CAREER: A Multidisciplinary Approach for the Discovery and Characterization of Hormone Inducers of Natural Product Biosynthetic Gene Clusters

在化学系生命过程化学项目的支持下，普渡大学的Elizabeth Parkinson正在研究激素及其阻遏物对链霉菌天然产物生物合成的调节。来自土壤中的链霉菌的天然产物是药物、农产品和用于操纵和研究生物过程的化学工具的邦蒂富尔来源。虽然基因组学数据表明，仍有数十万种可能具有有趣生物活性的新型天然产物有待于从链霉菌中发现，但许多在实验室中生长时并不产生。这可能是因为细菌没有暴露在土壤中遇到的相同化学刺激下。 不幸的是，调节这些天然产物生产的化学信号仍然是一个谜。了解这些信号对于获得这些天然产物并最终最大限度地发挥链霉菌的天然产物潜力至关重要。拟议的实验旨在开发改进的方法，以获得新的激素和确定它们调节的天然产物。该项目还将实施一项教育计划，以改善农村学生获得科学、科学认同和兴趣的机会。具体而言，将为农村高中开发一个基于项目的学习实验室，重点是从后院分离产寄生虫细菌。此外，还将制作一个适合年轻学生在农村儿童博物馆表演的修改版实验室。γ-丁内酯和丁烯内酯是两种已知的调节天然产物生物合成的链霉菌激素。预计超过一半的链霉菌菌株具有受这些激素调节的基因，但已知的例子很少，因为1）激素的产量非常低，2）没有快速，有效的检测方法来鉴定它们。该研究项目的中心假设是，序列相似性网络中的阻遏物基于其激素配体和DNA结合结构域的结构而聚集。为了测试这一点，将开发不同的合成路线，使人们能够轻松获得扩大的激素库。该文库包括基于受体基因组邻域内存在的激素生物合成基因簇预测的激素。然后将利用高通量方法（即GFP报告基因测定和DNA亲和纯化测序）来探索受体文库的配体和结合位点特异性。这些测定有望为激素影响天然产物生物合成的机制提供有价值的见解。它们还将允许测试受体的序列相似性网络预测配体和DNA结合特异性的假设。如果正确的话，这将使开发激素激活的预测模型成为可能，从而可能发现新的天然产物。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

With the support of the Chemistry of Life Processes program in the Division of Chemistry, Elizabeth Parkinson of Purdue University is studying the regulation of Streptomyces natural product biosynthesis by hormones and their repressors. Natural products from the soil-dwelling bacteria Streptomyces are a bountiful source of medicines, agricultural products, and chemical tools for manipulating and studying biological processes. While genomics data suggests that hundreds of thousands of novel natural products likely to have interesting bioactivities remain to be discovered from Streptomyces, many are not produced when grown in the laboratory. This is likely because the bacteria are not exposed to the same chemical stimuli that they encounter in the soil. Unfortunately, the chemical signals that regulate the production of the majority of these natural products remain a mystery. Understanding these signals is critical to accessing these natural products and ultimately maximizing the natural product potential from Streptomyces. The proposed experimentation is directed at developing improved methods for accessing novel hormones and determining the natural products they regulate. The project will also implement an education plan to improve rural student access to science, science identity and interest. Specifically, a project-based learning laboratory for rural high schools focused on isolation of antibiotic-producing bacteria from their backyard will be developed. Additionally, a modified version of the lab appropriate for younger students to perform at rural children’s museums will be generated.γ-Butyrolactones and butenolides are two of the known classes of Streptomyces hormones that regulate natural product biosynthesis. Over half of Streptomyces strains are predicted to have genes regulated by these hormones, but few examples are known because 1) hormones are produced at very low quantities and 2) no rapid, efficient assays exist to identify them. The central hypothesis of this research project is that repressors in sequence similarity networks cluster based on the structure of their hormone ligands and their DNA-binding domains. To test this, divergent synthetic routes that allow easy access to an expanded library of hormones will be developed. This library includes hormones predicted based on the hormone biosynthetic gene clusters that exist within the genomic neighborhood of the receptors. High throughput methods (i.e. GFP-reporter assays and DNA affinity purification sequencing) will then be utilized to explore ligand and binding-site specificity for a library of receptors. These assays are expected to provide valuable insight into the mechanisms by which hormones affect natural product biosynthesis. They will also allow for testing of the hypothesis that sequence similarity networks of receptors are predictive of ligand and DNA-binding specificity. If correct, this will enable development of a predictive model for hormone activation that will likely permit the discovery of novel natural products.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.