Collaborative Research: Molecular Genetic and Metabolic Analyses to Characterize Terpenoid Indole Alkaloid Pathways

合作研究：分子遗传和代谢分析以表征萜类吲哚生物碱途径Jacqueline Shanks爱荷华州立大学CBET-0729753生物化学工程和植物分子生物学专业知识将结合起来，采取跨学科方法，解决成功进行萜类吲哚生物碱工程(TIA)和其他复杂代谢途径工程所面临的一些基本问题。为了增加对TIA途径的基本了解，将开发和实施改进的代谢物分析方法。此外，代谢工程，特别是萜类途径和TIA转录因子的代谢工程，将被用来进一步探索这些途径的调控。本工作的具体目的是：(1)建立和鉴定高表达萜类和吲哚途径代谢基因策略组合的转基因玫瑰毛状根；(2)研究TIA基因转录激活子的高表达和TIA基因转录抑制子的低表达对TIA代谢的影响；(3)建立TIA和途径中间产物的代谢谱方法，并将该技术应用于所开发的转基因毛状根的分析。特别是，这些实验将有助于识别和量化途径中不同步骤的代谢中间产物。这些实验还将通过检测操纵不同组合的TIA生物合成或调控基因的表达的效果，提供关于这些途径调控的关键信息。更广泛的影响：从这项工作中获得的工具和知识是设计TIA和其他途径以生产更高水平的具有商业重要性的药品的必要前提，从而有助于降低处方药的成本。拟议的工作还将为本科生和研究生提供跨学科培训，并正在作出重大努力，以纳入任职人数不足的群体的成员。

Collaborative Research: Molecular Genetic and Metabolic Analyses to Characterize Terpenoid Indole Alkaloid PathwaysJacqueline ShanksIowa State UniversityCBET-0729753Expertise in biochemical engineering and plant molecular biology will be combined to take an interdisciplinary approach to addressing some of the fundamental issues facing successful engineering of terpenoid indole alkaloids (TIAs) and other complex metabolic pathways. To increase basic understanding of TIA pathways, improved methods of metabolite analysis will be developed and implemented. In addition, metabolic engineering, particularly of the terpenoid pathway and of TIA transcription factors, will be used to further explore regulation of these pathways. The Specific Aims to be carried out in this work are to: (1) generate and characterize transgenic C. roseus hairy root lines overexpressing strategic combinations of terpenoid and indole pathway metabolic genes, (2) characterize the effects of overexpressing transcriptional activators of TIA genes and of decreasing expression of transcriptional repressors of TIA genes on TIA metabolism, and (3) develop the methodology for metabolic profiling of TIAs and pathway intermediates and apply this technology to the analysis of the transgenic hairy root lines developed.Intellectual merit: The proposed experiments will significantly increase understanding of TIA and related pathways. In particular, these experiments will help identify and quantify the metabolic intermediates for different steps in the pathways. These experiments will also provide critical information about the regulation of these pathways by examining the effects of manipulating expression of different combinations of TIA biosynthetic or regulatory genes. Broader impacts: The tools and knowledge to be gained from this work represent a necessary prerequisite to engineering TIA and other pathways to produce higher levels of commercially important pharmaceuticals, thereby helping to lower the costs of prescription drugs. The proposed work will also provide interdisciplinary training for undergraduate and graduate students, with significant efforts being made to include members of underrepresented groups.