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

项目摘要/摘要 抗菌素耐药性在所有主要类别的抗生素中都很普遍，许多细菌都有耐药性 到多种，在某些情况下，所有可用的抗生素。为了对抗这种耐药性，需要新的抗生素。 放线菌的天然产物(NPs)传统上是抗生素的丰富来源，但发现 通过传统方法从这些菌株中提取新的NPs(即筛选粗制培养上清液中的抗生素 活动)很少见效，因为重新发现率很高。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，表明存在许多新的 NPS。梅特卡夫和凯莱赫实验室最近开发了一种基于 基于基因组和质谱学分析(称为通过质量鉴定天然产物 基于光谱的分析，灵活)。我建议通过整合计算结构来改进Nimble 预测和分子网络。我将使用改进的Nimble发现两个新的抗生素NPs和 阐明两者中生物活性较高的一种生物合成途径。 在目标1中，将使用计算结构预测来分析通过Nimble识别的20个新的NP 和分子网络。这种分析将允许使用新的化学支架(不是 已知NP的衍生品！)。10个具有独特结构的NPs将从它们的生产中部分提纯 并对一组临床相关细菌进行活性筛选。由于较高的百分比 抗生素NPs(放线菌NPs约50%)，我们预计将至少鉴定出两种抗生素NPs。对于 两个最活跃的NPs，NPs与其生物合成基因簇的灵活预测的关联将是 使用活体遗传分析进行了验证。然后这些化合物将被分离，它们的结构将被 已澄清。最后，将对纯化的纳米颗粒进行进一步的抗菌活性分析。在目标2中， 目标1中确定的最有效NP的生物合成途径将被破译。敏捷型的优势 方法是它允许同时识别新的NPs及其基因簇。了解…… 生物合成途径将允许未来对菌株进行工程改造，以提高NP和 可改善活性或药代动力学性质的衍生物的生产。世界的边界 生物合成基因簇和簇内基因的功能将通过生物信息学确定 对簇中编码的酶进行分析、遗传验证，如果需要，还可以进行体外分析。的顺序 该途径将通过交叉喂养研究来确定。总体而言，这项提议将允许确定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， 它将为发现更多具有生物活性的天然产物提供一个通用平台。

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.