Strategic Reprogramming of the Ergot Alkaloid Pathway

麦角生物碱途径的战略性重编程

• 批准号: 10513437
• 负责人: Daniel G. Panaccione
• 金额: $ 45.6万
• 依托单位: WEST VIRGINIA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10513437
• 关键词: Acids; Active Sites; Address; Affect; Alkaloids; Alzheimer' s Disease; Amides; Anabolism; Aspergillus; Biochemical; CRISPR/Cas technology; Candidate Disease Gene; Complex; Dementia; Development; Ergot Alkaloids; Ergot Fungus; Family; Future; Gene Cluster; Gene Combinations; Genes; Genetic Transcription; Goals; Health; Homologous Gene; Housekeeping; Human; Hydrolase; Hyperprolactinemia; Knock-out; Knowledge; Lead; Methods; Migraine; Mixed Function Oxygenases; Modification; Molecular Analysis; Non-Insulin-Dependent Diabetes Mellitus; Outcome; Oxidoreductase; POU domain factors; Pantetheine; Parkinson Disease; Pathway interactions; Pharmacologic Substance; Process; Regulation; Research; Role; Route; Scheme; Senile dementia; Site-Directed Mutagenesis; Structure; Technology; Testing; Training; Uncertainty; Variant; base; esterase; experience; experimental study; fungus; gene discovery; gene function; gene product; gene synthesis; genetic manipulation; genetic resource; graduate student; improved; mutant; novel; peptide synthase; programs; protein folding; tool; translational potential; undergraduate student

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.

七、项目概要 麦角生物碱作为强大且多功能的药物可改善人类健康，用于治疗多种疾病 包括老年痴呆症、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。可以发现结构和活性的巨大多样性 天然和半合成麦角生物碱。结构上的微小变化可能会带来巨大的变化 活动的变化。该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多种麦角生物碱 麦角生物碱的生产和多样化。这个特定项目的目标之一是了解和控制 麦角酰胺的生物合成。许多更重要的药用麦角生物碱是 麦角酰胺或可以衍生自麦角酰胺。这些化合物的合成 涉及从非核糖体肽上卸载酰胺的替代和竞争机制 合成酶。现在对参与制造这些酰胺麦角生物碱的基因有了更多的了解 由于他们最近在实验上可处理的真菌中发现，包括三个物种，因此很容易获得 曲霉属以及绿僵菌。该项目的第二个目标是测试调查 最近发现的基因被认为可以控制麦角酰酰胺的调节和分泌；两个都 这些过程具有转化意义。拟议项目的具体目标是：1) 了解和控制麦角酸的竞争性非核糖体肽合成酶卸载机制 酰胺； 2) 确定新型麦角生物碱合成基因在积累和 麦角酰胺的分泌。目标 1 将通过两个互惠但独立的项目来实现 用于产生麦角酰肽还原酶结构域突变体的替代方案的方法 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 酰胺。目标 2 的实验方法将涉及转化和 CRISPR/Cas9 的应用- 基于我们开发的技术来评估转录调节因子和主要的功能 家庭运输促进者。将分析通过这些基因操作产生的真菌菌株 通过分子和生化方法来确定候选基因的表达或改变 影响了真菌的麦角生物碱特征。拟议项目的结果将揭示具体的角色 基因并提供产生具有药学意义的分子的真菌菌株。额外的 好处包括为研究生和本科生提供有意义的经历，以及进一步 开发用于修饰和改进其他或新型麦角生物碱的平台。