Hospital acquired microbial infections are the fourth largest killer in America, taking 100,000 lives and adding $30B to hospital costs. The emergence of drug resistant microbes has further amplified public health concern. Fungi are prolific producers of anti-microbial secondary metabolites (SM) and since the turn of the century have provided 45% of bioactive molecules from all microbial sources. However, fungal secondary metabolic (SM) pathways remain largely untapped due to difficulties in efficiently expressing these SM pathways. This research proposal is to advance the science of functional SM metagenomics, to clone the entire set of intact SM pathways from sequenced fungal genomes, and to discover new antibiotics for pharmaceutical and clinical development. During Phase I research Dr. Wu's group at Intact Genomics (formerly Lucigen) and scientists at the University of Wisconsin Madison and Northwestern University applied numerous key technological breakthroughs that together resulted in the next generation functional metagenomic library. This library combined 1) an improved methodology for the isolation and purification of high molecular weight genomic DNA from fungi;2) a new E. coli- Aspergillus shuttle vector and an A. nidulans host for enhanced expression of cloned DNAs;3) a random shear BAC cloning method to produce unbiased very large insert sizes (>100 kb) for covering the entire set of intact SM pathways of a fungal genome (one BAC clone = one intact SM pathway);and 4) a rapid and improved small molecule identification method to identify unique compounds. In Phase I research, all of 56 SM pathways were identified in the unbiased Random Shear shuttle BAC library of A. terreus. Fourteen SM BAC clones where transformed into A. nidulans with at least two BAC clones showing strong antibacterial activities and three antifungal. Moreover, 6 new astechrome compounds were uncovered from the astechrome- pathway-containing BAC. We propose in Phase II study to create 5 additional unbiased large- insert shuttle BAC libraries from 5 sequenced fungi (~ 200 SM pathways) which will be extensively screened for small molecule compounds and antibiotics. We expect to uncover hundreds of 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 libraries produced from this research are a valuable genomic resource that may be screened for other bioactive compounds: for example antiviral, anticancer, and anti-inflammatory activities.
We are losing the battle in the fight against infectious diseases due to the alarming increasing number of multi-drug resistance microbes coupled with 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 BAC technology and tools for drug discovery that permits access to the ENTIRE set of INTACT 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.