A robust heterologous expression system of intact fungal secondary metabolite gene clusters for natural product discovery in Aspergillus nidulans

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 DESCRIPTION (provided by applicant): There is societal need for new therapeutic agents in our arsenal of defenses against bacterial and fungal pathogens, many (superbugs) of which are increasingly resistant to existing antibiotics. Filamentous fungi are considered promising resources for the development of novel bioactive compounds because of their great potential to produce various kinds of secondary metabolites (SM), however, natural product (NP) discovery and production in fungi lags far behind plants and bacteria. This research proposal advances sciences of fungal functional genomics to develop a robust heterologous expression system for intact SM gene clusters in the filamentous fungus Aspergillus nidulans by using the newly developed fungal artificial chromosomes (FACs). Our purpose is to discover novel antibiotics and identify the best lead candidates for clinical development. Scientists at Intact Genomics Inc, University of Wisconsin at Madison, Donald Danforth Plant Science Center, and Northwestern University will develop, utilize, and combine at least 6 aspects of novel technology innovation and genomic tools to enable NP discovery in fungi. Specifically, the proposed research will identify NP compounds using: i) the unbiased large-insert Random Shear Shuttle BAC libraries as FACs, ii) more than 200 large intact SM gene clusters (about 20~100 kb) in the completely sequenced genome of 6 fungal strains, iii) the knowledge of regulatory elements-strong promotors for high heterologous expression of SM gene clusters in Aspergillus, iv) the successfully engineered fungal host: A. nidulans to provide a robust background in which to search for new metabolites, v) in vitro BAC/FAC engineering, vi) advanced LC-MS analysis. The primary objectives of Phase I research are 1) to develop simple FAC transformation method in an optimized A. nidulans strain, 2) to activate at least 2 of 5 silent and or cryptic SM gene clusters (FACs) for proof-of-concept using the above technologies to discover novel NP compounds. Our long-term goals are to develop a high through-put small molecule discovery platform in fungi in order to discover novel natural products from at least 1,000 fungal intact SM pathways from completely sequenced fungal genomes. Moreover, we will characterize identified antimicrobial agents to determine the best lead candidates for clinical development. Lead candidates will have novel chemical structures, high potency against bacterial and or fungal pathogens, and minimal toxicity for eukaryotic cells. The combination of these novel technological innovations has a high probability of success and also represents a significant advancement for the science of natural product discovery. In addition, the 1,000 novel SM clusters and their metabolites produced from this research are a valuable resource that may be screened for other bioactive compounds (e.g., with anticancer or antiviral activities) in subsequent research.