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 most fungi. We report a scalable metabolomic profiling technology using untargeted liquid chromatography-mass spectrometry (LC-MS) with ultrahigh mass accuracy to systematically identify the secondary metabolite (SM) products of heterologously expressed fungal biosynthetic gene clusters (BGCs). This platform uses fungal artificial chromosomes (FACs) to capture full-length BGCs derived from unbiased large-insert libraries of genomic DNA [1] of Aspergillus terreus, A. aculeatus, and A. wentii (Fig. A). Host A. nidulans strains transformed with FACs (Fig. B) are then screened by LC-MS (Fig. C), and FAC-encoded products are identified by a robust scoring system to identify spectral features most likely associated with each FAC (Fig. D). 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 (Fig. E). 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. For example, the application of FAC-MS to a collection of over 200 Wisconsin Aspergillus strains collected by Dr. Martha Christensen, with an average of 50 BGCs per strain and an estimated hit rate of > 20%, would lead to validated assignments of at least 2,000 compounds to their BGCs.

Keywords: Heterologous expression, artificial chromosome, filamentous fungi, aspergillus, orphan natural products, mass spectrometry, LC-MS, FT-MS, biosynthesis, metabolomics.

References:

[1] Bok JW, Ye R, Clevenger KD, Mead D, Wagner M, Krerowicz A, Albright JC, Goering AW, Thomas PM, Kelleher NL, Keller NP, Wu CC. Fungal artificial chromosomes for mining of the fungal secondary metabolome. BMC Genomics. 2015; 16: 343