Collaborative Research: Elucidating the Molecular Architecture and Dynamics of Phenylalanine Biosynthesis in Plants

苯丙氨酸是植物体内合成8000种酚类化合物的前体，对植物的生长、发育、繁殖和防御有着深远的影响。苯丙氨酸是许多植物天然产物的前体，广泛用于医药和食品补充剂。本项目将描述一种在植物细胞壁中合成苯丙氨酸的新途径，确定该途径对苯丙氨酸总产量的贡献。这一结果将大大有助于了解植物主要代谢网络的基础知识，并为更好地理解芳香族氨基酸的生物合成提供依据。这些信息将允许对苯丙氨酸水平进行合理的代谢工程，以生产重要的苯丙氨酸衍生化合物，以改善农艺性状、生物燃料生产、农产品的适口性和观赏植物的商业价值。计划中的研究将为本科生和研究生提供多学科培训。该教育计划还将通过直接的课堂互动和促进实验室练习，将当地一所中学的学生介绍给以STEM为基础的研究，让学生生成和测试假设。长期以来，人们一直认为芳香氨基酸的生物合成只发生在叶绿体中，在进入网络的入口点和向单个氨基酸的承诺步骤中，芳香氨基酸的生物合成都受到复杂的反馈调节。最近的发现发现，胞质中也形成了苯丙氨酸，这对植物如何调节平行途径之间的碳通量分配提出了新的问题。该项目将采用一种综合策略，包括遗传学、分子生物学、代谢图谱、蛋白质和膜生物化学、稳定同位素标记和代谢通量分析，以确定(I)植物是否包含完整的细胞质途径，(Ii)这条途径受到不那么严格的反馈调节，以及(Iii)当对苯丙氨酸的生产和消费的需求增加时，通过这条途径的通量增加。矮牵牛花易于遗传改变，不会对植物的生命力产生有害影响，并且含有丰富的苯丙氨酸和苯丙氨酸衍生的挥发物，将被用作模式系统。具体地说，该项目将揭示胞质苯丙氨酸生物合成的途径结构和分子成员。此外，该项目还将揭示胞质路线对苯丙氨酸总产量的能力和相对贡献。

The amino acid phenylalanine is the precursor for the synthesis of 8000 phenolic compounds in plants, which have profound impacts on plant growth, development, reproduction and defense. Phenylalanine serves as a precursor for numerous plant natural products that are widely used in medicine and as food supplements. This project will characterize a new pathway for phenylalanie synthesis in the plant cytostol determine the contribution of this pathway to the total phenylalanine production. The results will significantly contribute to the fundamental knowledge of a major plant metabolic network and provide a better understanding of aromatic amino acid biosynthesis. Such information will allow rational metabolic engineering of phenylalanine levels for production of important phenylalanine-derived compounds to improve agronomic traits, biofuel production, palatability of produce, and commercial value of ornamentals. The plannned research will provide multidisciplinary training to undergraduate and graduate students. The educational program will also introduce students from a local middle school to STEM-based research through direct classroom interactions and facilitating laboratory exercises where students generate and test hypotheses. It has long been accepted that aromatic amino acid biosynthesis occurs exclusively in plastids where it is subject to complex feedback regulation both at the entry point into the network and at the committed steps towards individual amino acids. The recent finding that phenylalanine is also formed in the cytosol raises new questions about how plants regulate carbon flux distribution between the parallel pathways. This project will employ an integrative strategy comprised of genetics, molecular biology, metabolic profiling, protein and membrane biochemistry, stable-isotope labeling, and metabolic flux analysis to determine if (i) plants contain a complete cytosolic pathway, (ii) this route is subject to less stringent feedback regulation, and (iii) the flux through this pathway increases when the demand for phenylalanine production and consumption increases. Petunia flowers, which are amenable to genetic alterations without deleterious effects to plant vitality, and are rich in phenylalanine and phenylalanine-derived volatiles, will be used as a model system. Specifically, this project will uncover the pathway structure and the molecular players of the cytosolic phenylalanine biosynthesis. Moreover, the project will reveal the capacity and relative contribution of the cytosolic route to overall production of phenylalanine.