Secondary Metabolite Niches: Whole Genome Functional Analysis of Non-Ribosomal Peptide Synthetases

该项目的目标是对Cochliobolus Heterostrophus非核糖体多肽合成酶(NPS)基因及其编码的酶产生的小分子多肽进行详尽的表征，以确定在真菌细胞中(和为其)起作用的是什么多肽次生代谢产物。非核糖体肽合成酶(NRPS)是一种多模块的酶，它通过一种独立于蛋白质合成的细胞器核糖体的机制来合成小的非核糖体肽(NRP)。到目前为止，NRPS的多肽生物合成方法已经被描述用于丝状子囊菌真菌(在有限的程度上，用于担子菌真菌)和仅用于细菌。可能的NRP产品的多样性可能是无限的，因为NRP可以由D-和L-氨基酸、蛋白质和非蛋白质氨基酸、羟基、鸟氨酸和其他不寻常的成分组成。此外，NRP可以是线性的、环状的或支链的环状结构，并且可以通过糖基化、N-甲基化或酰化来修饰。除了结构多样性，NRPS还具有令人惊讶的广泛的生物活动范围。然而，尽管多肽产品与其他生物体相互作用的活性是有很好的记录的，而且肯定是了不起的，但认为这是它们的主要功能的说法可能是不正确的。例如，在寄主玉米上致病只需要12个异种线虫NPS基因中的一个。事实上，这些小肽对产生真菌的生理意义在很大程度上是未知的。这个项目的基础是假设NRPS酶是基础新陈代谢和特殊环境利基的小分子供给者。该项目的目标是创建一个异种梭菌NPS缺失菌株的文库，用于解决小分子、非核糖体肽在真菌细胞生物学中所起的作用。具体地说，实验的目的是：a.创造多个NPS基因缺失菌株。B.创造每一种NPS基因都过度表达的菌株。C.进行一系列表型分析以探索其功能。D.确定表达NPS基因的条件。E.对野生型和选定的具有有趣表型的突变菌株进行化学分析，以确定NRP结构。这项研究将允许开发一个受欢迎的本科生生物学课程的可选研究模块。这一外展合作项目将通过交织和传播对地球上第二大生物群体真菌的兴奋来展示并帮助学生进入科学管道。有兴趣的学生将被邀请参加一到两个课程，在那里他们将被介绍给NRPS的概念。然后，学生将被指派协助并最终负责表型分析。学生还将被要求使用当代分子遗传学/基因组学工具来回答有关重要真菌代谢物的起源和功能的问题，包括它们在农业中的应用及其社会意义。

The goal of this project is to undertake an exhaustive characterization of the set of Cochliobolus heterostrophus non-ribosomal peptide synthetase (NPS) genes, and the small molecule peptides produced by the enzymes they encode, to ascertain what peptide secondary metabolite products are doing in (and for) the fungal cell. Non-ribosomal peptide synthetases (NRPSs) are multimodular enzymes that make small non-ribosomal peptides (NRPs) through a mechanism, independent of organelles best known for protein synthesis, the ribosomes. To date, the NRPS method of peptide biosynthesis has been described for filamentous ascomycete fungi (and to a limited extent, for basidiomycete fungi) and for bacteria, only. The diversity of possible NRP products is potentially limitless as NRPs can be composed of D- and L-amino acids, protein and non-protein amino acids, hydroxy acids, ornithine, and other unusual constituents. Furthermore, NRPs can be linear, cyclic, or branched cyclic, and may be modified by glycosylation, N-methylation or acylation. In addition to structural diversity, NRPs have an astonishingly broad spectrum of biological activities. However, despite the fact that activities of the peptide products, with respect to interactions with other organisms, are well documented, and certainly remarkable, to suggest that this is their primary function is likely incorrect. For example, only one of the 12 C. heterostrophus NPS genes is required for virulence on the host, maize. In fact, the physiological significance of these small peptides to the producing fungi is largely unknown. The foundation of this project is the hypothesis that NRPS enzymes are purveyors of small molecules for both basal metabolism and for specialized environmental niches. The goals of this project are to create a library of C. heterostrophus NPS- deletion strains that will be used to resolve roles that small molecule, non-ribosomal peptides play in the biology of the fungal cell. Specifically, experiments are designed to: A. Create multiple NPS gene-deletion strains. B. Create strains in which each NPS gene is overexpressed. C. Perform a series of phenotypic assays to explore function. D. Determine conditions under which NPS genes are expressed. E. Perform chemical analyses on wild-type and selected mutant strains with interesting phenotypes, to identify NRP structure. This research will allow the development of an optional research module for a popular undergraduate biology course. This outreach collaboration program will expose and help move students into the science pipeline by interweaving and disseminating excitement about the second largest group of organisms on earth, the fungi. Interested students will be invited to one or two sessions where they will be introduced to the concept of NRPSs. Then, students will be assigned to assist with and eventually take charge of phenotypic assays. Students will also be challenged to use contemporary molecular genetics/genomics tools to answer questions about the genesis and function of important fungal metabolites including their applications in agriculture and their social implications.