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）：在我们对针对细菌和真菌病原体的防御措施中，有社会需要对新的治疗剂进行社会需求，其中许多人对现有抗生素具有抗药性。丝状真菌被认为是开发新型生物活性化合物的有希望的资源，因为它们具有生产各种二级代谢产物（SM）的巨大潜力，但是，真菌的抗生素发现和生产远远落后于细菌。这项研究建议使用包含整个SM途径及其调节元素的大型DNA的穿梭克隆来提高真菌功能基因组学的科学，以发现新型抗生素并确定最佳的临床发育层次候选者。露西根公司（Lucigen Corporation）和威斯康星大学麦迪逊（University of Madison）的科学家将开发，利用并结合新技术创新和基因组工具的四个方面，以使真菌的治疗剂发现能够发现。具体而言，拟议的研究将使用以下方式鉴定抗生素化合物：i）在完全测序的A. terreus的完全测序的基因组中，至少56个大型次级代谢途径（约20〜100 kb），至少56个大型次生代谢途径（约20〜100 kb），iii，iii），对全球次级代谢群体的自动构成的努力，Asered unig univ and univers and univer univ and unive niv），iv）IV）在其中寻找新的代谢产物。主要目标是构建两个穿梭BAC文库，并鉴定包含56个SM途径的BAC及其调节元素，用于使用上述技术进行概念验证，并将这些BAC克隆筛选为细菌和真菌测试剂菌株，以发现新型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的小分子发现平台，以从完全测序的真菌基因组中发现至少500个SM途径的新型天然产物。此外，我们将表征已鉴定的抗菌剂，以确定临床发育的最佳铅候选者。铅候选者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的技术都已被证明是分别有效的。因此，这些不同技术的组合具有很高的成功可能性，并且代表了抗生素发现科学的重大进步。此外，这项研究产生的库是一种有价值的基因组资源，可以在随后的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：自1940年代抗生素商业化以来，对新的抗菌剂的需求已经达到了强度，但许多传统上富有成果的化学来源已经停止产生新的化合物。拟议的研究将开发，利用和结合四种技术创新和基因组工具，以从真菌中发现治疗剂。工程的真菌宿主Aspergillus nidulans将直接用于收获和表达真菌二级代谢途径及其调节元素，而无需在实验室中培养和设计不同的真菌。这项技术将允许通过大量丝状真菌产生的各种新型小分子，其中许多分子目​​前是科学不明的。这项工作的最终目标是鉴定用于治疗细菌和真菌疾病的新型治疗化合物。 1