Mining the aspergillus nidulans secondary metabolome

描述(由申请人提供)：这是一份项目资助(5P01GM084077)的竞争性修订申请，题为“挖掘Nidulans曲霉次级代谢组”，它是针对通知编号NOT-OD-09-058，通知标题：NIH宣布恢复法案资金可用于竞争性修订申请而写的。该计划项目资助的目标是利用基因组学、分子遗传学和天然产品化学的协同组合来挖掘尼杜拉曲霉次级代谢组，以获得有用的天然产品。 尽管这项资助已经生效不到一年，但我们正在取得很好的进展，并希望将这种方法扩展到数百种其他真菌，这些真菌可能会产生医学上有用的次生代谢物，但缺乏为根瘤拟青霉开发的优秀遗传和分子遗传学工具。由于开发这些真菌的分子遗传系统可能是困难和非常耗时的，所以我们希望将根瘤拟青霉作为一种异源表达其他真菌次生代谢物基因簇的系统。在要求的两年支持期内，我们建议通过删除根瘤拟弧菌次级代谢物基因簇来改善其作为异源表达次生代谢物的宿主生物。 这在正常生长条件下表现为高水平。这可能会产生更多的目标次生代谢物，当然也会使目标次生代谢物更容易被质谱学检测到。然后，我们将开发两个基于粘粒的系统，用于表达来自其他真菌的基因簇。我们将使用土曲霉的基因簇来开发这个系统，土曲霉是一种经济和医学上都很重要的真菌，具有测序的基因组，但使用的分子遗传学工具远远不如尼杜拉曲霉。我们将开发一种将土曲霉的基因簇整合到多粒曲霉基因组中的系统，以及一种在自主复制的粘粒载体上维护土曲霉基因簇的系统。我们将通过液相色谱/质谱学分析次级代谢物的产生，并开发程序，使我们能够获得足够数量的次级代谢物的产生，用于纯化和结构分析。 公共卫生相关性：真菌次生代谢物是医学上有价值的化合物的丰富来源。尼杜拉曲霉的分子遗传学方法为鉴定新的次生代谢物和推断产生它们的生物合成途径的步骤提供了一个非常有效的系统。这项应用将使我们能够将同样的方法应用于其他缺乏可用于根结线虫的分子工具的真菌。

DESCRIPTION (provided by applicant): This is an application for a competitive revision of Program Project grant (5P01GM084077) entitled "Mining the Aspergillus nidulans Secondary Metabolome," It is written in response to Notice Number NOT-OD-09-058, Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The goal of the Program Project grant as funded is to use a synergistic combination of genomics, molecular genetics and natural products chemistry to mine the Aspergillus nidulans secondary metabolome for useful natural products. Although the grant has been in effect for less than one year, we are making excellent progress and wish to extend this approach to the hundreds of other fungi which likely make medically useful secondary metabolites, but lack the excellent genetic and molecular genetics tools that have been developed for A. nidulans. Since developing molecular genetic systems for these fungi is likely to be difficult and extremely time consuming, we wish, instead, to develop A. nidulans as a system for heterologous expression of secondary metabolite gene clusters from other fungi. In the requested two year period of support, we propose to improve A. nidulans as a host organism for heterologous expression of secondary metabolites by deleting A. nidulans secondary metabolite gene clusters that is expressed at high levels under normal growth conditions. This may allow greater production of target secondary metabolites, and should certainly allow target secondary metabolites to be detected more easily by mass spectroscopy. We will then develop two cosmid-based systems for expression of gene clusters from other fungi. We will develop the system using gene clusters of Aspergillus terreus, an economically and medically important fungus with a sequenced genome, but with a much less well developed set of molecular genetics tools than A. nidulans. We will develop a system for integrating gene clusters from A. terreus into the A. nidulans genome and a system for maintenance of A. terreus gene clusters on autonomously replicating cosmid vectors. We will analyze production of secondary metabolites by liquid chromatography/mass spectroscopy and develop procedures that allow us to obtain production of adequate quantities of secondary metabolites for purification and structural analysis. PUBLIC HEALTH RELEVANCE: Fungal secondary metabolites are a rich source of medically valuable compounds. Molecular genetics approaches in Aspergillus nidulans are providing a very efficient system for identifying new secondary metabolites and deducing the steps of the biosynthetic pathways that produce them. This application will allow us to apply the same approach to other fungi that lack the molecular tools available for A. nidulans.