Symbiont Models for Natural Product Pathway Manipulation

描述（由申请人提供）：通知编号（NOT-OD-09-058）NIH宣布恢复法基金的竞争性修订申请的可用性天然产品仍然是大多数治疗药物。然而，它们在创造衍生品方面遇到了困难，而且它们也很难供应。衍生物在优化性质（例如对开发治疗剂至关重要的毒性或溶解度）方面极其重要。最近，利用天然产物生物合成基因的基因工程来创建衍生物文库已经变得越来越流行。即使在最好的图书馆里，衍生品的数量也只有几百或几千个。最近，我们发现了一种进化途径，可以被收获以产生数百亿种衍生物。这些化合物可以通过直接挑战生产E.大肠杆菌与不同的新环境。我们希望开发一个系统，在这个系统中，新的活动可以在几天到几周的时间内进行优化，探索各种各样的非自然的天然产品。为了充分利用这种不寻常的进化特性，以满足人类健康的需求，在这次修订中，我们建议：1）表征约800万规模库的化学输出。我们将确定这种天然产物生物合成途径的化学输出是否有任何限制。2)应用检测，探索蛋白质-蛋白质相互作用，抗生素活性，和该库的诊断能力。我们将测试这些“不断发展”的系统对广泛和困难的治疗目标领域的适用性。3)优化文库复合表达和多样化。我们将探索在E.杆菌4)提供进一步的途径工具，以向化合物添加其他翻译后修饰。将探索新酶对文库开发的适用性。 公共卫生相关性：我们将利用最新的科学发现，包括抗生素，蛋白质相互作用疾病和诊断（如癌症和免疫疾病）的治疗领域。发现的化合物将被快速优化，以推进临床前测试的药物先导。

DESCRIPTION (provided by applicant): Notice Number (NOT-OD-09-058) NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications Natural products are still the majority of therapeutic agents. However, they suffer from a difficulty in the creation of derivatives, and they can also be hard to supply. Derivatives are extremely important in the optimization of properties, such as toxicity or solubility that are critical to development of therapeutics. Recently, it has become increasingly popular to create libraries of derivatives using genetic engineering of natural product biosynthetic genes. Even in the best libraries, the numbers of derivatives are in the hundreds or low thousands. Recently, we discovered an evolutionary pathway that could potentially be harvested to generate tens of billions of derivatives. The compounds can be "evolved" by directly challenging producing E. coli with different new environments. We hope to develop a system in which new activities can be pharmacologically optimized in a matter of days to weeks, exploring a huge diversity of unnatural natural products. To fully harness this unusual evolutionary property for application to human health needs, in this revision we propose to: 1) Characterize the chemical output of the ~8 million-scale library. We will determine whether there are any limits on the chemical output of this natural products biosynthetic pathway. 2) Apply assays that explore protein-protein interactions, antibiotic activity, and diagnostic capability of this library. We will test the suitability of these "evolving" systems for broad and otherwise difficult target areas of therapeutic interest. 3) Optimize library compound expression and diversification. We will explore renewable expression and improve stable yields in E. coli. 4) Provide further pathway tools to add other post-translational modifications to the compounds. The suitability of new enzymes to library development will be explored. PUBLIC HEALTH RELEVANCE: We will harness recent scientific discoveries to therapeutic areas including antibiotics, protein interaction diseases, and diagnostics (such as for cancers and immune diseases). Compounds discovered will be rapidly optimized to advance drug leads for preclinical testing.