Biosynthesis of a Novel Diketopiperazine Aspkyncin Incorporating a Kynurenine Unit from Aspergillus aculeatus

The simplest cyclo-peptides, also known as diketopiperazines (DKPs), are widespread in nature. The growing interest in these simplest cyclo-peptides is driven by their significant potential for therapeutic applications. In this study, we identified a biosynthetic gene cluster from <i>Aspergillus aculeatus</i> CRI323-04 through genome mining and heterologous expression in <i>Aspergillus nidulans</i>. The two core genes, <i>aacA</i> and <i>aacB</i>, within the gene cluster were characterized for their role in the biossoynthesis of aspkyncin, a novel DKP compound that incorporates a l-kynurenine (l-Kyn) unit. Furthermore, we successfully reconstituted the activities of the minimal bimodular non-ribosomal peptide synthetase (NRPS) AacA and the methyltransferase AacB both in vivo and in vitro. Our findings demonstrate that AacA catalyzes the condensation and cyclization of two non-proteinogenic amino acids, l-Kyn and <i>N</i>-methyl-l-alanine, to produce aspkyncin without the involvement of any release domain. Notably, the <i>N</i>-methyl-l-alanine is generated by a specialized l-alanine <i>N</i>-methyltransferase AacB prior to NRP assembly. This study reveals an unconventional pathway for the biosynthesis of fungal DKPs.