A robust heterologous expression system of intact fungal secondary metabolite gene clusters for natural product discovery in Aspergillus nidulans

完整真菌次生代谢物基因簇的强大异源表达系统，用于构巢曲霉天然产物的发现

• 批准号: 9120977
• 负责人: Chengcang Charles Wu
• 金额: $ 22.49万
• 依托单位: INTACT GENOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9120977
• 关键词: Antibiotics; Antiviral Agents; Artificial Chromosomes; Aspergillus; Aspergillus nidulans; Bacteria; Bacterial Artificial Chromosomes; Cells; Chemical Structure; Chemistry; Chromosomes, Human, 1-3; Clinical; Core Facility; DNA; Development; Disease; Electroporation; Elements; Engineering; Eukaryotic Cell; Freezing; Fungal DNA; Fungal Genome; Gene Cluster; Gene Expression; Genetic Transcription; Genomics; Goals; In Vitro; Knowledge; Laboratories; Lead; Libraries; Metabolic; Metabolic Pathway; Methods; Molds; Natural Products; Pathway interactions; Pharmaceutical Preparations; Phase; Plants; Probability; Production; Proteomics; Protoplasts; Regulatory Element; Research; Research Proposals; Resistance; Resource Development; Resources; Science; Scientist; Small Business Innovation Research Grant; Source; Spheroplasts; Structure; Superbug; System; Techniques; Technology; Therapeutic Agents; Toxic effect; Transcription Coactivator; Universities; Wisconsin; antimicrobial drug; cell transformation; commercialization; experience; functional genomics; fungus; genome sequencing; genomic tools; innovation; new technology; novel; novel therapeutics; pathogen; prevent; promoter; public health relevance; research study; small molecule; success; technological innovation; tool; transcription factor

 DESCRIPTION (provided by applicant): There is societal need for new therapeutic agents in our arsenal of defenses against bacterial and fungal pathogens, many (superbugs) 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, natural product (NP) discovery and production in fungi lags far behind plants and bacteria. This research proposal advances sciences of fungal functional genomics to develop a robust heterologous expression system for intact SM gene clusters in the filamentous fungus Aspergillus nidulans by using the newly developed fungal artificial chromosomes (FACs). Our purpose is to discover novel antibiotics and identify the best lead candidates for clinical development. Scientists at Intact Genomics Inc, University of Wisconsin at Madison, Donald Danforth Plant Science Center, and Northwestern University will develop, utilize, and combine at least 6 aspects of novel technology innovation and genomic tools to enable NP discovery in fungi. Specifically, the proposed research will identify NP compounds using: i) the unbiased large-insert Random Shear Shuttle BAC libraries as FACs, ii) more than 200 large intact SM gene clusters (about 20~100 kb) in the completely sequenced genome of 6 fungal strains, iii) the knowledge of regulatory elements-strong promotors for high heterologous expression of SM gene clusters in Aspergillus, iv) the successfully engineered fungal host: A. nidulans to provide a robust background in which to search for new metabolites, v) in vitro BAC/FAC engineering, vi) advanced LC-MS analysis. The primary objectives of Phase I research are 1) to develop simple FAC transformation method in an optimized A. nidulans strain, 2) to activate at least 2 of 5 silent and or cryptic SM gene clusters (FACs) for proof-of-concept using the above technologies to discover novel NP compounds. 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 1,000 fungal intact 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. The combination of these novel technological innovations has a high probability of success and also represents a significant advancement for the science of natural product discovery. In addition, the 1,000 novel SM clusters and their metabolites produced from this research are a valuable resource that may be screened for other bioactive compounds (e.g., with anticancer or antiviral activities) in subsequent research.

 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，社会需要新的治疗剂，其中许多（超级细菌）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物质的重要资源，具有产生多种次生代谢产物的巨大潜力，但真菌中天然产物的发现和生产远远落后于植物和细菌。本研究计划通过使用新开发的真菌人工染色体（FACs），推进真菌功能基因组学的科学，以开发一个强大的丝状真菌构巢曲霉中完整SM基因簇的异源表达系统。我们的目的是发现新的抗生素，并确定临床开发的最佳候选药物。Intact Genomics Inc、威斯康星州麦迪逊大学、Donald Danforth植物科学中心和西北大学的科学家将开发、利用和联合收割机至少6个方面的新技术创新和基因组工具，以实现真菌中的NP发现。具体而言，拟议的研究将使用以下方法识别NP化合物：i）作为FACs的无偏大插入随机剪切穿梭BAC文库，ii）超过200个大的完整SM基因簇，（约20~100 kb）的序列，iii）对曲霉属SM基因簇高效异源表达的调控元件-强启动子的了解，iv）成功工程化的真菌宿主：A. nidulans提供了一个强大的背景，在其中寻找新的代谢物，v）在体外BAC/FAC工程，vi）先进的LC-MS分析。第一阶段研究的主要目标是：1）在优化的A. 2）激活5个沉默和/或隐蔽SM基因簇（FACs）中的至少2个，用于使用上述技术发现新NP化合物的概念验证。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至少1,000个真菌完整的SM途径中发现新的天然产物。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。这些新技术创新的结合具有很高的成功概率，也代表了天然产物发现科学的重大进步。此外，从这项研究中产生的1,000种新型SM簇及其代谢物是一种有价值的资源，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）。