Comprehensive analysis of NRPS-derived metabolomes of three Aspergillus species

描述（由申请人提供）：丝状真菌产生大量次级代谢产物（SM），其显示出广泛的用于制药和农业目的的有用活性，例如抗生素、免疫抑制剂、降脂或抗微生物特性。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，其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（或不可检测），这在很大程度上是由于控制这些代谢物的生物合成的复杂内源性调控网络，其充其量仍然知之甚少。因此，许多含有NRPS或NRPS样基因的基因簇没有已知的代谢物（“孤儿基因簇”），即使在充分研究的物种如遗传模型构巢曲霉中也是如此。该R 01在目的1中提出鉴定与A中所有剩余孤儿NRPS和NRPS样基因簇相关的代谢物。nidulans和条件致病菌A.使用诱导型启动子激活策略，结合基于高分辨率MS和2D NMR光谱的比较代谢组学，对烟曲霉进行了研究。在目标2中，我们将利用细菌人工染色体（BAC）为基础的战略，以确定代谢产物的所有NRPS和NRPS样基因簇的A。terreus，一种工业上重要的真菌物种。这一目标的最终目标是检查的假设，即包含在真菌NRPS/NRPS样簇的特定基因可能是预测的生物活性-和药物发现潜力-的SM产品。总体而言，这种不同物种的平行研究和不同表达方法的比较将显着推进NRPS途径的理解和异源表达的范围。此外，这项工作将为SM簇表达的社区提供先进的真菌株系，全面评估真菌NRPS的A结构域底物偏好，并提供一种创新的方法来鉴定新的代谢产物进行生物学评价。

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.