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）：社会需要针对细菌和真菌病原体的新治疗剂，其中许多（超级细菌）对现有抗生素的耐药性越来越强，丝状真菌被认为是开发新型抗生素的有希望的资源。生物活性化合物因其产生各种次生代谢物（SM）的巨大潜力，然而，真菌中天然产物（NP）的发现和生产远远落后于植物和细菌。真菌功能基因组学科学，通过使用新开发的真菌人工染色体（FAC），为丝状真菌构巢曲霉中的完整 SM 基因簇开发强大的异源表达系统。我们的目的是发现新型抗生素并确定临床的最佳主要候选药物。 Intact Genomics Inc、威斯康星大学麦迪逊分校、唐纳德丹福斯植物科学中心和西北大学的科学家将开发、利用和结合至少 6 个方面的新颖性。具体来说，拟议的研究将使用以下方法识别 NP 化合物：i) 作为 FAC 的无偏大插入随机剪切穿梭 BAC 文库，ii) 超过 200 个大型完整 SM 基因簇（约 100 个）。 20~100 kb) 6个真菌菌株的完整测序基因组，iii) SM基因簇高异源表达的调控元件-强启动子的知识曲霉菌，iv) 成功改造的真菌宿主：构巢曲霉，为寻找新的代谢物提供了强大的背景，v) 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。 1) 在优化的构巢曲霉菌株中开发简单的 FAC 转化方法，2) 激活 5 个沉默和/或隐性 SM 基因簇 (FAC) 中的至少 2 个使用上述技术发现新化合物的概念验证我们的长期目标是开发一个真菌中的高通量小分子发现平台，以便从至少 1,000 个真菌完整的 SM 途径中发现新的天然产物。此外，我们还将对已鉴定的抗菌剂进行表征，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、对细菌和/或真菌病原体的高效力以及对真核生物的最小毒性。这些新颖的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，本研究产生的 1,000 个新颖的 SM 簇及其代谢物是一种宝贵的资源。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）。