Mining Actinomycetal Genomes for Natural Product Discovery and Biosynthesis

项目总结 天然产物(NPs)继续激发新的化学、生物学和医学的灵感，但 新的NPs已经显著减慢，因为大多数编码它们的生物合成的基因簇在 微生物是在标准的实验室条件下培养的。在此Mira应用程序中，我们建议 通过挖掘放线菌基因组发现新的NPs，开发使能技术来生产目标NPs 足够的数量用于结构鉴定和生物学评价，并表征它们的生物合成 发现新的化学和酶学的机械。我们的假设是：(I)基因组调查 斯克里普斯研究所(TSRI)收集的放线菌菌株将使我们能够识别潜在的 靶向NP支架的生产者，(Ii)靶向潜在生产者的基因组测序 生物合成基因簇(BGC)将使我们能够预测新NPs的结构新颖性，(Iii)遗传 最有希望的BGC在其本土生产者中的操纵或在异源基因中的表达 模型宿主将使我们能够生产和分离新的NPs，以及(Iv)生物合成的表征 这些新NPs的机械将使我们能够发现新的化学和酶学。来自过去两年的研究 目前的两笔NIGMS赠款已累计用于：(I)在TSRI建造天然产品图书馆 目前由约211,000个微生物菌株、约461,000个粗提物和部分纯化的馏分组成 (2)C-1027和替西霉素模型的建立 烯二炔NP生物合成体系、雷诺霉素和广南霉素作为生物合成的模型体系 雷诺霉素NPs家族(即具有史无前例硫磺特征的杂交肽-聚酮大内酰胺) 功能)，以及作为细菌二萜和硫代羧酸的模型体系的铂新霉素和铂菌素 含酸/衍生NP的生物合成，以及(Iii)190、43、66和264的鉴定不同 BGC，编码新的烯二炔类，雷诺霉素家族的新成员，新的细菌二萜类化合物，以及新的 分别含有硫代羧酸/衍生的NPs。这些发现为当前的 放线菌基因组挖掘在NP发现和生物合成中的应用。这样做的结果是 应用包括(I)对基因组挖掘和大BGC的激活的基本贡献，在本地 生产者和异源模式宿主，通过代谢途径产生NP和结构多样性 工程学，(Ii)发现新的NPs，具有特权的支架，以激发新的化学，生物学和 医学，以及(Iii)对生物合成机制和新的化学和酶学的新见解 生物合成烯二炔、雷诺霉素纳米粒家族、细菌二萜类化合物和硫代羧酸- 含有/派生的NP。我们研究的长期目标是在分子水平上了解 微生物合成复杂的NPs，并利用这一知识发现新的NPs并进行工程设计 它们的类似物用于药物发现。

PROJECT SUMMARY Natural products (NPs) continue to inspire novel chemistry, biology, and medicine, but the rate of discovery of novel NPs has slowed dramatically as most gene clusters encoding their biosynthesis are silent when the microorganisms are cultured under standard laboratory conditions. In this MIRA application, we propose to discover new NPs by mining actinomycetal genomes, develop enabling technologies to produce the target NPs in enough quantities for structural elucidation and biological evaluation, and characterize their biosynthetic machineries to discover new chemistry and enzymology. Our hypotheses have been: (i) genome survey of the actinomycetale strain collection at The Scripps Research Institute (TSRI) will allow us to identify potential producers of the targeted NP scaffolds, (ii) genome sequencing of the potential producers for the targeted biosynthetic gene clusters (BGCs) will allow us to predict the structural novelty of the new NPs, (iii) genetic manipulation of the most promising BGCs in their native producers or expression of them in heterologous model hosts will allow us to produce and isolate the new NPs, and (iv) characterization of the biosynthetic machinery of these new NPs will allow us to discover new chemistry and enzymology. Studies from two past and two current NIGMS grants have cumulated into (i) the construction of the Natural Products Library at TSRI that currently consists of ~211,000 microbial strains, ~461,000 crude extracts and partially purified fractions prepared from selected strains, and ~600 pure NPs, (ii) the establishment of C-1027 and tiancimycin as model systems for enediyne NP biosynthesis, leinamycin and guangnanmycin as model systems for biosynthesis of the leinamycin family of NPs (i.e., hybrid peptide-polyketide macrolactams featuring unprecedented sulfur functionalities), and platensimycin and platencin as model systems for bacterial diterpenoid and thiocarboxylic acid-containing/derived NP biosynthesis, respectively, and (iii) the identification of 190, 43, 66, and 264 distinct BGCs, encoding new enediynes, new members of the leinamycin family, new bacterial diterpenoids, and new thiocarboxylic acid-containing/derived NPs, respectively. These findings set the stage in the current application to mine the actinomycetal genomes for NP discovery and biosynthesis. The outcomes of this application include (i) fundamental contributions to genome mining and activation of large BGCs, in native producers and heterologous model hosts, for NP production and structural diversity by metabolic pathway engineering, (ii) discovery of new NPs, with privileged scaffolds, to inspire new chemistry, biology, and medicine, and (iii) new insights into biosynthetic machineries and novel chemistry and enzymology for the biosynthesis of enediynes, the leinamycin family of NPs, bacterial diterpenoids, and thiocarboxylic acid- containing/derived NPs. The long-term goal of our research is to understand at a molecular level how microorganisms synthesize complex NPs and to exploit this knowledge to discover novel NPs and engineer their analogues for drug discovery.