Expanding small molecule functional metagenomics through shuttle BAC expression i

描述（由申请人提供）：医院获得性微生物感染是美国的第四大杀手，夺走了10万人的生命，增加了300亿美元的医院成本。耐药微生物的出现进一步加剧了对公共卫生的关注。真菌是抗微生物次生代谢物（SM）的多产生产者，自世纪之交以来，真菌提供了所有微生物来源中45%的生物活性分子。然而，由于真菌次级代谢（SM）途径难以有效表达，这些途径在很大程度上仍未被开发。本研究计划旨在推进功能性SM宏基因组学的研究，从真菌基因组中克隆完整的SM通路，并为药物和临床开发发现新的抗生素。在第一阶段的研究中，Wu博士的团队在完整基因组（原Lucigen）和威斯康星大学麦迪逊分校和西北大学的科学家应用了许多关键技术突破，共同形成了下一代功能宏基因组库。该文库结合了1)从真菌中分离和纯化高分子量基因组DNA的改进方法；2)构建新的大肠杆菌-曲霉穿梭载体和假芽孢杆菌宿主，增强克隆dna的表达；3)随机剪切BAC克隆方法，产生无偏的非常大的插入片段（>100 kb），用于覆盖真菌基因组的整套完整SM通路（一个BAC克隆=一个完整的SM通路）；4)一种快速改进的小分子鉴定方法来鉴定独特的化合物。在I期研究中，我们在A. terreus的无偏随机剪切穿梭BAC文库中鉴定出56条SM通路。将14个SM BAC克隆转化为A. nidulans，其中至少2个BAC克隆具有较强的抗菌活性，3个BAC克隆具有抗真菌活性。此外，从含星形色素途径的BAC中发现了6个新的星形色素化合物。我们建议在II期研究中从5种已测序的真菌（约200个SM通路）中创建5个额外的无偏大插入穿梭BAC文库，这些文库将广泛筛选小分子化合物和抗生素。我们希望利用这种方法发现数百种新的化学实体，并领导具有高效抗多重耐药细菌和真菌病原体的候选物质。这些技术代表了天然产物发现科学的重要进步，特别是抗生素的发现。此外，本研究产生的文库是一种宝贵的基因组资源，可用于筛选其他生物活性化合物：例如抗病毒、抗癌和抗炎活性。

DESCRIPTION (provided by applicant): 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.