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

 描述(由申请人提供)：在我们对细菌和真菌病原体的防御方面，社会上需要新的治疗剂，其中许多(超级细菌)对现有抗生素的抗药性越来越强。丝状真菌因其产生多种次生代谢物(SM)的巨大潜力而被认为是开发新型生物活性物质的潜在资源，但在真菌中天然产物(NP)的发现和生产远远落后于植物和细菌。这项研究计划推动真菌功能基因组学科学的发展，利用新开发的真菌人工染色体(FACS)，在丝状真菌中为完整的SM基因簇建立一个强大的异源表达系统。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。完整基因组公司、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个方面的新技术创新和基因组工具，以在真菌中发现NP。具体地说，拟议的研究将利用以下方面来鉴定NP化合物：i)无偏大插入随机剪切穿梭BAC文库作为FAC，ii)在6个真菌菌株的全序列基因组中超过200个大的完整SM基因簇(约20~100kb)，iii)调控元件的知识-SM基因簇在曲霉中的高异源表达的强启动子，iv)成功的工程真菌宿主：A.nidulans为寻找新的代谢物提供强有力的背景，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是：1)在优化的弧菌菌株中开发简单的FAC转化方法；2)激活5个沉默和或隐蔽的SM基因簇(FAC)中的至少2个，用于概念验证，利用上述技术发现新的NP化合物。我们的长期目标是开发一个高吞吐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从至少1000个完整的真菌SM途径中发现新的天然产物。此外，我们将对已识别的抗菌剂进行表征，以确定临床开发的最佳候选药物。候选铅将具有新颖的化学结构，对细菌和/或真菌病原体具有高效力，对真核细胞的毒性最小。这些新技术创新的结合具有很高的成功几率，也代表着天然产品发现科学的重大进步。此外，这项研究产生的1000个新的SM簇及其代谢物是一个有价值的资源，可以在后续研究中筛选出其他生物活性化合物(例如，具有抗癌或抗病毒活性)。

 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.