Molecule-to-gene approaches to new natural products

新天然产物的分子到基因方法

• 批准号: 7684744
• 负责人: Jon Clardy
• 金额: $ 41.68万
• 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/7684744
• 关键词: Actinobacteria class; Actinomyces; Antibiotics; Bacteria; Base Sequence; Biological; Biological Assay; Biological Factors; Classification; Complement; Cosmids; DNA; DNA Library; Data; Enzymes; Escherichia coli; Gammaproteobacteria; Gene Cluster; Gene Expression; Genes; Genetic; Genome; Genomic Library; Genomics; Institutes; Knock-out; Lead; Life; Link; Methods; Molecular; Molecular Bank; Nematoda; Organism; Pantoea agglomerans; Pathway interactions; Pharmaceutical Preparations; Photorhabdus; Play; Principal Investigator; Production; Property; Reaction; Recording of previous events; Refractory; Research; Role; Scanning; Screening procedure; Source; Stream; Techniques; Testing; Therapeutic Agents; United States National Institutes of Health; Work; Xenorhabdus; antimicrobial; base; drug quality; gene discovery; genetic manipulation; genome sequencing; high throughput screening; interest; medical schools; novel; programs; public health relevance; small molecule; small molecule libraries; success; tool; virtual

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）：本申请对新天然产物方法和新天然产物本身的 RFA 作出回应，这些方法将增加 NIH 分子库路线图和其他筛选计划中的生物学相关分子多样性。两种方法构成了该应用程序的两个具体目标。两者都强调从细菌来源发现新天然产物的遗传起点。具体目标 1 侧重于 Gammaproteobacteria，具体目标 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尚未得到充分探索、它们的遗传可及性以及它们的培养能力和提供筛选计划所需的数毫克材料的能力。这两个目标不仅使用截然不同的细菌群，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。第一种方法利用光杆菌属基因组 DNA 的粘粒文库。和致病杆菌属，在代谢相容的宿主大肠杆菌中的异源表达，以及抗生素活性的功能测定，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。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。具体目标 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工作对特定目标 2 中 20 种放线菌菌株的二级生物合成途径进行测序和注释。该目标使用“基因敲除扫描”来识别小分子与预测基因簇相关的分子。对于难以进行遗传操作的菌株，将在适当的宿主中使用异源表达。 公共卫生相关性：高通量筛选 (HTS) 是在生物测定中测试大量小分子，是发现新药的主要工具，小分子库的质量在很大程度上决定了 HTS 方法的成功。天然产物是许多生物体产生的小分子，具有真正显着水平的分子多样性，并且在发现新药方面有着悠久的成功历史。但尽管有这样的历史，它们在当今的高温超导世界中只发挥了很小的作用。该项目描述了两种方法，它们最大限度地减少了传统天然产物发现技术的一些缺陷，这些技术可能为高温超导产生重要的小分子流。