Molecule-to-gene approaches to new natural products

描述（由申请人提供）：本申请对新天然产物和新天然产物本身的方法的RFA作出回应，这些方法将增加NIH分子文库路线图和其他筛选计划中生物学相关的分子多样性。两种方法构成了应用程序的两个特定目标。两者都强调从细菌来源发现新的天然产物的遗传起点。特异性目标1侧重于γ变形菌，特异性目标2侧重于放线菌。强调细菌来源是因为它们过去的贡献，最近的研究表明它们的潜力尚未得到充分的探索，它们的遗传可及性，以及它们的培养能力，并提供筛选程序所需的多毫克量的物质。这两个目标不仅使用了非常不同的细菌群，而且还使用了不同的起点，即未确定的基因组DNA和基因组序列，以发现具有生物活性的小分子。具体目标通过基因到分子的方法发现生物活性小分子。第一种方法利用photohabdus spp.和Xenorhabdus spp.基因组DNA中的cosmid文库，在代谢相容的宿主大肠杆菌中进行异源表达，并进行抗生素活性的功能测定，该功能测定可作为许多其他活性的替代测定。Photorhabdus和Xenorhabdus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，促进了这种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。在Specific Aim 1中，基于功能的方法将被基于序列的方法所补充，该方法利用正在进行的对20种放线菌菌株在Specific Aim 2中的次级生物合成途径进行测序和注释。该目标使用“敲除扫描”来识别与预测基因簇相关的小分子。对于难以遗传操作的菌株，将在适当的宿主中使用异源表达。

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.