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

项目总结/摘要 抗生素耐药性普遍存在于所有主要类别的抗生素中，许多细菌都具有耐药性 多种抗生素，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，但放线菌的发现， 通过传统方法（即筛选粗培养物上清液的抗生素）从这些菌株中获得新的NP 活动）由于再发现率高而很少有效。放线菌全基因组测序 揭示了许多未知的生物合成基因簇的存在，这表明存在许多新的生物合成基因簇。 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， 在基因组和质谱分析（称为通过质谱的天然产物鉴定）之后， 基于光谱的分析，NIMBLE）。我建议通过结合计算结构来改进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， 以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鉴定的20个新的NP 和分子网络。该分析将允许鉴定具有新的化学支架的NP（不包括纳米颗粒）。 已知NP的衍生物！）。其中10个具有独特结构的NP将从其生产过程中部分纯化， 微生物，并筛选针对一组临床相关细菌的活性。由于高比例的 抗生素NP（约50%用于放线菌NP），我们预期将鉴定至少两种抗生素NP。为 两个最活跃的NP，NIBLE预测的NP与其生物合成基因簇的关联将是 使用体内遗传分析验证。然后将化合物分离，并且将它们的结构进行表征。 阐明。最后，将进一步分析纯化的NP的抗菌活性。在目标2中， 目标1中鉴定的最有效的NP的生物合成途径将被破译。敏捷的优势 方法的另一个优点是它允许同时鉴定新的NP及其基因簇。知悉 该生物合成途径将允许将来工程改造该菌株以提高NP的生产， 生产可具有改善的活性或药代动力学性质的衍生物。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来确定 分析、遗传验证，以及如果需要，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。总的来说，这项建议将允许确定2个 新的抗生素天然产物，并阐明其中之一的生物合成途径。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.