Biosynthesis of Waspergillamide A Involves Unclustered Genes and a <i>depsi</i> -Tetrapeptide Diketopiperazine-Forming NRPS

Comparative genomics identified a nonribosomal peptide synthetase gene cluster (<i>wpa</i>), spanning two genomic scaffolds, responsible for waspergillamide A (<b>1</b>) in <i>Aspergillus</i> sp. CMB-W031. Pathway reconstitution revealed the cytochrome P450 WpaD as a gatekeeper: its presence directs biosynthesis toward the <i>depsi</i>-tetrapeptide diketopiperazine <b>2</b>, which spontaneously dehydrates to <b>1</b> under weak acid condition, whereas its absence yields linear tetrapeptide <b>4</b>. Pathway characterization further enabled structural revision of <b>1</b> and confirmed the conservation of <i>p</i>-nitrobenzoic acid biosynthesis in fungi.