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

PROJECT SUMMARY According to a CDC report antibiotic resistant infections are associated with deaths and million illnesses in the United States each year The estimated annual impact of antibiotic resistant infections on the national economy is $ billion in excess direct health care costs and in lost productivity from hospitalizations and sick days The emergence of drug resistant microbes the diminishing supply of novel classes of antibiotics and the dramatic reduction in discovery and development of anti infective anti proliferation and anti inflammation agents have further amplified public health concerns Fungi are prolific producers of anti microbial secondary metabolites SM and since the turn of the century have provided of bioactive molecules from all microbial sources However fungal SM pathways remain largely untapped due to difficulties in efficiently handling and expressing these SM pathways This research proposal is to advance the science of functional SM metagenomics to further advance our newly developed fungal artificial chromosome FAC technology precisely engineer and activate large intact silent SM pathways containing FAC clones and to discover new antibiotics for pharmaceutical and clinical development Ongoing research from Dr Wu s team at Intact Genomics and scientists at the University of Wisconsin Madison and Northwestern University applied numerous key technological breakthroughs that resulted in the next generation fungal SM discovery platform This discovery technology combined an improved methodology for the isolation and purification of high molecular weight genomic DNA from fungi a new E coli Aspergillus shuttle or FAC vector and an A nidulans host for enhanced expression of cloned large DNAs a random shear BAC FAC cloning method to produce unbiased very large insert sizes andgt kb for covering the entire set of intact SM pathways of a fungal genome one FAC clone one intact SM pathway precisely engineering and activating large intact silent SM gene clusters FACs by Red ET techniques and a rapid and improved small molecule identification method to identify unique compounds In Phase I research we tested FAC engineering by precisely cutting FAC sizes deleting individual SM genes and or inserting a strong promoter and generated more than engineered FAC constructs from the large SM FAC clones with a successful rate We have discovered the benzomalvin compounds and established the biosynthesis of this NRPS metabolites that has long eluded the field The FAC SM gene deletants not only allow us to see loss of their corresponding gene products but also accumulation of biosynthetic precursors Moreover the engineered silent SM gene clusters show at least a fold increase in expression in response to the strong promoter and analysis We believe that we are the first group to develop a superior BAC recombineering and transgenic animal system for fungal functional SM study We propose in Phase II study to engineered andgt silent SM pathways from sequenced fungi SM pathways which will be extensively screened for small molecule compounds and antibiotics We expect to uncover andgt novel chemical entities using this approach and lead candidates with high potency against multiple drug resistance bacterial and fungal pathogens These technologies represent an important advancement for the science of natural product discovery in general and antibiotic discovery in particular In addition the FACs produced from this research are a valuable genomic resource that may be screened for other bioactive compounds such as antiviral anticancer and anti inflammatory activities PROJECT NARRATIVE We are losing the battle against infectious diseases as evidenced by the alarming increase number of multi drug resistance microbes and our inability to find antibiotics with novel acting mechanisms The loss of life and the burden of treatment is a significant public health threat to American citizens The proposed research utilizes a new methodology advancing fungal artificial chromosome FAC technology and tools for drug discovery This novel technology permits access to the majority of fungal LARGE INTACT and SILENT natural product pathways in a sequenced fungal genome with the aim to eventually activate all silent and cryptic pathways for pharmaceutical discovery Our functional metagenomic approach will be used to identify and characterize novel antibiotic compounds to combat the threat of bacterial and fungal pathogens