Strategic Reprogramming of the Ergot Alkaloid Pathway

7.项目总结/摘要 麦角生物碱作为治疗多种疾病的强大和多功能药物改善人类健康 包括老年性痴呆、阿尔茨海默病、帕金森病、偏头痛， 高催乳素血症和2型糖尿病。在结构和活性上可以发现巨大的多样性 在天然和半合成麦角生物碱中。结构上的微小变化可能导致巨大的 活动的变化。这项研究计划的长期目标是了解并最终控制 通过确定基因和基因产物的功能， 生产和多样化麦角生物碱。要求的设备是沃茨Arc HPLC（高性能 液相色谱）系统，将取代一个老化的仪器，提出频繁的维护和 数据管理挑战。新仪器将提高研究小组的效率， 提高数据质量。使用新仪器将为本科生和研究生提供 接受相关培训的学生。该仪器将用于收集每种方法的数据 被用于父母奖的两个具体目标，题为“麦角生物碱的战略重编程” 路”。这两个具体目标都涉及通过基于CRISPR/Cas9的基因修饰真菌麦角生物碱途径。 方法或异源基因的表达，然后通过以下方法分析改变的麦角生物碱谱： HPLC和随后的质谱法。新的HPLC将在以下方面发挥不可或缺的作用： 为所有计划的实验方法收集数据。高效液相色谱法在鉴别 哪些候选菌株已经通过重组DNA方法突变， 代谢产物在这些构建的菌株中积累。HPLC还将用于收集定量 麦角生物碱积累的数据，将用于统计分析，以检验以下假设 基因功能拟议项目的结果将揭示特定基因的作用，并提供 产生具有药物意义的分子的真菌。其他好处包括有意义的 研究生和本科生的经验，并进一步发展平台， 另外的或新的麦角生物碱的修饰和改进。

7. Project Summary/Abstract Ergot alkaloids improve human health as powerful and versatile pharmaceuticals for treatment of multiple conditions including senile dementia, Alzheimer’s disease, Parkinson’s disease, migraines, hyperprolactinemia, and type 2 diabetes. Tremendous diversity in structure and activity can be found among the natural and semi-synthetic ergot alkaloids. Small changes in structure may lead to a great changes in activity. The long-term goal of this research program is to understand and ultimately control the biosynthesis of diverse ergot alkaloids by determining the functions of genes and gene products that produce and diversify ergot alkaloids. The requested equipment, a Waters Arc HPLC (high performance liquid chromatography) system, will replace an aging instrument that presents frequent maintenance and data management challenges. The new instrument will enhance efficiency of the research team and improve the quality of data. Working with the new instrument will provide undergraduate and graduate students with relevant training. The instrument will be used to gather data for each of the approaches being used for both specific aims of the parent award, titled “Strategic reprogramming of the ergot alkaloid pathway.” Both specific aims involve modifying fungal ergot alkaloid pathways by CRISPR/Cas9-based approaches or expression of heterologous genes, and then analyzing the altered ergot alkaloid profiles by HPLC and subsequent mass spectrometry approaches. The new HPLC will have an integral role in gathering data for all of the planned experimental approaches. The HPLC will be critical in identifying which candidate strains have been mutated by the recombinant DNA approaches and in identifying the metabolites accumulating in those constructed strains. The HPLC also will be used to collect quantitative data on ergot alkaloid accumulation which will be used in statistical analyses to test hypotheses about gene function. Results of the proposed project will reveal roles of specific genes and provide strains of fungi that produce molecules with pharmaceutical significance. Additional benefits include meaningful experiences for graduate and undergraduate students and further development of platforms for modification and improvement of additional or novel ergot alkaloids.