Molecule-to-gene approaches to new natural products

描述(由申请人提供)：本申请回应了RFA对新天然产品和新天然产品本身的研究，这将增加美国国立卫生研究院分子图书馆路线图和其他筛选倡议中与生物相关的分子多样性。两种方法形成了应用程序的两个具体目标。两者都强调从细菌来源发现新的天然产品的基因起点。特定目标1侧重于伽玛变形杆菌，而特定目标2侧重于放线菌。细菌来源之所以受到重视，是因为它们过去的贡献，最近的研究表明，它们的潜力只被很少地探索过，它们的基因可获得性，以及它们被培养和提供筛查计划所需的数百万克材料的能力。这两个目标不仅使用非常不同的细菌群体，他们还使用不同的起点，未确定特征的基因组DNA和基因组序列，以发现具有生物活性的小分子。具体目标1.通过基因到分子的方法发现具有生物活性的小分子。第一种方法利用Photorhabdus spp基因组DNA中的粘粒文库。和致病杆菌，在代谢相容的宿主大肠杆菌中异源表达，以及作为许多其他活性的替代试验的抗生素活性的功能测定。这两个属都可能产生大量的抗生素分子，它们与异源宿主大肠杆菌的密切关系有助于这种DNA文库方法中小分子的高水平表达。具体目标2.开发基于序列的方法，从20个新的放线菌基因组中发现天然产物。特定目标1的主要基于功能的方法将得到基于序列的方法的补充，该方法利用正在进行的努力来对特定目标2中的20个放线菌菌株的二级生物合成途径进行排序和注释。该目标使用“基因敲除扫描”来识别与预测的基因簇相关的小分子。对于对遗传操作不敏感的菌株，将使用在适当宿主中的异源表达。 与公众健康相关：高通量筛选(HTS)，即在生物检测中测试大量小分子，是发现新药的主要工具，而小分子文库的质量在很大程度上决定了HTS方法的成功。天然产物是许多活着的有机体产生的小分子，含有真正非凡的分子多样性，在发现新药方面有着悠久的成功历史。但尽管有这段历史，它们在当今的HTS世界中只扮演着微不足道的角色。该项目描述了两种方法，这两种方法可以最大限度地减少传统天然产物发现技术的一些负担，这些技术可能会为HTS带来重要的小分子流。

DESCRIPTION (provided by applicant): This application responds to an RFA for approaches to new natural products and the new natural products themselves that would increase the biologically relevant molecular diversity in the NIH Molecular Libraries Roadmap and other screening initiatives. Two approaches form the application's two Specific Aims. Both emphasize a genetic starting point for discovering new natural products from bacterial sources. Specific Aim 1 focuses on Gammaproteobacteria, and Specific Aim 2 focuses on actinomycetes. Bacterial sources are emphasized because of their past contributions, recent studies that show their potential has been only poorly explored, their genetic accessibility, and their ability to be cultured and provide the multimilligram amounts of material needed for screening programs. The two aims not only use very different groups of bacteria, they also use different starting points, uncharacterized genomic DNA and genome sequences, to discover bioactive small molecules. Specific Aim 1. Discover biologically active small molecules through gene-to- molecule approaches. The first approach utilizes cosmid libraries from genomic DNA of Photorhabdus spp. and Xenorhabdus spp., heterologous expression in the metabolically compatible host Escherichia coli, and a functional assay for antibiotic activity, which serves as a surrogate assay for many other activities. Both genera, Photorhabdus and Xenorhabdus, are likely to produce large numbers of antibiotic molecules, and their close relationship to the heterologous host, E. coli, facilitates high levels of expression of the small molecules in this DNA library approach. Specific Aim 2. Develop sequence-based approaches to discovering natural products from 20 new actinomycete genomes. The largely function-based approach of Specific Aim 1 will be complemented by a sequence-based approach that leverages ongoing efforts to sequence and annotate the secondary biosynthetic pathways of 20 actinomycete strains in Specific Aim 2. This aim uses `knockout scanning' to identify small molecules associated with predicted gene clusters. For strains refractory to genetic manipulation, heterologous expression in appropriate hosts will be used. PUBLIC HEALTH RELEVANCE: High-throughput screening (HTS), the testing of large numbers of small molecules in biological assays, is the major tool for discovering new drugs, and the quality of the small molecule libraries largely determines the success of the HTS approach. Natural products, the small molecules produced by many living organisms, contain truly remarkable levels of molecular diversity and have a long history of success in discovering new drugs. But in spite of this history, they play only a minimal role in today's HTS world. This project describes two approaches, which minimize some of the liabilities of traditional natural products discovery techniques that could lead to an important small molecule stream for HTS.