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）：在我们的细菌和真菌病原体防御武器库中，社会需要新的治疗药物，其中许多对现有抗生素的耐药性越来越强。丝状真菌是开发新型生物活性化合物的重要资源，可产生多种次级代谢产物，但真菌的抗生素发现和生产远远落后于细菌。这项研究计划使用穿梭克隆含有整个SM途径及其调控元件的大DNA来推进真菌功能基因组学科学，以发现新型抗生素并确定临床开发的最佳候选药物。Lucigen公司和威斯康星州麦迪逊大学的科学家将开发，利用和联合收割机的四个方面的新技术创新和基因组工具，使治疗剂发现真菌。具体而言，拟议的研究将利用：i）大插入无偏随机剪切穿梭BAC文库，ii）在完全测序的A. terreus，iii）曲霉属中全局次级代谢物簇调控的知识，iv）工程化真菌宿主A.为寻找新的代谢物提供了一个强大的背景。主要目标是构建两个穿梭BAC文库，并使用上述技术鉴定含有56个SM途径及其调控元件的BAC用于概念验证，并针对细菌和真菌测试菌株筛选这些BAC克隆以发现新的抗细菌和抗真菌特性。 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发现至少500个SM途径的新天然产物。此外，我们将对已鉴定的抗菌药物进行表征，以确定临床开发的最佳候选药物。先导候选物将具有新颖的化学结构，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。拟议研究所需的每种不同技术都已被证明是有效的;因此，这些不同技术的组合具有很高的成功概率，也代表了抗生素发现科学的重大进步。此外，从这项研究中产生的文库是一种有价值的基因组资源，可以筛选其他生物活性化合物（例如，具有抗癌或抗病毒活性）。1 公共卫生关系：对新的抗菌剂的需求已经达到了自20世纪40年代抗生素商业化以来从未经历过的强度，但许多传统上富有成效的化学来源已经停止产生新的化合物。拟议的研究将开发，利用和联合收割机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。工程化的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控元件，而不需要在实验室中培养和工程化不同的真菌。这项技术将允许获得由多种丝状真菌产生的各种各样的新型小分子，其中许多是目前科学未知的。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