Fungal Artificial Chromosomes for Mining of the Fungal Secondary Metabolome

Jin Woo Bok1, Jessica C. Albright2, Rosa Ye3,4, David Mead4, Megan Wagner4, Amanda Krerowicz4, Anthony W. Goering5, Kenneth D. Clevenger5, Thomas Velk1, Paul M. Thomas5, Neil L. Kelleher2,5, Nancy Keller1, and Chengcang Wu3,4

1 Departments of Medical Microbiology and Immunology and Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA, 2 Department of Chemistry, Northwestern University, Evanston, Illinois, USA, 3 Intact Genomics Inc., St. Louis, Missouri, USA, 4 Lucigen Corporation, Middleton, Wisconsin, USA, 5 Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois, USA

With thousands of fungal genomes being sequenced, each genome containing up to 70 secondary metabolite (SM) clusters 30 – 80 kb in size, breakthrough techniques are needed to characterize this SM wealth. Here we describe a novel system-level methodology for unbiased cloning of intact SM clusters from a single fungal genome for one-step transformation and expression in a model host. All 56 intact SM clusters from Aspergillus terreus were individually captured in self-replicating fungal artificial chromosomes (FACs) containing both E. coli F replicon and an Aspergillus autonomously replicating sequence (AMA1). Candidate FACs were successfully shuttled between E. coli and the heterologous expression host A. nidulans. As proof-of-concept, an A. nidulans FAC strain was characterized in a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) and data analysis pipeline leading to the discovery of the A. terreus astechrome machinery.