Discovery and characterization of new natural products from uncultured bacteria

描述（由申请人提供）：天然产物是大多数FDA批准的药物的来源，特别是微生物产生的代谢物是当今使用的许多抗生素和抗癌药物的来源。在过去的二十年里，尽管微生物天然产物作为小分子治疗药物的先导结构在历史上取得了成功，并且对新的抗菌剂和化疗药物的临床需求持续不断，但在很大程度上，大型筛选项目已经不再重视微生物提取物。据估计，环境中存在的细菌中只有不到1%可以在实验室中培养，因此可以用于传统的小分子发现策略。未培养的细菌可能是生物合成多样性的最大剩余池，尚未被用于生产生物活性天然产品。虽然在环境中容易培养大多数细菌仍然是不可能的，但可以直接从环境样品（环境DNA， eDNA）中提取微生物DNA并将其克隆到容易培养的细菌中，从而首次可以对其进行功能表征。在这项研究中，从美国西南部四个主要沙漠（索诺兰沙漠、奇瓦瓦沙漠、莫哈韦沙漠和大盆地沙漠）收集的土壤中构建的eDNA大型文库将被构建和排列，以允许筛选新的天然产物生物合成基因簇。然后将使用两种相关但互补的基于dna的筛选方法从这些eDNA大型文库中恢复新的小分子生物合成基因簇。在第一种方法中，我们将重点关注含有具有生产结构多样化生物活性天然产物历史的生物合成系统的克隆的鉴定和恢复。在第二个策略中，我们将侧重于鉴定和恢复含有特定类别的重要药理天然产物的新变体的克隆。由新的环境dna衍生基因簇编码的分子将在系统发育不同的培养细菌中通过异源表达进行功能访问，这些代谢物将在全细胞抗菌、抗真菌和抗癌实验中进行测试。天然产物作为发现治疗方法的起点的历史成功为我们的假设提供了支持，即在这些研究中发现的代谢物将具有对人类健康有益的高潜力。

DESCRIPTION (provided by applicant): Natural products are the source of the majority of FDA approved drugs and, in particular, metabolites produced by microorganisms are the source of many of the antibiotics and anticancer agents used today. Over the past two decades large screening programs have largely deemphasized microbial extracts in spite of the historical success of microbial natural products as lead structures for the development of small molecule therapeutics and the continuing clinical need for new antimicrobials and chemotherapeutics. It is estimated that less than one percent of the bacteria present in the environment is readily cultured in the laboratory and therefore accessible to use in traditional small molecule discovery strategies. Uncultured bacteria are likely the largest remaining pool of biosynthetic diversity that has not yet been examined for the production of bioactive natural products. While it is still not possible to easily culture most bacteria in the environment, it is possible to extract microbial DNA directly from environmental samples (environmental DNA, eDNA) and clone this DNA into easily cultured bacteria where, for the first time, it can be functionally characterized. In this study a collection of eDNA mega-libraries constructed from soils collected in the four major deserts of the American Southwest (Sonoran Desert, Chihuahuan Desert, Mojave Desert and the Great Basin Desert) will be constructed and arrayed to permit screening for novel natural product biosynthetic gene clusters. Two related but complementary DNA-based screening approaches will then be used to recover novel small molecule biosynthetic gene clusters from these eDNA mega-libraries. In the first approach we will focus on the identification and recovery of clones containing biosynthetic systems with a history of producing structurally diverse bioactive natural products. In the second strategy we will focus on the identification and recovery of clones containing new variants of specific classes of pharmacologically important natural products. Molecules encoded by new environmental DNA-derived gene clusters will then be functionally accessed using heterologous expression in phylogenetically diverse cultured bacteria, and these metabolites will be tested in whole cell antibacterial, antifungal and anticancer assays. The historical success of natural products as starting points for the discovery of therapeutics provides support for our hypothesis that the metabolites found in these studies will have a high potential of being beneficial to human health.