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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

基本信息

批准号：
8846537
负责人：
Jin Woo Bok
金额：
$ 86.1万
依托单位：
INTACT GENOMICS, INC.
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-15 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8846537
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 Health 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 small molecule tool vector

项目摘要

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 100,000 条生命，并增加了 30B 美元的医院费用。耐药微生物的出现进一步加剧了公共卫生问题。真菌是抗微生物次生代谢物 (SM) 的多产者，自世纪之交以来，真菌已从所有微生物来源提供了 45% 的生物活性分子。然而，由于难以有效表达这些 SM 途径，真菌次级代谢 (SM) 途径在很大程度上仍未开发。该研究计划旨在推进功能性 SM 宏基因组学科学，从测序的真菌基因组中克隆整套完整的 SM 通路，并发现用于药物和临床开发的新抗生素。在第一阶段研究中，Intact Genomics（前身为 Lucigen）吴博士的团队以及威斯康星大学麦迪逊分校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) 改进的从真菌中分离和纯化高分子量基因组 DNA 的方法； 2) 一种新的大肠杆菌-曲霉穿梭载体和构巢曲霉宿主，用于增强克隆 DNA 的表达； 3) 随机剪切 BAC 克隆方法，产生无偏的非常大的插入片段大小 (>100 kb)，用于覆盖真菌基因组的整套完整 SM 途径（一个 BAC 克隆 = 一个完整 SM 途径）； 4) 一种快速且改进的小分子鉴定方法，用于鉴定独特的化合物。在第一阶段研究中，所有 56 条 SM 途径均在土曲霉的无偏随机剪切穿梭 BAC 文库中得到鉴定。十四个 SM BAC 克隆转化为构巢曲霉，其中至少两个 BAC 克隆显示出强抗菌活性，三个 BAC 克隆显示出抗真菌活性。此外，从含有astechrome途径的BAC中发现了6种新的astechrome化合物。我们建议在第二阶段研究中，从 5 种测序真菌（约 200 个 SM 途径）中创建 5 个额外的无偏大插入穿梭 BAC 文库，这些文库将被广泛筛选小分子化合物和抗生素。我们期望利用这种方法发现数百种新型化学实体，并引导候选者对多重耐药细菌和真菌病原体具有高效能。这些技术代表了天然产物发现科学，特别是抗生素发现科学的重要进步。此外，本研究产生的文库是宝贵的基因组资源，可用于筛选其他生物活性化合物：例如抗病毒、抗癌和抗炎活性。