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

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，气候变化威胁着粮食供应。光合作用工程 在提高作物产量方面取得了成功，但碳固定的核心酶rubisco， 作为分子进化的目标仍然难以解决。用异源植物测试的少数植物 Rubiscos比它们的野生型对应物均匀地生长得慢。本提案的目的是 在植物中产生叶绿体基因组文库以探索Rubisco功能的限制， 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-功能景观， rubisco酶和一个新的，连接的变体我已经产生。我生成了一个深度变异的 扫描（OMS：所有可能的点突变体）文库，并将在一个独特适合的 依赖Rubisco的E. coli菌株。我已经证明，这些连接的Rubiscos在体内和体内发挥作用， 体外，并已经构建了突变体库;在这个项目的K99阶段， 在此库上执行选择并使用机器学习模型分析数据。在R 00 第一阶段将产生更多的文库，以探索预测的序列景观区域， 富含改良的rubiscos，目的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. 烟草为了做到这一点，我将产生序列特异性TALEN核酸酶，其将从 核基因组并运输到叶绿体，在那里它们将切割和破坏基因组， 优选转基因供体ONA的同源重组。在演示了TALEN切割后， K99第一阶段将把这项技术发展成一种细胞内基因驱动器， 叶绿体基因组编辑 在目标3中，利用目标2中开发的技术和目标1中发现的rubisco变体， 将产生具有改变的rubisco基因和启动子的植物文库， rubisco工程改造提高植物生长的潜力。此外，在R 00阶段，我将 建立一个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。 作为一名独立研究者，我的目标是研究模式植物的细胞器生物学， 探索单个蛋白质和代谢途径工程的局限性。我的训练 K99期间的细胞器转化、蛋白质文库生成和实验室管理 阶段将为R 00阶段做好准备。加州大学伯克利分校的创新基因组学研究所， 奥斯的实验室特别是奥维德·萨维奇和布莱恩·斯塔斯卡维奇， 为了我的职业转型，将蛋白质和蛋白质的必要混合物 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.