Biochemical and genetic basis of oligocyclic aromatic polyketide formation in basidiomycetes

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Regarding structural diversity and their useful or toxic bioactivities, the polyketides are an outstanding group of natural products. They are produced by polyketide synthases (PKSs), i.e. complex, multifunctional Enzymes. The genetic and biochemical basis of polyketide biosynthesis in bacteria and ascomycetes is well understood. Conversely, the knowledge for basidiomycetes (mushroom-type fungi) is very scarce. The formation of oligocyclic polyketides (e.g. pigments and toxins with this structural feature) in them has remained completely uninvestigated. This knowledge gap is even more surprising, as the basidiomycetes represent a phylum of some 40,000 species and an abundant source of bioactive natural products.The genus Cortinarius (Webcap Mushroom) is one of the largest genera of the basidiomycetes. Species within this genus produce numerous oligocyclic polyketides. Using the Fragrant Webcap (Cortinarius odorifer) as a model, the applicant investigated its genome. This mushroom produces phlegmacins A1 and B1, which are dimeric, methylated oligocyclic polyketides. Six highly similar genes (pks1-pks6) were identified which encode PKSs of a hitherto unknown, evolutionarily separate group. It is hypothesized that these genes/enzymes control the formation of oligocyclic polyketides. This hypothesis is supported by adjacent genes for two methyl transferases and an aromatic peroxidase that may catalyze methylation and dimerization during phlegmacin biosynthesis.This project aims at functional characterization of the enzymes PKS1-PKS6 to test the above hypothesis. To reach this goal, the enzymes will be produced heterologously in a fungal host (Aspergillus niger), followed by product formation in vivo and in vitro. The products will be analyzed by liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy. In addition, the two methyl transferases will be produced heterologously and characterized. The successfully completed project has model character and lays the foundation for future work to generally understand how structural diversity of toxic or pharmaceutically relevant basidiomycete polyketides is generated.