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，普渡大学的伊丽莎白·帕金森正在研究激素及其抑制物对链霉菌天然产物生物合成的调控。来自土壤细菌链霉菌的天然产物是操纵和研究生物过程的丰富的药物、农产品和化学工具的来源。虽然基因组学数据表明，有数十万种可能具有有趣生物活性的新型天然产物仍有待从链霉菌中发现，但许多产品在实验室培养时并不会产生。这很可能是因为细菌没有暴露在土壤中遇到的相同的化学刺激下。不幸的是，控制这些天然产物生产的化学信号仍然是个谜。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。这项拟议的实验旨在开发获得新激素并确定其调节的天然产品的改进方法。该项目还将实施一项教育计划，以提高农村学生接触科学的机会、科学认同感和兴趣。具体地说，将为农村高中开发一个以项目为基础的学习实验室，重点是从他们的后院分离产生抗生素的细菌。此外，还将推出适合年轻学生在农村儿童博物馆表演的改良版本的实验室。γ-丁内酯和丁烯内酯是调节天然产品生物合成的两类已知链霉菌荷尔蒙。据预测，超过一半的链霉菌菌株具有受这些激素调控的基因，但鲜为人知的例子是：1)激素产生的数量非常少，2)没有快速、有效的检测方法来鉴定它们。本研究项目的中心假设是，序列相似性网络中的抑制子基于其激素配体和DNA结合域的结构而聚在一起。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荷尔蒙资料库。该文库包括根据存在于受体基因组邻域内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报告分析和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。这些分析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。它们还将允许对受体序列相似性网络预测配体和DNA结合特异性的假设进行检验。如果正确，这将有助于开发荷尔蒙激活的预测模型，可能会允许发现新的天然产品。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.