Expanding small molecule functional metagenomics through shuttle BAC expression i

通过穿梭 BAC 表达扩展小分子功能宏基因组

• 批准号: 8123947
• 负责人: Chengcang Charles Wu
• 金额: $ 22.64万
• 依托单位: LUCIGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8123947
• 关键词: Anti-Bacterial Agents; Antibiotics; Antifungal Agents; Antiviral Agents; Artificial Chromosomes; Aspergillus; Aspergillus nidulans; Bacteria; Biological Factors; Chemical Structure; Chemistry; Clinical; Cloning; DNA; Development; Disease; Engineering; Escherichia coli; Eukaryotic Cell; Fungal Genome; Gene Cluster; Genome; Genomics; Goals; Gram-Positive Bacteria; Harvest; Knowledge; Laboratories; Lead; Libraries; Metabolic; Metabolic Pathway; Metagenomics; Molds; Pathway interactions; Pharmaceutical Preparations; Phase; Probability; Production; Property; Regulation; Regulatory Element; Research; Research Proposals; Resistance; Resource Development; Resources; Science; Scientist; Screening procedure; Sequence Alignment; Source; System; Techniques; Technology; Therapeutic Agents; Toxic effect; Universities; Wisconsin; Work; antimicrobial; antimicrobial drug; commercialization; experience; functional genomics; fungus; genome sequencing; innovation; mutant; new technology; novel; novel therapeutics; pathogen; prevent; small molecule; success; tool; vector

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

描述（由申请人提供）：社会需要我们的防御细菌和真菌病原体的新治疗剂，其中许多病原体对现有抗生素的耐药性日益增强。丝状真菌因其产生各种次生代谢物（SM）的巨大潜力而​​被认为是开发新型生物活性化合物的有前途的资源，然而，真菌中抗生素的发现和生产远远落后于细菌。该研究提案利用包含整个 SM 途径及其调控元件的大 DNA 的穿梭克隆来推进真菌功能基因组学科学，以发现新型抗生素并确定临床开发的最佳先导候选药物。 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，拟议的研究将使用以下方法识别抗生素化合物：i）大插入无偏随机剪切穿梭BAC文库，ii）完整测序的土曲霉基因组中至少56个大型次级代谢途径（约20~100 kb），iii ) 曲霉属中全局次级代谢物簇调控的知识，iv) 工程真菌宿主构巢曲霉，为寻找新代谢物提供强大的背景。主要目标是建立两个穿梭 BAC 文库，并使用上述技术鉴定包含 56 个 SM 途径及其调控元件的 BAC，以进行概念验证，并针对细菌和真菌测试菌株筛选这些 BAC 克隆，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通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的至少 500 个 SM 途径中发现新型天然产物。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主要候选药物。主要候选药物将具有新颖的化学结构、针对细菌和/或真菌病原体的高效力以及对真核细胞的最小毒性。拟议研究所需的每项不同技术均已分别被证明是有效的；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宝贵的基因组资源，可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 公共卫生相关性：对新型抗菌药物的需求已达到自 20 世纪 40 年代抗生素商业化以来从未有过的强度，但许多传统上卓有成效的化学来源已不再产生新化合物。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，以实现从真菌中发现治疗剂。工程真菌宿主构巢曲霉将用于直接收获和表达真菌次级代谢途径及其调节元件，而无需在实验室中培养和改造不同的真菌。这项技术将允许获得由多种丝状真菌产生的各种新型小分子，其中许多目前科学界尚不清楚。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型治疗化合物。 1