Comprehensive analysis of NRPS-derived metabolomes of three Aspergillus species

三种曲霉属 NRPS 衍生代谢组的综合分析

• 批准号: 8986191
• 负责人: NANCY P KELLER
• 金额: $ 27.94万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-15 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8986191
• 关键词: Agriculture; Anabolism; Antibiotics; Aspergillus; Aspergillus fumigatus; Aspergillus nidulans; Bacterial Artificial Chromosomes; Biological; Biological Models; Biology; Chemicals; Communities; Complement; Complex; Coupled; Drug Design; Engineering; Evaluation; Fungal Genome; Future; Gene Cluster; Genes; Genetic; Genetic Models; Genetic Recombination; Genome; Goals; Grant; Immunosuppressive Agents; Knowledge; Laboratories; Lipids; Methodology; Modeling; Molds; NMR Spectroscopy; Orphan; Pathway interactions; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Play; Production; Property; Research; Resolution; Resources; Role; Source; Sterigmatocystin; Structure; Substrate Domain; Surveys; Techniques; Virulence Factors; Work; Yeasts; antimicrobial; base; comparative; drug discovery; fungus; genome sequencing; innovation; interest; member; metabolome; metabolomics; overexpression; pathogen; peptide synthase; preference; promoter; public health relevance; small molecule; tool; vector

DESCRIPTION (provided by applicant): Filamentous fungi produce a vast universe of secondary metabolites (SM), which display a broad range of useful activities for pharmaceutical and agricultural purposes, e.g. antibiotic, immunosuppressant, lipid lowering, or antimicrobial properties. One group of metabolites of particular interest are non-ribosomal peptide synthetase- (NRPS-) derived metabolites which have provided important leads for drug design. However, production of NRPS-based pathways is often very low (or undetectable) under laboratory conditions, in large part due to complex endogenous regulatory networks that control the biosyntheses of these metabolites, which remain, at best, poorly understood. As a result, many gene clusters containing NRPS or NRPS-like genes have no known metabolites ("orphan gene clusters"), even in well-studied species such as the genetic model Aspergillus nidulans. This R01 proposes in Aim 1 to identify metabolites associated with all remaining orphan NRPS and NRPS-like gene clusters in A. nidulans and the opportunistic pathogen A. fumigatus using an inducible promoter activation strategy coupled with comparative metabolomics based on high-resolution MS and 2D NMR spectroscopy. In Aim 2 we will utilize a Bacterial Artificial Chromosome (BAC)-based strategy to identify metabolites for all NRPS and NRPS-like gene clusters of A. terreus, an industrially important fungal species. A final goal of this aim is to examine the hypothesis that specific genes contained within a fungal NRPS/NRPS-like cluster may be predictive of the biological activity - and drug discovery potential - of the SM products. Overall, this parallel study of different species and comparison of different expression approaches will significantly advance understanding of NRPS-pathways and the scope of heterologous expression. Moreover, this work will provide advanced fungal lines for the community for SM cluster expression, a comprehensive assessment of A-domain substrate preference for fungal NRPS and an innovative methodology to identify new metabolites for biological evaluation.

描述（由申请人提供）：丝状真菌产生大量次生代谢物（SM），这些代谢物在制药和农业领域具有广泛的有用活性，例如抗生素、免疫抑制剂、降脂或抗菌特性。一组特别感兴趣的代谢物是非核糖体肽合成酶（NRPS）衍生的代谢物，它们为药物设计提供了重要的线索。然而，在实验室条件下，基于nrps的途径的产生通常非常低（或无法检测到），这在很大程度上是由于控制这些代谢物生物合成的复杂内源性调节网络，而这些调节网络充其量也只是知之甚少。因此，许多含有NRPS或类似NRPS基因的基因簇没有已知的代谢物（“孤儿基因簇”），即使是在遗传模式的细粒曲霉等研究充分的物种中也是如此。该R01在Aim 1中提出，利用诱导启动子激活策略结合基于高分辨率质谱和二维核磁共振光谱的比较代谢组学，鉴定与A. nidulans和条件致病菌A. fumigatus中所有剩余孤儿NRPS和NRPS样基因簇相关的代谢物。在目标2中，我们将利用基于细菌人工染色体（BAC）的策略来鉴定a . terreus（一种重要的工业真菌物种）所有NRPS和NRPS样基因簇的代谢物。这一目的的最终目标是检验一种假设，即真菌NRPS/NRPS样簇中包含的特定基因可能预测SM产品的生物活性和药物发现潜力。总之，这种不同物种的平行研究和不同表达方式的比较将显著促进对nrps通路和异源表达范围的理解。此外，这项工作将为真菌界提供SM簇表达的高级真菌系，全面评估真菌NRPS的a结构域底物偏好，以及鉴定新的代谢物用于生物学评价的创新方法。