Molecule-to-gene approaches to new natural products

新天然产物的分子到基因方法

• 批准号: 7559105
• 负责人: Jon Clardy
• 金额: $ 40.54万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-09 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7559105
• 关键词: Actinobacteria class; Actinomyces; Antibiotics; Bacteria; Base Sequence; Biological; Biological Assay; Biological Factors; Classification; Compatible; Complement; Cosmids; DNA; DNA Library; Data; Enzymes; Escherichia coli; Gammaproteobacteria; Gene Cluster; Gene Expression; Genes; Genetic; Genome; Genomic Library; Genomics; Institutes; Lead; Life; Link; Methods; Molecular; Molecular Bank; Nematoda; Numbers; Object Attachment; Organism; Pantoea agglomerans; Pathway interactions; Pharmaceutical Preparations; Photorhabdus; Play; Production; Property; Public Health; Purpose; Reaction; Recording of previous events; Refractory; Research; Role; Scanning; Screening procedure; Source; Stream; Techniques; Testing; Therapeutic Agents; Today; United States National Institutes of Health; Work; Xenorhabdus; antimicrobial; base; drug quality; genetic manipulation; genome sequencing; high throughput screening; interest; medical schools; novel; programs; small molecule; small molecule libraries; success; tool

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.

描述（由申请人提供）：此申请对新天然产品的方法以及新天然产品本身的RFA做出响应，这些产品将增加NIH分子库路线图和其他筛选计划中与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的。两者都强调了从细菌来源发现新天然产品的遗传起点。特定的目标1专注于γ-细菌，而特定的目标2专注于放线菌。由于它们过去的贡献，最近的研究表明，细菌来源的强调仅探索了，其遗传可及性以及培养的能力并提供了筛选程序所需的多毫米材料量。这两个目的不仅使用非常不同的细菌组，而且还使用不同的起点，未表征的基因组DNA和基因组序列来发现生物活性的小分子。特定目标1。通过基因到分子方法发现生物活性的小分子。第一种方法利用了Photorhabdus spp的基因组DNA的宇宙文库。和Xenorhabdus spp。，代谢兼容的宿主大肠杆菌中的异源表达，以及用于抗生素活性的功能测定，它是许多其他活动的替代测定。属，Photorhabdus和Xenorhabdus都可能产生大量的抗生素分子，并且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。特定目标2。开发基于序列的方法来发现20种新的放线菌基因组中的天然产物。基于特定目标1的基于功能的方法将得到一种基于序列的方法的补充，该方法利用了持续的努力来序列和注释20个在特定目标中的20个静脉菌株菌株的二次生物合成途径。此目标使用“敲除扫描”来识别与预测的基因簇相关的小分子。对于对遗传操纵的菌株的菌株，将使用适当宿主中的异源表达。 公共卫生相关性：高通量筛查（HTS），在生物测定中测试大量小分子，是发现新药物的主要工具，并且小分子库的质量在很大程度上决定了HTS方法的成功。天然产物是由许多活​​生物体产生的小分子，含有真正显着的分子多样性，并且在发现新药方面具有悠久的成功历史。但是，尽管有这段历史，但他们在当今的HTS世界中只发挥了最小的作用。该项目描述了两种方法，可以最大程度地减少传统天然产品发现技术的某些责任，这可能会导致HTS重要的小分子流。