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

合成生物学和细胞器基因组学：rubisco 文库作为进化景观和细胞器工程的案例研究

• 批准号: 10441273
• 负责人: Noam Prywes
• 金额: $ 9.39万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-12 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10441273
• 关键词: Address; Basic Science; Biochemical; Biochemistry; Biological Assay; Biological Models; Biology; Carbon; Career Mobility; Case Study; Chloroplasts; Data; Engineering; Environment; Enzymes; Escherichia coli; Evolution; Food Supply; Future; Gene Expression; Gene Library; Gene Mutation; Generations; Genes; Genome; Genomic Library; Genomics; Goals; Growth; Health; Human; In Vitro; Individual; Institutes; Knowledge; Laboratories; Libraries; Link; Maps; Mentors; Metabolic Control; Metabolic Pathway; Modeling; Molecular Evolution; Molecular Target; Mutate; Nuclear; Nutritional; Organelles; Outcome; Phase; Photosynthesis; Plant Model; Plants; Population; Process; Protein Isoforms; Proteins; Research Personnel; Ribulose-Bisphosphate Carboxylase; Science; Series; Technology; Testing; Tobacco; Training; Transgenic Organisms; Transgenic Plants; Translating; Variant; Work; carbon fixation; career; climate change; design; experimental study; food security; genetic selection; genetic variant; genome editing; homologous recombination; improved; in vivo; innovation; insight; knockout gene; machine learning model; metabolic engineering; mutant; mutation screening; novel; nuclease; plant growth/development; preference; promoter; synthetic biology; technology development; transcription activator-like effector nucleases

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.

随着全球人口数量的增加，粮食安全是21世纪人类健康面临的重大问题 人口接近 100 亿，气候变化威胁着粮食供应。光合作用工程有 在提高作物产量方面取得了成功，但固碳的核心酶 Rubisco 却已 作为分子进化的目标仍然是棘手的。少数经过异源试验的植物 rubiscos 的生长速度均比野生型慢。该提案的目标是 生成植物叶绿体基因组文库以探索 rubisco 功能的极限 细胞器基因组组织。 在本提案的目标 1 中，我将探索同二聚体模型的序列功能景观 rubisco 酶和我生成的一种新型连锁变体。我已经生成了一个深度突变 扫描（OMS：所有可能的点突变体）库，并将以独特的方式测定其功能 rubisco依赖性大肠杆菌菌株。我已经证明这些连接的 rubiscos 在体内和体内发挥作用 体外并已构建突变体库；在这个项目的K99阶段我将 对该库进行选择并使用机器学习模型分析数据。在R00 第一阶段将生成更多的文库来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 叶绿体编辑模型植物 N. 的叶绿体基因组文库规模空前。 烟草。为此，我将生成序列特异性 TALEN 核酸酶，其表达自 核基因组并被运输到叶绿体，在那里它们将切割和破坏基因组， 偏好转基因供体ONA的同源重组。演示 TALEN 切割后 K99一期将把这项技术发展成细胞内基因驱动器，这将加速 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，我 将生成具有改变的 rubisco 基因和启动子的植物文库，以证明 Rubisco 工程改善植物生长的潜力。另外，在R00阶段，我会 产生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，我的目标是研究模型植物中的细胞器生物学，以便 探索单个蛋白质和代谢途径的工程局限性。我的训练在 K99 期间的细胞器转化、蛋白质库生成和实验室管理 阶段将为我的 R00 阶段做好准备。加州大学伯克利分校创新基因组学研究所和 Ors的实验室。 Oavid Savage 和 Brian Sttaskawicz 尤其是理想的环境 为了我的职业转型，精确地结合了必要的专业知识组合 - 蛋白质 分别是进化和植物转化。