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表征萜类吲哚生物碱途径将结合生物化学工程和植物分子生物学方面的专业知识，采取跨学科的方法来解决萜类吲哚生物碱（TIAs）和其他复杂代谢途径成功工程所面临的一些基本问题。为了增加对TIA通路的基本了解，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，将用于进一步探索这些途径的调控。本工作的具体目标是：(1)生成并鉴定过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系；(2)鉴定过表达TIA基因转录激活因子和TIA基因转录抑制因子表达降低对TIA代谢的影响；(3)发展TIAs和途径中间体的代谢谱分析方法，并将该技术应用于转基因毛状根系的分析。智力优势：提出的实验将显著增加对TIA和相关途径的理解。特别是，这些实验将有助于确定和量化代谢途径中不同步骤的代谢中间体。这些实验还将通过检查操纵TIA生物合成或调节基因的不同组合表达的影响，提供有关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商业重要药物的必要先决条件，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，并作出重大努力，纳入代表性不足的群体的成员。

Collaborative Research: Molecular Genetic and Metabolic Analyses to Characterize Terpenoid Indole Alkaloid PathwaysKa-yiu SanWilliam Marsh Rice UniversityCBET-0729622Expertise 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.