Heterologous expression of the Metarhizium anisopliae barc gene cluster reveals a diphenyl ether-to-xanthene transformation in barceloneic acid biosynthesis.

Filamentous fungi generate chemical diversity through several evolutionary strategies, including the elaboration of conserved biosynthetic intermediates into structurally distinct secondary metabolites. Emodin and its corresponding anthrone are central intermediates in fungal aromatic polyketide metabolism, yet the enzymatic logic that remodels these precursors into more complex scaffolds remains incompletely understood. To investigate this process, we examined an infection-responsive non-reducing polyketide synthase biosynthetic gene cluster (barc) from the entomopathogenic fungus Metarhizium anisopliae predicted to encode an emodin-derived aromatic polyketide pathway. Heterologous reconstruction of the barc cluster in Aspergillus nidulans led to the production of the previously reported diphenyl ether barceloneic acid A (1) and the xanthene barceloneic acid C2 (2), together with two new compounds derived from 2, barceloneic acids D and E (3 and 4). Compound 3 is a cyclopentanone adduct of the xanthene 2, whereas 4 is a polyketide-indole hybrid, highlighting the chemical reactivity of the xanthene 2 scaffold. Through combinatorial heterologous expression and feeding assays, we identified a predicted glucose-methanol-choline oxidoreductase (BarcL) as the enzyme responsible for converting diphenyl ether 1 to xanthene 2. This defines a previously uncharacterised oxidative scaffold-remodelling step in barceloneic acid biosynthesis, revealing how an emodin-derived pathway can access a xanthene scaffold. Deletion of the backbone gene barcB in M. anisopliae did not significantly alter virulence towards Tenebrio molitor (mealworm) under the conditions tested, suggesting that barceloneic acids are not essential for pathogenicity in this insect model.