Expanding small molecule functional metagenomics through shuttle BAC expression i

描述（由申请人提供）：社会需要新的治疗药物来防御细菌和真菌病原体，其中许多细菌和真菌病原体对现有抗生素的耐药性越来越强。丝状真菌由于具有产生多种次生代谢物（SM）的巨大潜力，被认为是开发新型生物活性化合物的有前景的资源，但真菌抗生素的发现和生产远远落后于细菌。本研究计划通过穿梭克隆含有整个SM通路及其调控元件的大DNA来推进真菌功能基因组学科学，以发现新的抗生素并确定临床开发的最佳候选药物。Lucigen公司和威斯康星大学麦迪逊分校的科学家们将开发、利用和结合新技术创新和基因组工具的四个方面，以实现真菌治疗剂的发现。具体来说，该研究将使用以下方法来鉴定抗生素化合物：i)大插入无偏随机剪切穿梭BAC文库；ii)在完整测序的a . terreus基因组中至少56个大的次级代谢途径（约20~100 kb）； iii)曲霉的全球次级代谢产物簇调控知识；iv)工程真菌宿主a . nidulans为寻找新的代谢物提供坚实的背景。主要目标是建立两个穿梭BAC文库，鉴定含有56个SM通路及其调控元件的BAC，并利用上述技术对这些BAC克隆进行细菌和真菌测试菌株的筛选，以发现新的抗菌和抗真菌特性。我们的长期目标是在真菌中开发一个高通量的小分子发现平台，以便从完全测序的真菌基因组中从至少500个SM途径中发现新的天然产物。此外，我们将表征已确定的抗菌药物，以确定临床开发的最佳候选药物。主要候选药物将具有新颖的化学结构，对细菌和/或真菌病原体具有高效力，对真核细胞的毒性最小。拟议研究所需的每项不同技术已分别证明是有效的；因此，这些不同技术的结合具有很高的成功概率，也代表了抗生素发现科学的重大进步。此外，本研究产生的文库是一种宝贵的基因组资源，可以在后续研究中筛选其他生物活性化合物（例如，具有抗癌或抗病毒活性）。1

DESCRIPTION (provided by applicant): There is societal need for new therapeutic agents in our arsenal of defenses against bacterial and fungal pathogens, many of which are increasingly resistant to existing antibiotics. Filamentous fungi are considered promising resources for the development of novel bioactive compounds because of their great potential to produce various kinds of secondary metabolites (SM), however, antibiotic discovery and production in fungi lags far behind bacteria. This research proposal advances sciences of fungal functional genomics using shuttle cloning of large DNA containing the entire SM pathways and their regulatory elements in order to discover novel antibiotics and identify the best lead candidates for clinical development. Scientists at Lucigen Corporation and the University of Wisconsin at Madison will develop, utilize, and combine four aspects of novel technology innovation and genomic tools to enable therapeutic agent discovery in fungi. Specifically, the proposed research will identify antibiotic compounds using: i) large-insert unbiased Random Shear Shuttle BAC libraries, ii) at least 56 large secondary metabolic pathways (about 20~100 kb) in the completely sequenced genome of A. terreus, iii) the knowledge of global secondary metabolite cluster regulation in Aspergillus, iv) an engineered fungal host A. nidulans to provide a robust background in which to search for new metabolites. The primary objectives are to build two shuttle BAC libraries and identify BACs containing 56 SM pathways and their regulatory elements for proof-of-concept using the above technologies and to screen these BAC clones against bacterial and fungal tester strains to discover novel antibacterial and antifungal properties. Our long-term goals are to develop a high through-put small molecule discovery platform in fungi in order to discover novel natural products from at least 500 SM pathways from completely sequenced fungal genomes. Moreover, we will characterize identified antimicrobial agents to determine the best lead candidates for clinical development. Lead candidates will have novel chemical structures, high potency against bacterial and or fungal pathogens, and minimal toxicity for eukaryotic cells. Each of the different technologies necessary for the proposed research has been proven effective separately; therefore, the combination of these different techniques has a high probability of success and also represents a significant advancement for the science of antibiotic discovery. In addition, the libraries produced from this research are a valuable genomic resource that may be screened for other bioactive compounds (e.g., with anticancer or antiviral activities) in subsequent research. 1 PUBLIC HEALTH RELEVANCE: The need for new antimicrobial agents has reached an intensity not experienced since the commercialization of antibiotics in the 1940s, but many traditionally fruitful sources of chemistry have ceased to yield new compounds. The proposed research will develop, utilize, and combine four technology innovations and genomic tools to enable therapeutic agent discovery from fungi. An engineered fungal host, Aspergillus nidulans, will be used for harvesting and expressing fungal secondary metabolic pathways and their regulatory elements directly, without the need to cultivate and engineer the different fungi in a laboratory. This technology will allow access to a wide variety of novel small molecules produced by a great diversity of filamentous fungi, many of which are currently unknown to science. The ultimate goal of this work is to identify novel therapeutic compounds for use in treating bacterial and fungal diseases. 1