1 Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, 3330 Hospital Drive, Calgary, Alberta, T2N 4N1, Canada.
2 Centre for Structural and Functional Genomics, Concordia University, 7141 Rue Sherbrooke Ouest, Montreal, Quebec, H4B 1R6, Canada.

Genomics analysis confirmed the status of filamentous fungi as a rich source of novel secondary metabolites; however, the discovery of these compounds is hampered by the cryptic nature of their biosynthetic pathways under laboratory conditions. Consequently, despite substantial research effort over the past decades, much of the secondary metabolome remains uncharacterized in fungal organisms. Our manual curation of biosynthetic gene clusters (BGCs) in the Aspergillus niger NRRL3 genome revealed that only 13 of 86 BGCs have had their cognate secondary metabolite products confirmed or reliably inferred. We also identified 60 transcription factors (TFs) associated with cryptic BGCs. To further characterize A. niger secondary metabolism, we created a collection of strains each overexpressing a single BGC-associated TF. We analyzed the strain collection using a standardized pipeline where we monitored phenotypic changes and compound production using mass spectrometry. Strains showing evidence of secondary metabolism activation were selected for gene expression analysis. Our approach resulted in the production of multiple potentially novel secondary metabolites and linked a specific BGC to tensidol production in A. niger. More broadly, this study found evidence counter to the existing paradigm of BGC expression controlled by colocalized TFs, lending credence to the emerging picture of a complex regulatory network governing fungal secondary metabolism.