Research Project
9315098
PROJECT SUMMARY According to a CDC report, antibiotic-resistant infections are associated with 23,000 deaths and 2 million illnesses in the United States each year. The estimated annual impact of antibiotic- resistant infections on the national economy is $55 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 45% 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: 1) an improved methodology for the isolation and purification of high molecular weight genomic DNA from fungi; 2) a new E. coli- Aspergillus shuttle or FAC vector and an A. nidulans host for enhanced expression of cloned large DNAs; 3) a random shear BAC/FAC 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 FAC clone = one intact SM pathway); 4) precisely engineering and activating large intact silent SM gene clusters-FACs by Red/ET techniques; and 5) 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 20 engineered FAC constructs from the 5 large SM-FAC clones with a 100% 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 100-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 >100 silent SM pathways from 6 sequenced fungi (263 SM pathways) which will be extensively screened for small molecule compounds and antibiotics. We expect to uncover >50 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.
