Combining Stable Isotope Labeling and Molecular Networking for Biosynthetic Pathway Characterization.

Filamentous fungi are a rich source of bioactive compounds, ranging from statins over immunosuppressants to antibiotics. The coupling of genes to metabolites is of large commercial interest for production of the bioactives of the future. To this end, we have investigated the use of stable isotope labeled amino acids (SILAAs). SILAAs were added to the cultivation media of the filamentous fungus Aspergillus nidulans for the study of the cyclic tetrapeptide nidulanin A. Analysis by UHPLC-TOFMS confirmed that the SILAAs were incorporated into produced nidulanin A, and the change in observed m/z could be used to determine whether a compound (known or unknown) incorporated any of the added amino acids. Samples were then analyzed using MS/MS and the data used to perform molecular networking. The molecular network revealed several known and unknown compounds that were also labeled. Assisted by the isotope labeling, it was possible to determine the sequence of several of the compounds, one of which was the known metabolite fungisporin, not previously described in A. nidulans. Several novel analogues of nidulanin A and fungisporin were detected and tentatively identified, and it was determined that these metabolites were all produced by the same nonribosomal peptide synthase. The combination of stable isotope labeling and molecular network generation was shown to very effective for the automated detection of structurally related nonribosomal peptides, while the labeling was effective for determination of the peptide sequence, which could be used to provide information on biosynthesis of bioactive compounds.