Strategic reprogramming of the ergot alkaloid pathway

 描述(申请人提供)：麦角生物碱可改善人类健康，是治疗多种疾病的强大而通用的药物，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。不同类型的麦角生物碱是由远亲的真菌群产生的。某些种类的Clavicep和Eichloë会产生从麦角酸衍生的麦角生物碱，这些生物碱具有很强的药理活性。另外两种Clavicep可以制造二氢麦角生物碱，其中略有不同的化学成分会产生不同但同样强大的活动。第三类真菌，包括烟曲霉，产生一种麦角生物碱家族，不是从麦角酸或二水解丝氨酸中提取的，不用于临床；然而，烟曲霉菌生长更快，更容易操纵，具有更大的遗传改良麦角生物碱的潜力。所有这些真菌在它们的麦角生物碱途径中都有共同的早期步骤，然后分化成它们独特的最终产品。通过在特定的点上阻断烟曲霉菌的途径，并添加从一种麦角酸产生菌中选择的两个基因，烟曲霉菌已经被改造成生产麦角酸。拟议项目的长期目标是修改和扩大烟曲霉菌表达系统，以回答有关麦角酸和相关化合物起源的基本问题。除了回答基本的遗传学问题外，这项工作还将具有实际的医疗保健相关影响，因为经过改良的真菌菌株将产生重要的麦角生物碱，这些生物碱在自然界中很少见，但在药物方面是相关的化合物。具体目的是(1)澄清生产麦角酸的步骤，并阐明交替的麦角生物碱雀麦草酸和麦角豆醇的生物合成来源，以及(2) 确定二氢麦角生物碱的生物合成来源。将从产生这些独特麦角生物碱的真菌中分离出单个基因或两个基因的组合，并在烟曲霉菌中表达，以产生两种不同底物麦角生物碱中的一种。转化了这些基因的烟曲霉菌株将通过分子和生化方法进行分析，以确定候选基因的表达如何影响真菌的麦角生物碱特征。拟议项目的结果将揭示特定基因的作用并阐明 几种重要麦角生物碱的来源。该项目将生产烟曲霉菌株， 生产出具有药学意义的分子。其他好处包括为研究生和本科生提供有意义的经验，以及进一步开发一个平台，用于修改和改进额外或新的麦角生物碱。

 DESCRIPTION (provided by applicant): 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. Different types of ergot alkaloids are produced by distantly related groups of fungi. Certain species of Claviceps and Epichloë produce ergot alkaloids derived from lysergic acid, and these have powerful pharmacological activity. Two other species of Claviceps make dihydroergot alkaloids, in which slightly different chemistry results in different but equally powerful activitis. A third group of fungi, including Aspergillus fumigatus, makes a family of ergot alkaloids that is no derived from lysergic acid or dihydrolysergic acid and is not used clinically; however, A. fumigatus grows faster, is more readily manipulated, and has greater potential for genetically improving ergot alkaloids. All these fungi share common early steps in their ergot alkaloid pathways before diverging to make their unique end products. By blocking the A. fumigatus pathway at a specific point and adding two selected genes from a lysergic acid producer, A. fumigatus has been modified to produce lysergic acid. The long-term goals of the proposed project are to modify and expand the A. fumigatus expression system to answer basic questions about the origins of lysergic acid and related compounds. In addition to answering basic genetic questions, the work will have practical healthcare-related implications, because modified fungal strains will produce important ergot alkaloids that are rare in nature but are pharmaceutically relevant compounds. Specific aims are to (1) Clarify steps in the production of lysergic acid and elucidate the biosynthetic origins of alternate ergot alkaloids paspalic acid and lysergol, and (2) Determine the biosynthetic origin of dihydroergot alkaloids. Individual genes or combinations of two genes will be isolated from fungi that make these unique ergot alkaloids and expressed in A. fumigatus modified to produce one of two different substrate ergot alkaloids. The A. fumigatus strains transformed with these genes will be analyzed by molecular and biochemical methods to determine how expression of the candidate genes has affected the fungus's ergot alkaloid profile. Results of the proposed project will reveal roles of specific genes and elucidate origins of several important ergot alkaloids. The project will produce strains of A. fumigatus that produce molecules with pharmaceutical significance. Additional benefits include meaningful experiences for graduate and undergraduate students and further development of a platform for modification and improvement of additional or novel ergot alkaloids.