Expanding small molecule functional metagenomics through shuttle BAC expression i

通过穿梭 BAC 表达扩展小分子功能宏基因组

• 批准号: 8781067
• 负责人: Jin Woo Bok
• 金额: $ 94.13万
• 依托单位: INTACT GENOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-15 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8781067
• 关键词: American; Americas; Anti-Bacterial Agents; Anti-Inflammatory Agents; Anti-inflammatory; Antibiotics; Antifungal Agents; Antiviral Agents; Applications Grants; Aspergillus; Aspergillus nidulans; Bacteria; Biochemical; Biochemistry; Biological Factors; Businesses; Characteristics; Chemical Structure; Chemicals; Clinical; Cloning; Communicable Diseases; Coupled; DNA; Data; Development; Drug resistance; Engineering; Escherichia coli; Freedom; Fungal Genome; Fungi Model; Genetic; Genetic Variation; Genomic DNA; Genomics; Goals; Hospital Costs; Hospitals; Infection; Lead; Libraries; Life; Metabolic Pathway; Metagenomics; Methodology; Methods; Microbe; Molecular Weight; Multi-Drug Resistance; Multiple Bacterial Drug Resistance; Pathway interactions; Pharmacologic Substance; Phase; Preparation; Public Health; Publishing; Research; Research Proposals; Resources; Science; Scientist; Services; Shuttle Vectors; Small Business Innovation Research Grant; Source; Structure; System; Technology; Therapeutic; Time; Universities; Wisconsin; Work; Workplace; antimicrobial; chemical genetics; combat; drug discovery; expression cloning; fight against; fungus; improved; innovation; microbial; mutant; next generation; novel; pathogen; phase 2 study; public health relevance; small molecule; tool; vector

DESCRIPTION (provided by applicant): Hospital acquired microbial infections are the fourth largest killer in America, taking 100,000 lives and adding $30B to hospital costs. The emergence of drug resistant microbes has further amplified public health concern. Fungi are prolific producers of anti-microbial secondary metabolites (SM) and since the turn of the century have provided 45% of bioactive molecules from all microbial sources. However, fungal secondary metabolic (SM) pathways remain largely untapped due to difficulties in efficiently expressing these SM pathways. This research proposal is to advance the science of functional SM metagenomics, to clone the entire set of intact SM pathways from sequenced fungal genomes, and to discover new antibiotics for pharmaceutical and clinical development. During Phase I research Dr. Wu's group at Intact Genomics (formerly Lucigen) and scientists at the University of Wisconsin Madison and Northwestern University applied numerous key technological breakthroughs that together resulted in the next generation functional metagenomic library. This library combined 1) an improved methodology for the isolation and purification of high molecular weight genomic DNA from fungi; 2) a new E. coli- Aspergillus shuttle vector and an A. nidulans host for enhanced expression of cloned DNAs; 3) a random shear BAC cloning method to produce unbiased very large insert sizes (>100 kb) for covering the entire set of intact SM pathways of a fungal genome (one BAC clone = one intact SM pathway); and 4) a rapid and improved small molecule identification method to identify unique compounds. In Phase I research, all of 56 SM pathways were identified in the unbiased Random Shear shuttle BAC library of A. terreus. Fourteen SM BAC clones where transformed into A. nidulans with at least two BAC clones showing strong antibacterial activities and three antifungal. Moreover, 6 new astechrome compounds were uncovered from the astechrome- pathway-containing BAC. We propose in Phase II study to create 5 additional unbiased large- insert shuttle BAC libraries from 5 sequenced fungi (~ 200 SM pathways) which will be extensively screened for small molecule compounds and antibiotics. We expect to uncover hundreds of novel chemical entities using this approach, and lead candidates with high potency against multiple-drug-resistance bacterial and fungal pathogens. These technologies represent an important advancement for the science of natural product discovery in general and antibiotic discovery in particular. In addition, the libraries produced from this research are a valuable genomic resource that may be screened for other bioactive compounds: for example antiviral, anticancer, and anti-inflammatory activities.

描述（由申请人提供）：医院获得性微生物感染是美国第四大杀手，夺走了10万人的生命，增加了300亿美元的医院成本。抗药性微生物的出现进一步加剧了公众对健康的担忧。真菌是抗微生物次级代谢物（SM）的多产者，并且自世纪之交以来已经提供了来自所有微生物来源的45%的生物活性分子。然而，真菌次级代谢（SM）途径仍然在很大程度上未开发，由于在有效地表达这些SM途径的困难。这项研究计划旨在推进SM功能宏基因组学的研究，从测序的真菌基因组中克隆完整的SM通路，并发现用于制药和临床开发的新抗生素。在第一阶段研究中，Wu博士在Intact Genomics（前身为Lucigen）的团队以及威斯康星州麦迪逊大学和西北大学的科学家应用了许多关键技术突破，共同产生了下一代功能宏基因组文库。该文库结合了1）从真菌中分离和纯化高分子量基因组DNA的改进方法; coli-Aspergillus穿梭载体和A. 3）随机剪切BAC克隆方法，以产生无偏倚的非常大的插入片段大小（>100 kb），用于覆盖真菌基因组的完整SM途径的整个集合（一个BAC克隆=一个完整SM途径）;和4）快速和改进的小分子鉴定方法，以鉴定独特的化合物。在第一阶段的研究中，所有56个SM途径都在无偏随机剪切穿梭BAC文库中被鉴定。terreus将14个SM BAC克隆转化到A.至少有两个BAC克隆显示出较强的抗菌活性和三种抗真菌活性。此外，还从含染料途径的BAC中发现了6个新的染料化合物。我们建议在II期研究中从5种测序的真菌（~ 200个SM途径）中创建5个额外的无偏倚大插入穿梭BAC文库，其将被广泛筛选小分子化合物和抗生素。我们希望使用这种方法发现数百种新的化学实体，并领导对多重耐药细菌和真菌病原体具有高效力的候选物。这些技术代表了天然产物发现科学的重要进步，特别是抗生素发现。此外，从这项研究中产生的文库是一种有价值的基因组资源，可以筛选其他生物活性化合物：例如抗病毒，抗癌和抗炎活性。