Evaluation of Novel Antimicrobial Agents and a Novel Shuttle Vector

描述（由申请人提供）：抗生素是我们抵御细菌性病原体的第一道防线，这些病原体在历史上一直是人类文明的瘟疫。由于抗生素耐药性，曾经被视为可治疗的传染原的人类病原体日益成为严重的问题。在制药管道中迫切需要新型抗生素，但由于许多原因，制药行业尚未在抗生素发现过程中投入大量资金。抗生素发现的最佳资源是探索微生物群落，复杂的化学物质在那里自然进化。为了从不同的微生物中获取复杂的化学物质，本提案使用了一种社区基因组方法，其中来自不同微生物的生物合成基因直接从土壤中回收并在大肠杆菌或其他细菌宿主中表达。已经从这些社区基因组文库中分离出了44个产生抗生素活性的克隆。拟议的研究将确定这些收集的抗生素化合物的化学结构，并确定与这些化合物相关的效力和任何毒性。除了表达抗生素活性的克隆外，另一组克隆已被分离出来，其中包含一类特定天然产物的基因，即聚酮。聚酮类化合物具有多种结构和多种活性，如抗菌和抗癌特性。从一个土壤微生物群落文库中发现的聚酮生物合成途径的遗传分析表明，34个含聚酮的克隆与已知的聚酮生物合成途径有很大的不同。我们的研究工作将把这些聚酮途径转移到一种新的克隆载体上，旨在允许在革兰氏阴性菌之间转移遗传途径，然后通过遗传和生物测定筛选来确定这些聚酮途径是否在不同的细菌物种中表达。这项研究的结果将是收集具有抗菌活性的先导候选化合物，新的聚酮生物合成途径，用于工程新型具有治疗性质的聚酮结构，新的革兰氏阴性穿梭载体的评估，以及下一代微生物群落基因组文库，用于该技术的未来发展。公共卫生相关性：抗生素对我们抗击传染病的能力至关重要，特别是在抗生素耐药性发病率上升的时代，有必要发现和开发临床有用的抗生素。利用克隆和表达不同微生物遗传途径的新技术，已分离出一系列产生抗菌活性或编码生物合成途径的克隆。拟议的研究将在遗传和生化水平上评估这些克隆的收集，以确定哪些主要候选药物具有作为新型治疗剂的最佳潜力。

DESCRIPTION (provided by applicant): Antibiotics are our first line of defense against bacterial pathogens that have historically been a plague on human civilization. Increasingly, human pathogens that had been regarded as treatable infectious agents are becoming serious problems as a result of antibiotic resistance. There is an urgent need for novel antibiotics in the pharmaceutical pipeline, yet for many reasons the pharmaceutical industry has not invested heavily in the antibiotic discovery process. The best possible resource for antibiotic discovery is to explore microbial communities, where complex chemistries have naturally evolved. To access the complex chemistries from diverse microorganisms, this proposal uses a community genomic approach in which biosynthetic genes from diverse microorganisms are directly recovered from soils and expressed in E. coli or other bacterial hosts. A collection of fourty-four clones that produce antibiotic activity has already been isolated from these community genomic libraries. The proposed research will determine the chemical structure of antibiotic compounds from this collection, and determine the potency and any toxicity associated with these compounds. In addition to clones that express antibiotic activity, another collection of clones has been isolated that contain the genes for a specific class of natural product, the polyketides. Polyketide compounds have diverse structures, and diverse activities, such as antimicrobial and anticancer properties. A genetic analysis of polyketide biosynthesis pathways discovered from a soil microbial community library indicates that the 34 polyketide-containing clones are very different from known polyketide pathways. Our research efforts will transfer these polyketide pathways into a novel cloning vector, designed to allow transfer of genetic pathways between Gram-negative bacteria, and then genetic and bioassay screens will determine whether these polyketide pathways are expressed in different bacterial species. The results of this research will be a collection of lead candidate compounds with antimicrobial activity, novel polyketide biosynthetic pathways useful in engineering novel polyketide structures with therapeutic properties, an evaluation of a novel Gram- negative shuttle vector, and the next generation of microbial community genomic library for future development of this technology. PUBLIC HEALTH RELEVANCE: Antibiotics are critical to our ability to combat infectious disease, and especially in an era in which the incidence of antibiotic resistance is on the rise there is a need for discovery and development of clinically useful antibiotics. Using new technology for cloning and expressing genetic pathways from diverse microorganisms, a collection of clones that either produce an antimicrobial activity or encode a biosynthetic pathway have been isolated. The proposed research would evaluate this collection of clones at a genetic and biochemical level to determine which lead candidates have the best potential as novel therapeutic agents.