Challenging Aromaticity: Revealing a Thioesterase Domain in a Fungal Nonreducing Polyketide Synthase Governing the Production of 3‑Methylene Isochromanone

Thioesterase (TE) domain exerts a great influence over the structure of the final product and TE-released nonreduced polyketides (nrPKs) retain aromaticity. 3-Methylene isochromanones are lactones with a unique olefin at C3 that disrupts the aromaticity, whose biosynthetic details are speculative. Our study unveils the complete biosynthesis of ascochin, in which the construction of the 3-methylene isochromanone backbone is achieved by a nonreducing polyketide synthase (nrPKS) alone and two subsequent oxidations are involved. Intriguingly, the TEAscD serves as a gatekeeper to direct the product release toward formation of nonaromatic 3-methylene isochromanone, rather than the typical aromatic product.