Activating silent fungal secondary metabolite gene clusters and studying their biosynthetic pathways using CRISPR/Cas9

Filamentous fungi are a well known source of secondary metabolites (SMs) which are compounds that have valuable bioactivities and potential applications in medicine and agriculture. Many attempts have been made, using traditional methods, to exploit these fungal compounds with limited success. Genomics reveal many fungal biosynthetic gene clusters (BGCs) which are silent under laboratory growth conditions¹ hindering the discovery of novel SMs. Activation of these BGCs and studying their biosynthetic pathways is garnering increased attention which are being hailed as the beginning of a golden era in fungal SMs.

In this work, we overexpressed the transcription factor NRRL3_09035 which was predicted to regulate a fatty acid synthase and polyketide synthase gene cluster of Aspergillus niger NRRL3. Mass spectra revealed novel compounds compared to the parental strain. Nuclear magnetic resonance (NMR) spectra suggested these compounds belong to fatty acid class which is consistent with the presence of a fatty acid synthase backbone enzyme in the BGC. Each gene of the cluster was inactivated in order to study the biosynthetic pathway as well as to accumulate the intermediate compounds for structural elucidation by NMR. Overexpression and gene inactivation was carried out using CRISPR/Cas9 system for A. niger; a recent technique developed in our lab².

1. Inglis DO, Binkley J, Skrzypek MS, Arnaud MB, Cerqueira GC, Shah P, Wymore F, Wortman JR, Sherlock G (2013). Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae. BMC Microbiology. 13: 91.
2. Recruitment of tRNA PolIII Promoters in CRISPR/Cas9 System to Edit Genome of Aspergillus niger. Letian Song, Jean-Paul Ouedraogo, Magdalena Kolbusz, Thi Truc Minh Nguyen, Adrian Tsang. Submitted.