Esterification and dipeptide offsite uploading are mediated by fungal nonribosomal peptide synthetases

Amino acid esters are a group of structurally diverse natural products with pronounced activities. Some compounds in this group are catalysed by nonribosomal peptide synthetases (NRPSs) featuring inter-molecular esterification of linear peptidyl precursors in the biosynthesis. Bacteria usually form intra-molecular ester bond which is catalysed by thioesterase domains of NRPSs, while how fungal NRPSs perform this process remains limited. Here, we described that two NRPSs ApmA and ApmB are essential for biosynthesis of an amino acid ester asperphenamate by genome mining and targeted gene disruption in Penicillium brevicompactum. Unprecedented, we show, using heterologous system in Aspergillus nidulans, that ApmB not only uploads directly linear dipeptide but also catalyses the formation of inter-molecular ester bond and releases the asperphenamate product. The offsite uploading for linear dipeptide by ApmB was confirmed by feeding of isotope-labelled N-Benzoylphenylalanine. Furthermore, ApmA was rarely revealed to mediate the transitions from acid-form to alcohol-form in a NRPS. Our finds provided the insights into fungal NRPS machinery for the formation of amino acid esters.