Mining Actinomycetal Genomes for Natural Product Discovery and Biosynthesis

项目摘要 天然产物（NPs）继续激发新的化学，生物学和医学，但天然产物的发现率 新的纳米颗粒已经大大减缓，因为当它们的生物合成时，编码它们的生物合成的大多数基因簇是沉默的。 在标准实验室条件下培养微生物。在这个MIRA应用程序中，我们建议 通过挖掘放线菌基因组发现新的纳米粒子，开发生产目标纳米粒子的技术 以足够的量进行结构解析和生物学评价，并表征其生物合成 发现新的化学和酶学的机器。我们的假设是：（i）基因组调查 斯克里普斯研究所（TSRI）的放线菌菌株收集将使我们能够确定潜在的 （ii）靶向NP支架的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NP的结构新颖性，（iii）遗传 在其天然生产者中操纵最有前途的BGC或在异源生产者中表达它们。 模型宿主将使我们能够生产和分离新的NP，和（iv）生物合成的表征 这些新纳米粒子的机制将使我们能够发现新的化学和酶学。过去两个研究 和两个目前的NIGMS赠款已累积到（i）在TSRI天然产品图书馆的建设 目前包含约211，000种微生物菌株，约461，000种粗提物和部分纯化的组分 从选定的菌株制备的，和~600个纯NP，（ii）C-1027和天赐霉素作为模型的建立 用于烯二炔NP生物合成的系统，莱那霉素和广南霉素作为用于 NP的来那霉素家族（即，具有前所未有的硫的杂合肽-聚酮大环内酰胺 功能），以及作为细菌二萜类化合物和硫代羧酸的模型系统的铂霉素和铂霉素 含酸/衍生的NP生物合成，分别，和（iii）鉴定190，43，66，和264个不同的 BGC，编码新的烯二炔，莱那霉素家族的新成员，新的细菌二萜类化合物，和新的 含硫代羧酸/衍生的NP。这些发现奠定了当前 应用于挖掘放线菌基因组用于NP发现和生物合成。这个结果 应用包括（i）对基因组挖掘和大BGC的激活的基本贡献，在天然的 生产者和异源模式宿主，通过代谢途径进行NP生产和结构多样性 工程，（ii）发现新的纳米粒子，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化学和酶学的新见解， 烯二炔的生物合成、NP的莱那霉素家族、细菌二萜类和硫代羧酸- 含有/衍生的NP。我们研究的长期目标是在分子水平上了解 微生物合成复杂的纳米颗粒，并利用这一知识来发现新的纳米颗粒和工程 用于药物研发的类似物。

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.