Expanding small molecule functional metagenomics through shuttle BAC expression i

描述(申请人提供)：医院获得性微生物感染是美国第四大杀手，夺走了10万人的生命，增加了300亿美元的医院费用。抗药性微生物的出现进一步放大了公众对健康的担忧。真菌是抗微生物次级代谢物(SM)的高产生产者，自世纪之交以来，真菌提供了所有微生物来源的45%的生物活性分子。然而，真菌次生代谢(SM)途径由于难以有效表达这些SM途径而在很大程度上仍未被开发。本研究旨在推进功能SM元基因组学的研究，从已测序的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，吴博士在完整基因组公司(原Lucigen)的团队以及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，共同产生了下一代功能元基因组文库。该文库结合了1)用于分离和纯化真菌高分子量基因组DNA的改进方法；2)新的大肠杆菌-曲霉穿梭载体和用于增强克隆DNA表达的嗜酸曲霉宿主；3)随机剪切BAC克隆方法，用于产生覆盖真菌基因组完整SM途径(一个BAC克隆=一个完整SM途径)的无偏非常大的插入片段(&gt；100kb)；以及4)用于鉴定独特化合物的快速和改进的小分子鉴定方法。在第一阶段的研究中，所有56条SM途径都在A.terreus的无偏随机剪切穿梭BAC文库中被鉴定。将14个SM-BAC克隆转化到根瘤拟青霉中，至少有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，从含有黄芪色素途径的BAC中还发现了6个新的黄芪内酯化合物。我们建议在第二阶段的研究中，从5种已测序的真菌(~200条SM途径)中额外创建5个无偏大插入穿梭BAC文库，这些文库将广泛用于小分子化合物和抗生素的筛选。我们预计将使用这种方法发现数百种新的化学实体，并引领具有高效力的候选药物对抗多重耐药细菌和真菌病原体。这些技术总体上代表着天然产物发现科学的重要进步，尤其是抗生素的发现。此外，本研究获得的文库是一种有价值的基因组资源，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.