Employing untargeted metabolomics we interrogated the effects HDACi has on the NP profile of the mold Aspergillus nidulans. We quantitatively monitored ~2,000 mass spectral features and found that NP synthesis was not unilaterally upregulated after HDACi as many assumed. Rather, many abundant NPs are no longer expressed after HDACi. Yet despite this, many novel NPs are induced. Among these we discovered 2 lipopeptides that are part of a recently discovered family, the aspercryptins.
Using stable-isotope incorporation experiments, MS/MS-networking and interpretation, we were able to annotate an additional 11 aspercryptin family members. Strikingly, because the aspercryptins have a C-terminal lipid, they appear backward compared to most lipopeptides. The unusual location of the aspercryptin lipid moiety indicates that it does not follow the standard lipopeptide biosynthetic logic. Fittingly, it appears that the lipid moiety is installed as an amino acid through canonical non-ribosomal peptide synthetase (NRPS) logic. The installation of fatty acids in this manner appears to be unique to a small group of fungal NPs.
The genome of A. nidulans contains a gene cluster that encodes proteins likely responsible for converting fatty acids into amino acids to then be loaded by the NRPS protein. We are now experimentally verifying this proposal through targeted bottom-up proteomics to show pathway intermediates covalently bound to their biosynthetic enzymes.