posted on 2015-07-07, 00:00authored byAndreas Klitgaard, Jakob
B. Nielsen, Rasmus J. N. Frandsen, Mikael R. Andersen, Kristian F. Nielsen
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.