Strategic Reprogramming of the Ergot Alkaloid Pathway

7.项目总结 麦角生物碱作为治疗多发性硬化症的强效药物改善人类健康 包括老年痴呆症、阿尔茨海默氏症、帕金森氏症、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。结构上的小变化可能会导致大的 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来生物合成不同的麦角生物碱 生产和多样化麦角生物碱。这个特定项目的一个目标是理解和控制 麦角酸酰胺的生物合成。许多更重要的药用麦角生物碱是 麦角酸酰胺或可由麦角酸酰胺衍生而成。这些化合物的合成 涉及从非核糖体肽中卸载酰胺的交替和竞争机制 合成酶。现在对制造这些酰胺麦角生物碱的基因的了解更多了 因为他们最近在一种实验上易驯化的真菌中发现了包括三个物种在内的 曲霉属和褐变绿僵菌属。这个项目的第二个目标是测试和调查 最近发现的基因被假设为控制麦角酸酰胺的调节和分泌；两者 这些过程中的一部分具有翻译意义。拟议项目的具体目标是：1) 了解和控制竞争的非核糖体多肽合成酶的卸载机制 以及，2)确定新的麦角生物碱合成基因在积累和 麦角酸酰胺的分泌。目标1将通过两个相互但独立的 用替代方案产生谷氨酰肽还原酶域突变体的方法 合成酶与其他麦角生物碱合成基因配对以了解释放麦角酸的步骤 阿米德。目标2的实验方法将涉及应用变换和CRISPR/CAS9- 基于我们开发的技术来评估转录调节因子和主要的 协助者家庭运输机。这些基因操作产生的真菌菌株将被分析 通过分子和生化方法来确定候选基因的表达或改变 已经影响了这种真菌的麦角生物碱分布。拟议项目的结果将揭示特定的角色 基因，并提供产生具有药学意义的分子的真菌菌株。其他内容 好处包括为研究生和本科生提供有意义的体验，并进一步 开发用于修饰和改进额外或新型麦角生物碱的平台。

7. Project Summary 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. One goal of this particular project is to understand and control the biosynthesis of lysergic acid amides. Many of the more important pharmaceutical ergot alkaloids are lysergic acid amides or could be derived from lysergic acid amides. The synthesis of these compounds involves alternate and competitive mechanisms for off-loading amides from a non-ribosomal peptide synthetase. An understanding of the genes involved in making these amide ergot alkaloids is now more accessible because of their recent discovery in an experimentally tractable fungi, including three species of Aspergillus as well as Metarhizium brunneum. A second goal of this project is to test investigate recently discovered genes hypothesized to control regulation and secretion of lysergic acid amides; both of these processes have translational significance. Specific aims of the proposed project are to: 1) Understand and control competing non-ribosomal peptide synthetase offloading mechanisms for lysergic acid amides; and, 2) Determine roles of novel ergot alkaloid synthesis genes in accumulation and secretion of lysergic acid amides. Aim 1 will be pursued through two reciprocal but independent approaches with alternate schemes for producing mutants of the reductase domain of a lysergyl peptide synthetase to pair with other ergot alkaloid synthesis genes to understand steps in liberating lysergic acid amides. The experimental approach to Aim 2 will involve application of transformation and CRISPR/Cas9- based technologies we have developed to assess functions of a transcriptional regulator and a major facilitator family transporter. The fungal strains produced by these genetic manipulations will be analyzed by molecular and biochemical methods to determine how expression or alteration of the candidate genes has affected the fungus's ergot alkaloid profile. 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.