Mapping the Secondary Metabolomes of Marine Cyanobacteria

描述（由申请人提供）：从天然来源中发现药物先导化合物的关键是有效获取和表征高度多样化的分子结构。在这方面，对具有生物活性的天然产物的海洋环境的探索正在揭示天然产物化学多样性的新前景。在本应用中，我们建议开发创新技术和知识，主要基于LC-MS/MS数据和“分子定位”，这将提高天然产物药物发现工作的有效性。这将大大提高在期望的结构类别中发现新的分子多样性或类似物的能力。我们将对培养菌株中天然产物途径的表达程度有所了解，并将开发新的方法来上调低表达或不表达的生物合成基因簇。作为这些研究的结果，新的海洋蓝藻天然产物将被发现，其生物医学特性将被表征。为了实现这些目标，我们有以下四个具体目标：1)使用LC-MS/MS分析蓝藻提取物和纯化合物，然后进行分子定位，以创建我们样品的化学宇宙的表示。2)利用QPCR和基因组测序技术评价我国培养的海洋蓝藻中天然产物通路的表达程度，并将天然产物超产菌株与其基因型联系起来。这后一种信息可以用来寻找遗传标记，可以迅速部署，以定位这种表型在新的蓝藻培养和收集。3)利用一套富有想象力的方法转录激活菌株中隐藏的天然产物生物合成基因簇

DESCRIPTION (provided by applicant): Key to the process of pharmaceutical lead compound discovery from natural sources is the effective access and characterization of highly diverse molecular structures. In this regard, exploration of the marine environment for bioactive natural products is revealing new vistas in natural products chemical diversity. In this application we propose the development of innovative technologies and knowledge, principally based on LC-MS/MS data and 'molecular mapping', which will improve the effectiveness of natural products drug discovery efforts. This will enable a much improved capacity to discover new molecular diversity or analogs in desired structure classes. We will develop an understanding of the degree of expression of natural product pathways in cultured strains, and will develop novel methods by which to upregulate low or non-expressing biosynthetic gene clusters. As a result of these studies, new marine cyanobacterial natural products will be discovered and their biomedical properties will be characterized. To accomplish these goals we have the following four specific aims: 1) To use LC-MS/MS profiling of cyanobacterial extracts and pure compounds, followed by molecular mapping, to create a representation of the chemical universe of our samples. 2) To use QPCR and genome sequencing technologies to evaluate the degree of expression of natural product pathways in our cultured marine cyanobacteria, and to connect Natural Product Super-producing strains of cyanobacteria with their genotypes. This latter information can be used to find genetic markers that can be rapidly deployed to locate this phenotype in new cyanobacterial cultures and collections. 3) To use a suite of imaginative methods to transcriptionally activate cryptic natural product biosynthetic gene clusters in strains determined in Aim 2 to possess un-expressed natural products capacity, and to analyze the resulting elicited secondary metabolomes by mass spectrometry and molecular mapping. 4) To isolate members of new families of compounds detected in Aims 1, as well as newly expressed natural products from Aim 3, and rigorously establish molecular structures using advanced analytical methods. Through the course of these four specific aims, this collaborative group will explore a number of innovative methods and approaches in the natural products sciences, including MS/MS molecular mapping, genomic analysis of natural products expression, elicitation of new natural products expression, connection of natural product-rich phenotypes to their corresponding genotypes, imaging mass spectrometry of complex consortiums of species wherein natural product pathways are activated, and novel automated MS approaches to natural products characterization. All of these methods are focused on improving the detection and characterization of the molecular diversity present in microorganisms, in this case, marine cyanobacteria. This molecular diversity continues to be an important source of inspirational molecules for biomedical research and drug discovery.