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The hancockiamides are an unusual new family ofN-cinnamoylated piperazines from the Australian soil fungusAspergillus hancockii. Genomic analyses ofA. hancockiiidentified a biosynthetic gene cluster (hkm) of 12 genes, including two single-module nonribosomal peptide synthetase (NRPS) genes. Heterologous expression of thehkmcluster inA. nidulansconfirmed its role in the biosynthesis of the hancockiamides. We further demonstrated that a novel cytochrome P450, Hkm5, catalyses the methylenedioxy bridge formation, and that the PAL genehkm12is dispensable, but contributes to increased production of the cinnamoylated hancockiamides.In vitroenzymatic assays and substrate feeding studies demonstrated that NRPS Hkm11 activates and transferstrans-cinnamate to the piperazine scaffold and has flexibility to accept bioisosteric thienyl and furyl analogues. This is the first reported cinnamate-activating fungal NRPS. Expression of a truncated cluster lacking the acetyltransferase gene led to seven additional congeners, including an unexpected family of 2,5-dibenzylpiperazines. These pleiotropic effects highlight the plasticity of the pathway and the power of this approach for accessing novel natural product scaffolds.