PlantSynBio: Regulatory Systems to Tune Gene Expression in Synthetic Chloroplast Operons

在过去的五十年里，对基础研究的投资在理解基本生物过程方面取得了显著进展，包括遗传、代谢和有机体发育的机制。合成生物学学科将这种理解应用于工程设计原则框架内的实际问题。该项目将利用这一框架，应用了解植物基因表达机制的最新进展，以改善农艺和营养性状，并促进植物作为药物和其他有益生物化学物质的“绿色”工厂的使用。从基础研究中推断出的基本原理将用于设计基因“部分”，以优化赋予有益特性的基因表达。该项目还将为本科生和博士后学生提供研究培训，并将让有风险的高中生参加营养生物化学的实践研讨会，作为俄勒冈大学一个更广泛的项目的一部分，旨在鼓励这些学生入学并在大学取得成功。该项目的总体目标是开发用于代谢工程、生物技术和提高植物农艺性状的工具。许多这样的应用需要多个基因的表达，最佳结果取决于它们的适当平衡表达。叶绿体遗传系统为多蛋白组合和多酶代谢途径的表达提供了一个特别的基础，因为叶绿体基因以操纵子样多顺反子转录单位自然表达，它们不经历表观遗传效应，并且它们可以达到非常高的表达水平。然而，目前的限制是缺乏特征遗传元件来编程在大动态范围内的可预测表达，特别是对于多基因应用的目的。该项目旨在通过利用以下方面的最新进展来填补这一空白：(i)了解决定天然叶绿体操纵子表达水平的机制，（ii）阐明五肽重复蛋白序列特异性RNA识别的基础，这是该调控的关键，以及（iii）编目天然叶绿体基因的翻译效率。这个深厚的知识库将用于合理设计顺式元件和同源反式因子的调色板，以实现合成多基因转录单元中可预测、可调节和适当调整的基因输出。该奖项由整合有机系统部门的植物基因组研究计划和分子与细胞生物科学部的系统与合成生物学集群共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Investments in basic research over the past five decades have been rewarded with remarkable advances in understanding fundamental biological processes, including mechanisms of heredity, metabolism, and organismal development. The discipline of synthetic biology applies this understanding to practical problems within a framework of engineering design principles. This project will use this framework to apply recent advances in understanding mechanisms of gene expression in plants to improve agronomic and nutritional traits, and to foster the use of plants as “green” factories for pharmaceuticals and other beneficial biochemicals. Foundational principles inferred from basic research will be used to design genetic “parts” to optimize the expression of genes that confer beneficial properties. This project will also provide research training for undergraduate and postdoctoral students, and it will engage at-risk high school students in hands-on workshops in nutritional biochemistry as part of a broader program at the University of Oregon that aims to encourage such students to enroll and succeed in college.The overarching goal of this project is to develop tools for metabolic engineering, biotechnology, and enhancement of agronomic traits in plants. Many such applications require expression of multiple genes, with optimal outcomes depending upon their suitably balanced expression. The chloroplast genetic system offers particular promise as a chassis for the expression of multiprotein assemblies and multi-enzyme metabolic pathways because chloroplast genes are naturally expressed in operon-like polycistronic transcription units, they do not experience epigenetic effects, and they can achieve very high expression levels. A current limitation, however, is a paucity of characterized genetic elements to program predictable expression over a wide dynamic range, especially for the purpose of multigene applications. This project aims to fill this gap by leveraging recent advances in (i) understanding mechanisms that determine expression levels in natural chloroplast operons, (ii) elucidating the basis for sequence-specific RNA recognition by pentatricopeptide repeat proteins, which are key to this regulation, and (iii) cataloging translational efficiencies of native chloroplast genes. This deep knowledge base will be used for the rational design of a palette of cis-elements and cognate trans-factors to achieve predictable, regulatable, and suitably tuned gene outputs in synthetic multigene transcription units.This award was co-funded by the Plant Genome Research Program in the Division of Integrative Organismal Systems and the Systems and Synthetic Biology Cluster in the Division of Molecular and Cellular Biosciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.