Discovery of Novel Natural Products via Characterization of LC/HRMS associated Gene Cluster Families

通过 LC/HRMS 相关基因簇家族的表征发现新型天然产物

• 批准号: 9467222
• 负责人: Elizabeth Ivy Parkinson
• 金额: $ 2.05万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-16 至 2018-08-12
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9467222
• 关键词: Actinobacteria class; Address; Analytical Chemistry; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Antifungal Agents; Bacteria; Bacterial Antibiotic Resistance; Bacterial Drug Resistance; Bioinformatics; Biological; Centers for Disease Control and Prevention (U.S.); Cessation of life; Chemicals; Complex; Crude Extracts; Data; Development; Drug Kinetics; Engineering; Enzymes; Family; Future; Gene Cluster; Gene Deletion; Genes; Genetic; Genomics; Goals; Health; Healthcare; In Vitro; Individual; Infection; Knowledge; Laboratories; Mass Spectrum Analysis; Methods; Mind; Molecular; Natural Products; Organism; Pathway Analysis; Pathway interactions; Production; Property; Resistance; Running; Scourge; Societies; Source; Structure; Techniques; Testing; Therapeutic; United States; Validation; Work; World Health Organization; anti-cancer; arm; base; bioactive natural products; chemical synthesis; clinically relevant; combat; experimental study; feeding; gene function; genetic analysis; genetic approach; genome sequencing; improved; in vivo; microorganism; mutant; novel; pathogen; professor; programs; scaffold; screening; skills; whole genome

Project Summary/Abstract Antibacterial resistance is widespread for all major classes of antibiotics with many bacteria being resistant to multiple, and in some cases all, available antibiotics. To combat this resistance, novel antibiotics are needed. Natural products (NPs) from actinomycetes have traditionally been rich sources for antibiotics, but discovery of novel NPs from these strains via traditional means (i.e. screening crude culture supernatants for antibiotic activity) is rarely effective due to high rates of rediscovery. Whole genome sequencing of actinomycetes has revealed the presence of many unknown biosynthetic gene clusters suggesting the existence of many novel NPs. The Metcalf and Kelleher laboratories recently developed a method for untargeted NP discovery based upon genomic and mass spectrometric analyses (referred to as Natural Product Identification via Mass Spectrometry Based Analyses, NIMBLE). I propose to improve NIMBLE by incorporating computational structure predictions and molecular networking. I will use the improved NIMBLE to discover two novel antibiotic NPs and to elucidate the biosynthetic pathway for the more biologically active of the two. In Aim 1, twenty novel NPs identified via NIMBLE will be analyzed using computational structure prediction and molecular networking. This analysis will allow for identification of NPs with novel chemical scaffolds (not derivatives of known NPs!). Ten of the NPs with unique structures will be partially purified from their producing organisms and screened for activity against a panel of clinically relevant bacteria. Due to the high percentage of antibiotic NPs (~50% for actinomycete NPs), we expect that at least two antibiotic NPs will be identified. For the two most active NPs, the NIMBLE-predicted associations of the NPs with their biosynthetic gene clusters will be validated using in vivo genetic analysis. The compounds will then be isolated, and their structures will be elucidated. Finally, the purified NPs will be further analyzed for their antibacterial activity. In Aim 2, the biosynthetic pathway for the most potent NP identified in Aim 1 will be deciphered. An advantage of the NIMBLE method is that it allows simultaneous identification of novel NPs and their gene clusters. Having knowledge of the biosynthetic pathway will allow for future engineering of the strain for enhanced production of the NP and production of derivatives that could have improved activity or pharmacokinetic properties. The boundaries of the biosynthetic gene cluster and the functions of the genes within the cluster will be determined via bioinformatics analyses, genetic validation, and if needed, in vitro analysis of the enzymes encoded in the cluster. The order of the pathway will be determined via cross-feeding studies. Overall, this proposal will allow for identification of 2 novel antibiotic natural products and elucidation of the biosynthetic pathway for one of them. More importantly, it will provide a general platform for discovering many more bioactive natural products.

项目概要/摘要 所有主要类别的抗生素都普遍存在细菌耐药性，许多细菌都具有耐药性 多种可用的抗生素，在某些情况下是所有可用的抗生素。为了对抗这种耐药性，需要新型抗生素。 放线菌的天然产物 (NP) 传统上是抗生素的丰富来源，但发现 通过传统方法（即筛选粗培养物上清液中的抗生素）从这些菌株中提取新的纳米颗粒 由于重新发现率很高，因此很少有效。放线菌全基因组测序 揭示了许多未知生物合成基因簇的存在，表明许多新的生物合成基因簇的存在 NP。 Metcalf 和 Kelleher 实验室最近开发了一种基于非靶向 NP 发现的方法 根据基因组和质谱分析（称为通过质量进行天然产物鉴定） 基于光谱分析，NIMBLE）。我建议通过合并计算结构来改进 NIMBLE 预测和分子网络。我将使用改进的 NIMBLE 来发现两种新型抗生素 NP 阐明两者中更具生物活性的生物合成途径。 在目标 1 中，将使用计算结构预测来分析通过 NIMBLE 识别的 20 个新型 NP 和分子网络。该分析将允许识别具有新型化学支架的纳米颗粒（不是 已知 NP 的衍生物！）。十种具有独特结构的纳米粒子将从其生产中部分纯化 生物体并筛选针对一组临床相关细菌的活性。由于比例很高 抗生素 NP（放线菌 NP 的~50%），我们预计至少会鉴定出两种抗生素 NP。对于 两个最活跃的 NP，NIMBLE 预测的 NP 与其生物合成基因簇的关联将是 使用体内遗传分析进行验证。然后将分离化合物，其结构将是 阐明了。最后，纯化的纳米颗粒将进一步分析其抗菌活性。在目标 2 中， 目标 1 中确定的最有效的 NP 的生物合成途径将被破译。 NIMBLE 的优势 方法的优点是它可以同时识别新型纳米颗粒及其基因簇。具备以下知识 生物合成途径将允许未来对该菌株进行工程改造，以增强 NP 和 生产具有改善活性或药代动力学特性的衍生物。的边界 生物合成基因簇以及簇内基因的功能将通过生物信息学确定 分析、遗传验证，如果需要，还可以对簇中编码的酶进行体外分析。的顺序 该途径将通过交叉喂养研究来确定。总体而言，该提案将允许确定 2 新型抗生素天然产物及其生物合成途径的阐明。更重要的是， 它将为发现更多生物活性天然产品提供一个通用平台。