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For many fungi the number of known secondary metabolites is surprisingly small compared to the -astonishingly large number of terpene cyclase, polyketide synthase (PKS), and non-ribosomal peptide synthetase (NRPS) secondary metabolite gene clusters found in their genomes. Correspondingly, the majority of fungal secondary metabolite genes have not yet been associated with the biosynthesis of any known small molecules, and it seems likely that for many more PKS and NRPS known small molecule products represent but a fraction of the entire spectrum of metabolites produced by the associated pathways. Comparative metabolomics based on differential analysis by 2D NMR spectroscopy (DANS) in conjunction with LC-MS analyses is emerging as a highly effective tool for pursuing small molecule structures and biosynthetic pathways associated with orphan PKS and NRPS gene clusters. Here we describe the use of DANS paired with LC-MS analyses for the comparison of the metabolomes of various fungal strains including wild-type (WT), PKS/NRPS overexpressing, and/or corresponding PKS/NRPS knock-out (KO) strains.

Correlating Secondary Metabolite Production with Genetic Changes Using Differential Analysis of 2D NMR Spectra