Activate Silent and Cryptic Intact Fungal SM Gene Clusters through FACs for Novel Antibiotics Discovery

Wu, Chengcang; Keller, Nancy

Abstract

There is societal need for new therapeutic agents in our arsenal of defenses against bacterial and fungal pathogens, many 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, antibiotic discovery and production in fungi lags far behind bacteria. This research proposal advances sciences of fungal functional genomics to activate fungal silent SM clusters 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. and the University of Wisconsin at Madison will develop, utilize, and combine three aspects of novel technology innovation and genomic tools to enable therapeutic agent discovery in fungi. Specifically, the proposed research will identify antibiotic compounds using: 1) genetically enhanced A. nidulans strains, 2) in vitro BAC/FAC engineering, and 3) culture conditions with epigenetic modifications and bacterial co-culture. The primary objectives are to activate at least 5 of 40 silent and or cryptic SM gene clusters (FACs) of A. terreus for proof-of-concept using the above technologies and to screen these activated FACs against bacterial and fungal tester strains to discover novel antibacterial and antifungal properties. 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 silent 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. Each of the different technologies necessary for the proposed research has been proven effective separately; therefore, the combination of these different techniques has a high probability of success and also represents a significant advancement for the science of antibiotic discovery. In addition, the 1,000 activated silent 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. 1

Public Health Relevance

The need for new therapeutic agents has reached an intensity not experienced since the commercialization of antibiotics in the 1940s, but many traditionally fruitful sources of chemistry have ceased to yield new compounds. This research will develop, utilize, and combine three aspects of novel technology innovation and genomic tools to enable therapeutic agent discovery in the successfully engineered fungal host Aspergillus nidulans by activating and expressing fungal silent secondary metabolic pathways directly, without the need to cultivate and engineer the different fungi in a laboratory. This technology will access the nove small molecules produced by a great diversity of filamentous fungi, many of which are unknown to science, and will identify the best novel therapeutic compounds for use in treating bacterial and fungal diseases.