Large scale discovery and deorphanization of natural products using fungal artificial chromosomes and untargeted metabolomics (FAC-MS)

The vast majority of fungal-encoded chemical space is uncharted due to difficulties culturing and genetically manipulating many fungi.  We report a scalable technology using fungal artificial chromosomes (FACs) to capture secondary metabolite biosynthetic gene clusters (BGCs) derived from random-shear libraries of genomic DNA from Aspergillus terreus, A. aculeatus, and A. wentii.  Host A. nidulans strains transformed with FACs are screened by untargeted liquid chromatography-mass spectrometry with ultrahigh mass accuracy, and FAC-encoded products are identified by a robust scoring system to identify spectral features most likely associated with each FAC.  Deletions of specific backbone and tailoring genes within FACs (achieved through facile E. coli genetics) then confirm assignment of products to BGCs and facilitate analyses of biosynthesis.  We use this “FAC-MS” platform to screen 56 FACs and report detection of SM products from 17/56 (30% hit rate), including an A. terreus FAC encoding three distinct products identified as a novel lipopeptide, a terpenoid from the ophiobolin family of compounds, and the orphan benzodiazepine benzomalvin A, which is 300-fold overexpressed relative to endogenous levels in A. terreus.  Deletants of benzomalvin A backbone and tailoring genes allow us to propose and test the first biosynthetic model for this molecule.  Wide application of the FAC-MS pipeline should have a major impact on fungal natural products research in the mid-term future.