Molecular Genetic Mining of the Aspergillus Nidulans Secondary Metabolome

次级代谢产物是医学上有用的化合物的非常丰富的来源。因此， 对鉴定新的次生代谢产物和开发提高次生代谢产物的方法具有重要价值 生产在真菌中，特定次级代谢物生物合成途径的基因通常是 聚集在一起，并对几种丝状真菌曲霉菌的基因组进行测序 已经揭示了有许多次生代谢基因簇（曲霉属中大约有46个 nidulans单独，一个更大的数目比之前已经预测的测序的 基因组）。这些途径中的绝大多数的产物是未知的，并且A.因此，nidulans是一种 潜在的有用的次级代谢产物的丰富来源，以及一个很好的模式生物， 开发挖掘真菌次级代谢物的程序。该提案利用了 基因打靶在A. nidulans创造一套分子遗传工具和菌株， 有效地利用了A。构巢菌次级代谢组。我们的第一个具体目标是创造一个 一组转化体，将允许所有A.拟将nidulans次级代谢基因簇 鉴定我们将采取两种办法。大约15个未探索的基因簇含有转录 预测控制簇中其它基因表达的因子基因。我们将取代 这些转录因子基因的启动子与诱导型启动子。这将允许过表达的 靶簇的基因和增加簇产物的合成。这将反过来使我们的 合作者，Clay Wang博士，鉴定和纯化特定簇的产物。我们的第二个目标是 删除选定的次级代谢途径的基因， 澄清和分流产品隔离。在某些情况下，分流产品可能与 而不是正常的代谢产物。我们的第三个目标是开发启动子替代菌株， 将允许增加次级代谢途径的表达。这将是特别重要的， 不包含转录因子基因的基因簇。这一目标的一部分将是开发一个系列 启动子替换程序，这将允许靶途径中所有基因的启动子被 更换为了实现所有这些目标，我们将与我们的合作者王博士和南希·凯勒博士密切合作。

Secondary metabolites are a remarkably rich source of medically useful compounds. It is, consequently, of great value to identify new secondary metabolites and to develop methods to increase secondary metabolite production. In fungi, the genes of particular secondary metabolite biosynthetic pathways are usually clustered together, and sequencing of the genomes of several species of the filamentous fungus Aspergillus has revealed that there are many secondary metabolism gene clusters (approximately 46 in Aspergillus nidulans alone, a much larger number than would have been predicted prior to the sequencing of the genome). The products of the great majority of these pathways are unknown and A. nidulans is, thus, a potentially rich source of useful secondary metabolites as well as an excellent model organism for developing procedures to mine the secondary metabolomes of fungi. This proposal takes advantage of recent progress in gene targeting in A. nidulans to create a set of molecular genetic tools and strains that will allow the efficient exploitation of the A. nidulans secondary metabolome. Our first specific aim is to create a set of transformants that will allow the products of all A. nidulans secondary metabolism gene clusters to be identified. We will pursue two approaches. Approximately 15 unexplored gene clusters contain transcription factor genes that are predicted to control expression of the other genes in the cluster. We will replace the promoters of these transcription factor genes with inducible promoters. This will allow overexpression of the genes of target clusters and increased synthesis of the product(s) of the cluster. This will, in turn, allow our collaborator, Dr. Clay Wang, to identify and purify the products of particular clusters. Our second aim will be to delete genes of selected secondary metabolism pathways to allow the steps of the pathways to be clarified and shunt products isolated. In some cases, the shunt products may be as useful as, or more useful than, the normal products of the pathway. Our third aim will be to develop promoter replacement strains that will allow increased expression of secondary metabolism pathways. This will be particularly important in gene clusters that do not contain transcription factor genes. A portion of this aim will be to develop a serial promoter replacement procedure that will allow the promoters of all the genes in target pathways to be replaced. For all of these aims we will work closely with our collaborators, Dr. Wang and Dr. Nancy Keller.