Bacterial-Like Nonribosomal Peptide Synthetases Produce Cyclopeptides in the Zygomycetous Fungus Mortierella alpina

Fungi are traditionally considered a reservoir of biologically active natural products. However, an active secondary metabolism has long not been attributed to early-diverging fungi such as Mortierella. Here, we report on the biosynthesis of two series of cyclic pentapeptides, the malpicyclins and malpibaldins, as products of Mortierella alpina ATCC 32222. The molecular structures of malpicyclins were elucidated by high-resolution tandem mass spectrometry (HR-MS/MS), Marfey's method, and one-dimensional (1D) and 2D nuclear magnetic resonance (NMR) spectroscopy. In addition, malpibaldin biosynthesis was confirmed by HR-MS. Genome mining and comparative quantitative real-time PCR (qRT-PCR) expression analysis pointed at two pentamodular nonribosomal peptide synthetases (NRPSs), malpicyclin synthetase MpcA and malpibaldin synthetase MpbA, as candidate biosynthetic enzymes. Heterologous production of the respective adenylation domains and substrate specificity assays confirmed the existence of promiscuous substrate selection and also confirmed their respective biosynthetic roles. In stark contrast to known fungal NRPSs, MpbA and MpcA contain bacterial-like dual epimerase/condensation domains allowing the racemization of enzyme-tethered I.-amino acids and the subsequent incorporation of ID-amino acids into the metabolites. Phylogenetic analyses of both NRPS genes indicated a bacterial origin and a horizontal gene transfer into the fungal genome. We report on the as-yet-unexplored nonribosomal peptide biosynthesis in basal fungi which highlights this paraphylum as a novel and underrated resource of natural products. IMPORTANCE Fungal natural compounds are industrially produced, with applications in antibiotic treatment, cancer medications, and crop plant protection. Traditionally, higher fungi have been intensively investigated for their metabolic potential, but reidentification of already known compounds is frequently observed. Hence, alternative strategies to acquire novel bioactive molecules are required. We present the genus Mortierella as representative of the early-diverging fungi as an underestimated resource of natural products. Mortierella alpina produces two families of cyclopeptides, designated malpicyclins and malpibaldins, via two pentamodular nonribosomal peptide synthetases (NRPSs). These enzymes are much more closely related to bacterial than to other fungal NRPSs, suggesting a bacterial origin of these NRPS genes in Mortierella. Both enzymes were biochemically characterized and are involved in as-yet-unknown biosynthetic pathways of natural products in basal fungi. Hence, this report establishes early-diverging fungi as prolific natural compound producers and sheds light on the origin of their biosynthetic capacity.

Automated summary

This study reports the biosynthesis of cyclic pentapeptides in Mortierella alpina, highlighting early-diverging fungi as an underestimated resource of natural products. The enzymes involved in this biosynthesis are more closely related to bacterial NRPSs, suggesting a bacterial origin of these NRPS genes in Mortierella. This discovery establishes early-diverging fungi as prolific natural compound producers and sheds light on the origin of their biosynthetic capacity.