Symbiont Models for Natural Product Pathway Manipulation

说明(由申请人提供)：通知号(NOT-OD-09-058)NIH宣布恢复法资金可用于竞争性修订申请天然产品仍然是治疗药物的主要成分。然而，它们在创造衍生品方面遇到了困难，而且它们也可能很难供应。衍生物在优化性质方面非常重要，例如毒性或溶解性，这些性质对治疗学的发展至关重要。最近，利用天然产物生物合成基因的基因工程建立衍生物库已经变得越来越流行。即使在最好的图书馆里，衍生词的数量也只有数百或更少的数千。最近，我们发现了一条进化路径，有可能被收获来产生数百亿美元的衍生品。这些化合物可以通过在不同的新环境中直接挑战生产的大肠杆菌来“进化”。我们希望开发一种系统，在这种系统中，新的活动可以在几天到几周内进行药理优化，探索各种非天然天然产品。为了充分利用这种不同寻常的进化特性来应用于人类健康需求，在这次修订中，我们建议：1)表征~800万规模的文库的化学输出。我们将确定这一天然产物生物合成途径的化学产出是否有任何限制。2)应用研究该文库的蛋白质-蛋白质相互作用、抗生素活性和诊断能力的分析。我们将测试这些“不断发展的”系统对治疗兴趣的广泛和其他困难的靶区的适用性。3)优化文库复合表达和多样化。我们将探索在大肠杆菌中的可再生表达和提高稳定产量。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。将探索新酶对图书馆发展的适应性。 公共卫生相关性：我们将利用最新的科学发现来治疗领域，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)。已发现的化合物将被迅速优化，以推进临床前试验的药物先导。

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.