A Structural and Functional Investigation of "red" Rubisco

“红色”Rubisco 的结构和功能研究

• 批准号: 2281187
• 金额: --
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2281187
• 关键词: Structural; Functional; Investigation; red; Rubisco

Estimates indicate that crop production needs to increase by 50% in 2050 in order to sustain the future population (Simkin et al. 2015). Many aspects of crop growth are being studied to enhance performance, including improving water use efficiency and drought resistance, but engineering photosynthesis will be the focus of this project (Parry et al. 2013; Sharwood 2017).Objectives of this project are:1. Genomic characterisation Griffithsia monilis.Sequencing the genome of G. monilis would enable the discovery of chaperone proteins and associated factors enabling the transformation of functional GmRubisco into higher plants. We will sequence the genome using PacBio long-read sequencing and will subsequently annotate the genome.2. Homology based search for Rubisco associated proteins.Identifying chaperones is necessary to exploit the improved kinetics offered by "red" Rubiscos. Once we have sequenced the genome of Griffithsia monilis we will use a homology-based approach, combined with RNAseq data previously gathered by our group (unpublished), to identify chaperone candidates. Candidates will be co-expressed with GmRubisco in a C. reinhardtii strain lacking native Rubisco and Rubisco biogenesis proteins, and in E. coli following the techniques established by Aigner et al. (2018). Proteins that enable successful folding and assembly of Rubisco will then be studied further.3. Structural characterisation of novel "red" chaperones.Any chaperones found in our investigations will be recombinantly expressed and purified in E. coli for structural investigations, both as individual proteins and in complex with individual Rubisco subunits and L8S8 Rubisco. X-ray crystallography and Cryo-EM will be used for these investigations.4. Design of a sophisticated cloning system to produce Rubisco chimeras using sequences from multiple species of both "red" and "green" Rubisco.A cloning system will be designed to enable assembly of multiple iterations of Rubisco sequences using subdivided rbcL and rbcS genes, allowing loops and helices to be interchanged between species. We will use a combination of cloning strategies including Golden-Gate, Start-Stop and Gibson assembly to ensure we do not produce scars in the protein sequence. A general Rubisco sequence library will be established to allow "pick-and-mix" assembly across species. Success will allow the identification of necessary motifs for C. reinhardtii chaperone interactions and the potential to express "red" Rubiscos from a number of understudied species without the species-specific chaperones.5. Optimisation of heterologous expression of Rubisco in C. reinhardtii.The main chassis for mutant Rubisco expression and investigation will be C. reinhardtii; transformation for recombinant expression of Rubisco will be optimised in our lab as will culturing techniques and strain library maintenance.6. Development of a high-throughput screen for Rubisco quantification and kinetics.A major addition to the field would be the development of a high-throughput screen for mutant Rubisco expression and function. Mutants will be expressed at varying levels due to folding differences and there will be inevitable kinetic differences; a high-throughput tool would be invaluable to quickly monitor which variants have improved properties and are well expressed. We want to use a screen that does not rely on 14C so we will explore potential with fluorescent antibodies and downstream 3-PGA reactions.

估计表明，到2050年，作物产量需要增加50%，以维持未来的人口（Simkin等人，2015）。目前正在研究作物生长的许多方面以提高性能，包括提高水分利用效率和抗旱性，但工程光合作用将是该项目的重点（Parry et al. 2013; Sharwood 2017）。念珠格里菲思菌的基因组特征。monilis将能够发现伴侣蛋白和相关因子，从而能够将功能性GmRubisco转化到高等植物中。我们将使用PacBio长读测序对基因组进行测序，并随后对基因组进行注释。2.基于同源性的Rubisco相关蛋白的搜索。识别分子伴侣对于利用“红色”Rubisco提供的改进动力学是必要的。一旦我们对Griffithsia monilis的基因组进行了测序，我们将使用基于同源性的方法，结合我们小组先前收集的RNAseq数据（未发表），以确定伴侣候选人。候选物将与GmRubisco在C. reinhardtii菌株缺乏天然Rubisco和Rubisco生物合成蛋白;根据艾格纳等人（2018）建立的技术，使Rubisco能够成功折叠和组装的蛋白质将进一步研究。新的“红色”分子伴侣的结构特征：在我们的研究中发现的任何分子伴侣都将在大肠杆菌中重组表达和纯化。大肠杆菌的结构研究，无论是作为单独的蛋白质，并在与个别Rubisco亚基和L 8 S8 Rubisco的复合物。X射线晶体学和Cryo-EM将用于这些调查。设计复杂的克隆系统以使用来自“红色”和“绿色”Rubisco的多个物种的序列产生Rubisco嵌合体将设计克隆系统以使得能够使用细分的rbcL和rbcS基因组装Rubisco序列的多次迭代，从而允许环和螺旋在物种之间互换。我们将使用一系列的克隆策略，包括Golden-Gate、Start-Stop和吉布森组装，以确保我们不会在蛋白质序列中产生疤痕。将建立一个通用的Rubisco序列文库，以允许跨物种的“挑选和混合”组装。成功将允许识别C的必要基序。reinhardtii分子伴侣的相互作用和潜在的表达“红色”Rubiscos从一些未充分研究的物种没有物种特异性分子伴侣. Rubisco在C.突变体Rubisco表达和研究的主要底盘将是C. reinhardtii;本实验室将优化Rubisco重组表达的转化以及培养技术和菌株库维护。Rubisco定量和动力学的高通量筛选的发展：该领域的一个主要补充是突变体Rubisco表达和功能的高通量筛选的发展。由于折叠差异，突变体将以不同的水平表达，并且将存在不可避免的动力学差异;高通量工具对于快速监测哪些变体具有改善的特性并且表达良好将是非常宝贵的。我们希望使用不依赖于14 C的筛选，因此我们将探索荧光抗体和下游3-PGA反应的潜力。