Evidencing 98 secondary metabolites of Penicillium verrucosum using substrate isotopic labeling and high-resolution mass spectrometry

Industrial applications of fungal compounds, coupled with the emergence of fungal threats to natural ecosystems and public health, have increased interest in filamentous fungi. Among all pathogenic fungi, Penicillium verrucosum is one of the most common mold-infecting stored cereals in temperate regions. However, it is estimated that 80% of fungal secondary metabolites remain unknown. To detect new P. verrucosum compounds, an untargeted metabolomic approach was applied to fungus grown on wheat grains labeled with stable isotopes: (i) natural grains (99% ¹²C); (ii) grains enriched with 97% of ¹³C; and (iii) grains enriched with 53% of ¹³C and 97% of ¹⁵N. Analyses performed by high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) enabled the specific detection of fungal metabolites, and the unambiguous characterization of their chemical formulas. In this way, 98 secondary metabolites were detected and their chemical formulas were determined. Of these, only 18 identifications could be made based on databases, the literature and mass spectrometry fragmentation experiments, with the result that 80 were totally unknown. Molecular networks were generated to analyze these results, leading to the characterization by MSⁿ experiments of a new fungisporin produced by P. verrucosum. More generally, this article provides precise mass spectrometric data about all these compounds for further studies of the Penicillium metabolome.