Symbiont Models for Natural Product Pathway Manipulation

描述（由申请人提供）：通知号（no - od -09-058） NIH宣布为竞争性修订申请提供恢复法案基金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存在困难，而且它们也难以供应。衍生物在优化性质方面非常重要，例如毒性或溶解度，这对治疗学的发展至关重要。近年来，利用天然产物生物合成基因的基因工程技术创建衍生物文库已变得越来越流行。即使在最好的图书馆中，衍生品的数量也只有几百个或几千个。最近，我们发现了一条进化途径，可以用来产生数百亿的衍生品。这些化合物可以通过在不同的新环境中直接挑战大肠杆菌的产生而“进化”。我们希望开发一种系统，在这种系统中，新的活性可以在几天到几周内进行药理学优化，探索各种非天然的天然产品。为了充分利用这一不寻常的进化特性，将其应用于人类健康需求，在本次修订中，我们建议：1)表征约800万规模文库的化学输出。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)应用检测方法探索蛋白质-蛋白质相互作用、抗生素活性和该文库的诊断能力。我们将测试这些“不断发展的”系统在治疗兴趣的广泛和其他困难的目标领域的适用性。3)优化库的复合表达和多样化。我们将探索可再生表达并提高大肠杆菌的稳定产量。4)提供进一步的途径工具，为化合物添加其他翻译后修饰。探讨新酶在文库建设中的适用性。

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.