Synthetic biology and organelle genomics: A rubisco library as a case study in evolutionary landscapes and organellar engineering

作为全球人口，粮食安全是21世纪人类健康面临的一个关键问题 接近100亿，气候变化威胁到粮食供应。光合作用工程有 在提高作物产量方面取得了成功，但固碳的中心酶Rubisco 作为分子进化的目标仍然难以处理。用异源植物测试的为数不多的植物 红宝石的生长速度都比野生红宝石慢得多。这项提议的目标是 在植物中建立叶绿体基因组文库，以探索Rubisco功能的极限和 细胞器基因组组织。 在本提案的目标1中，我将探索同源二聚体模型的序列功能图景 Rubisco酶和我产生的一种新的连锁变体。我已经产生了一种深度突变 扫描(OMS：所有可能的点突变)文库，并将在唯一适合的情况下分析它们的功能 依赖Rubisco的大肠杆菌菌株。我已经证明了这些相连的Ruiscos在体内和体内发挥作用 已经构建了突变体库；在这个项目的K99阶段，我将 对该库执行选择，并使用机器学习模型分析数据。在R00中 第一阶段将生成更多的文库，以探索预计将 富含改良的红宝石，目标是在植物中测试它们。 在目标2中，我将改进叶绿体转化技术，以便能够产生 在叶绿体编辑模式植物N。 烟草属。为了做到这一点，我将产生序列特异性的TALEN核酸酶，它将从 核基因组并被运送到叶绿体，在那里它们将切割和破坏基因组， 倾向于转基因供体ONA的同源重组。在演示了TALEN-切入 K99第一阶段将把这项技术发展成细胞内的基因驱动，它将加速 叶绿体基因组编辑。 在目标3中，随着目标2中开发的技术和目标1中发现的Rubisco变体，我 将生成具有改变的Rubisco基因和启动子的植物文库，以便证明 Rubisco工程在改善植物生长方面的潜力。此外，在R00阶段，我将 制作全面的叶绿体基因敲除文库，以回答基本问题 关于基因从细胞器基因组到核基因组的进化流动。 作为一名独立研究员，我的目标是研究模式植物的细胞器生物学，以便 探索单个蛋白质和代谢途径的工程学极限。我受过的培训 K99期间细胞器的转化、蛋白质文库的生成和实验室管理 阶段将使我为R00阶段做好准备。加州大学伯克利分校创新基因组研究所和 奥斯的实验室。Oavid Savage和Brian Staskawicz尤其是理想的环境 在我的职业生涯过渡中，精确地结合了必要的专业知识-蛋白质 分别是进化和植物转化。

Food security is a critical issue facing human health in the 21st century as the global population approaches 10 billion and climate change threatens the food supply. Photosynthetic engineering has been successful in improving crop yields but the central enzyme of carbon fixation, rubisco, has remained intractable as a target of molecular evolution. The few plants tested with heterologous rubiscos grow uniformly slower than their wild-type counterparts. The goal of this proposal is to generate a chloroplast genome library in plants to explore the limits of rubisco function and organellar genome organization. In Aim 1 of this proposal I will explore the sequence-function landscape of a model, homodimeric rubisco enzyme and a novel, linked variant I have generated. I have generated a deep-mutational scan (OMS: all possible point-mutants) library and will assay their function in a uniquely suited rubisco-dependent E. coli strain. I have shown that these linked rubiscos function in vivo and in vitro and have already constructed the mutant library; in the K99 phase of this project I will perform selections on this library and analyze the data with a machine learning model. In the R00 phase I will generate further libraries to explore regions of the sequence landscape predicted to be rich in improved rubiscos with the goal of testing them in plants. In Aim 2 I will improve chloroplast transformation technology in order to enable the generation of chloroplast genome libraries of unprecedented size in the chloroplast-editing model plant N. tabacum. To do this I will generate sequence-specific TALEN nucleases that will be expressed from the nuclear genome and trafficked to the chloroplast where they will cut and disrupt the genome, preferencing homologous recombination of transgenic donor ONA. After demonstrating TALEN-cutting in the K99 phase I will develop this technology into an intracellular gene drive which will accelerate chloroplast genome editing. In Aim 3, with the technology developed in Aim 2 and the rubisco variants discovered in Aim 1, I will generate libraries of plants with altered rubisco genes and promoters in order to demonstrate the potential of rubisco engineering to improve plant growth. In addition, in the R00 phase, I will produce a comprehensive chloroplast gene knockout library in order to answer fundamental questions about the evolutionary flow of genes from organellar genomes to nuclear genomes. My goal as an independent investigator is to study organellar biology in model plants in order to explore the limits of engineering in individual proteins and metabolic pathways. My training in organellar transformation, protein library generation and laboratory management during the K99 phase will prepare me well for the R00 phase. The Innovative Genomics Institute at UC Berkeley and the laboratories of Ors. Oavid Savage and Brian Staskawicz in particular are the ideal environment for my career transition, combining precisely the necessary mixture of expertise - protein evolution and plant transformation, respectively.