Expanding small molecule functional metagenomics through shuttle BAC expression i

描述(申请人提供)：在我们对细菌和真菌病原体的防御方面，社会上需要新的治疗剂，其中许多对现有抗生素的抗药性越来越强。丝状真菌具有产生多种次生代谢物的巨大潜力，被认为是开发新型生物活性物质的潜在资源，然而，真菌中抗生素的发现和生产远远落后于细菌。这项研究计划通过穿梭克隆包含整个SM通路及其调控元件的大DNA来推进真菌功能基因组学的科学，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将开发、利用和结合四个方面的新技术创新和基因组工具，以实现真菌治疗剂的发现。具体地说，拟议的研究将使用：i)大插入无偏随机剪切穿梭BAC文库，ii)在Terreus全序列基因组中至少56个大的次生代谢途径(约20~100kb)，iii)曲霉全球次生代谢产物簇调控的知识，iv)工程真菌宿主A.nidulans为寻找新的代谢物提供强大的背景。主要目标是构建两个穿梭BAC文库，鉴定包含56个SM通路及其调控元件的BAC，以利用上述技术进行概念验证，并筛选这些BAC克隆以对抗细菌和真菌测试菌株，以发现新的抗菌和抗真菌特性。我们的长期目标是开发一个高吞吐量的真菌小分子发现平台，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，我们将对已识别的抗菌剂进行表征，以确定临床开发的最佳候选药物。候选铅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。拟议研究所需的每一种不同技术都已被单独证明是有效的；因此，这些不同技术的组合具有很高的成功概率，也代表着抗生素发现科学的重大进步。此外，本研究建立的文库是一种有价值的基因组资源，可以在后续研究中筛选出其他生物活性化合物(例如，具有抗癌或抗病毒活性的化合物)。1 与公共健康相关：自20世纪40年代抗生素商业化以来，对新抗菌剂的需求达到了前所未有的程度，但许多传统上富有成效的化学来源已经不再生产新的化合物。拟议的研究将开发、利用和结合四项技术创新和基因组工具，以使从真菌中发现治疗剂成为可能。工程真菌宿主nidulans将被用来直接收获和表达真菌次生代谢途径及其调控元件，而不需要在实验室培养和工程不同的真菌。这项技术将使人们能够获得由多种多样的丝状真菌产生的各种新型小分子，其中许多目前尚不为科学所知。这项工作的最终目标是确定用于治疗细菌和真菌疾病的新的治疗化合物。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