified that the apt M beta L-TE is a dimanganese enzyme and requires both Mn2+ cations for the observed activities. The M beta L-TE is the first example of a thioesterase in polyketide biosynthesis that catalyzes the Claisen-like condensation without an alpha/beta hydrolase fold and forms no covalent bond with the substrate. These mechanistic features should be general to the biosynthesis of tetracyclic naphthacenedione compounds in fungi.