Mining the cultured and uncultured biosphere for new drugs

项目总结/摘要 这项研究计划的重点是在发现新的药物分子的基本障碍， 这就是所谓的供应问题。新的生物活性分子并不缺乏， 我们可以用它作为药物，因为这些分子已经进化了数十亿年， 数万亿个微生态体在这个程序中，我们将（1）使用序列分析和定向进化来设计 从实验室培养的菌株中未培养的细菌中制备有希望的分子的方法，以及（2）提高 从分离的可培养细菌菌株中发现新分子的速度， 它们的小分子途径是受调控的，并利用这些知识来打开这些途径。 1)据估计，有1万亿种细菌存在。在实验室里培育的物种数量 相比之下是微不足道的，但这微不足道的一部分给了我们目前大多数种类的抗生素 以及许多其他药物。我们知道，我们正在失去一个令人难以置信的数量的化学多样性， 未培养的生物圈，因为我们可以观察到小分子的生物合成途径， 非培养物依赖性测序。通常，这些基因存在于生活在环境中的细菌共生体的基因组中。 另一种动物，如海生无脊椎动物或昆虫。目前，这一序列数据只是一个学术 好奇心，因为将从未培养的共生体的途径转移到实验室菌株是非常具有挑战性的 它们之间可能相隔超过十亿年的进化。我们将继续揭露重要的小 共生体中的分子途径，但我们也将努力通过两个途径来供应这些化合物。 战略布局首先，我们将设计新的技术来搜索基因组中的相关途径， 活的细菌，这可能是以前错过了由于不完整的基因组组装。在第二 策略，我们将使用进化来优化新宿主的蛋白质序列，模仿途径如何 在不同的细菌之间水平转移了数十亿年。 2)当细菌菌株被分离用于药物发现时，它们所具有的大多数小分子途径 在标准培养条件下不表达，我们必须依赖于 在实验室生产的。这是因为大多数途径都受到严格控制，因此它们是在 具体的环境条件。许多由细菌产生的小分子被认为可以抑制 竞争对手的物种，因此条件表达式最大限度地发挥其影响，同时减少的机会， 阻力就会产生。虽然小分子途径是通过物种之间的水平 转移时，它们被整合到新主机的预先存在的监管网络中。我们建议确定 在一个特定的细菌群体的小分子的全球调节机制，使用的技术， 一般化，并操纵它们产生以前在实验室中无法获得的小分子。这将 克服了药物发现的一个主要障碍，允许在分离菌株中充分利用生物合成。

PROJECT SUMMARY/ABSTRACT This research program focuses on a fundamental barrier in the discovery of new drug molecules from the environment, known as the supply problem. There is no shortage of new bioactive molecules in the environment that we can use as drugs, because such molecules have been evolving for billions of years within trillions of microniches. In this program, we will (1) use both sequence analysis and directed evolution to devise ways of making promising molecules from uncultured bacteria in laboratory-grown strains, and (2) improve the discovery rate of new molecules from isolated culturable bacterial strains by determining the shared ways that their small molecule pathways are regulated, and exploiting that knowledge to turn on these pathways. 1) It is estimated that 1 trillion species of bacteria exist. The number of species that have been grown in the lab is minuscule by comparison, but that minuscule portion has given us most of the classes of antibiotics currently known as well as many other drugs. We know that we are missing an incredible amount of chemical diversity in the uncultured biosphere because we can observe the biosynthetic pathways for small molecules through culture-independent sequencing. Often these are found in the genomes of bacterial symbionts that live within another animal, such a marine invertebrate or insect. Currently, this sequence data is simply an academic curiosity because it is incredibly challenging to move pathways from uncultured symbionts to laboratory strains which might be separated by more than a billion years of evolution. We will continue to uncover important small molecule pathways in symbionts, but we will also work towards the supply of these compounds through two strategies. In the first we will devise new techniques to search for related pathways in the genomes of free- living bacteria, that might have been previously missed due to incomplete genome assembly. In the second strategy, we will use evolution to optimize protein sequences for the new host, mimicking how pathways have been horizontally transferred between different bacteria for billions of years. 2) When bacterial strains are isolated for drug discovery, most of the small molecule pathways they possess are not expressed under standard culture conditions, and we have to rely on the small subset that are produced in the lab. This is because most pathways are tightly controlled so that they are expressed under specific environmental conditions. Many small molecules made by bacteria are thought to inhibit the growth of rival species, and therefore conditional expression maximizes their impact while reducing the chance that resistance will develop. While small molecule pathways are passed between species through horizontal transfer, they become integrated into the pre-existing regulatory network of a new host. We propose to identify the global regulatory mechanisms for small molecules in a specific group of bacteria, using techniques that are generalizable, and manipulate them to produce small molecules previously inaccessible in the lab. This will overcome a major roadblock in drug discovery, allowing the full exploitation of biosynthesis in isolated strains.