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

PROJECT SUMMARY The economic and social burden for the treatment of chronic and infectious diseases is enormousandgt $BThe emergence of drug resistant microbesthe diminishing supply of novel classes of antibioticsand the dramatic reduction in discovery and development of anti infectiveanti proliferation and anti inflammation agents have further amplified public health concernFungi are prolific producers of anti microbial secondary metabolitesSMand since the turn of the century have providedof bioactive molecules from all microbial sourcesHoweverfungal SM pathways remain largely untapped due to difficulties in efficiently handling and expressing these SM pathwaysThis research proposal is to advance the science of functional SM metagenomicsto further advance our newly developed fungal artificial chromosomeFACtechnologyprecisely engineer and activate large intact silent SM pathways containing FAC clonesand to discover novel natural productsNPsfor pharmaceutical and clinical developmentOngoing research at Intact GenomicsUniversity of Wisconsin Madison and Northwestern University have orchestrated key technological breakthroughs that together resulted in the next generation fungal SM discovery platformThis discovery technology combinedan improved methodology for the isolation and purification of high molecular weight genomic DNA from fungia new Ecoli Aspergillus shuttle or FAC vector and an Anidulans host for enhanced expression of cloned large DNAsa random shear BAC FAC cloning method to produce unbiased very large insert sizesandgtkbfor covering the entire set of intact SM biosynthetic gene clustersBGCsof a fungal genomeone FAC cloneone intact SM pathwayprecisely engineering and activating large intact silent SM gene clusters FACs by Red ET techniques and BGC refactoring via yeastanda rapid and improved small molecule identification method through both function screening and chemical analysisIn Phase I and ongoing researchwe have achieved phenomenalcompound hit rate by directly transferring fungal genome sequence through FACs first timeWe propose in this Phase II study to further increase the compound hit rate to aboutWe will also engineer at leastsilent SM pathways fromsequenced fungiSM BGCswhich will be extensively screened for antimicrobial and anticancer agents and insecticidesWe expect to uncover andgtnovel chemical entities using this approachand lead candidates with high potency against multipledrug resistance bacterial and fungal pathogensanticancer and insecticide leadsThese technologies represent an important advancement for the science of NP discovery in generalIn additionthe FACs and compounds produced from this research are a valuable genomic resource that may be screened for other bioactive compoundsfor exampleantidepressantsantiviraland anti inflammatory activities PROJECT NARRATIVE We are potentially losing the battle in the fight against both chronic and infectious diseases due to the alarming increasing number of chronic problems and multi drug resistance microbes coupled with our inability to find drug leads with novel acting mechanismsthe loss of life and the burden of treatment is a significant public health threat to American citizensThe proposed research further develops a robust methodology advancing fungal artificial chromosomeFACtechnology and tools for drug discoverythis novel technology permits access to the majority of fungal cryptic biosynthetic gene clusters of natural products in a sequenced fungal genome with the aim to eventually activate all silent and cryptic pathways for pharmaceutical discoveryOur functional metagenomic approach will be used to identify and characterize novel anticancerantibioticand insecticidial compounds to combat the threat of cancersmosquitoesand microbial pathogens