A Structural and Functional Investigation of "red" Rubisco

估计表明，为了维持未来的人口，2050年作物产量需要增加50% （Simkin et al. 2015）。作物生长的许多方面正在被研究以提高性能，包括提高水分利用效率和抗旱性，但工程光合作用将是这个项目的重点（Parry et al. 2013; Sharwood 2017）。这个项目的目标是：1。毛鼠格里菲斯的基因组特征。G. monilis的基因组测序将有助于发现伴侣蛋白和相关因子，使功能性GmRubisco转化为高等植物。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。基于同源性的Rubisco相关蛋白搜索。识别伴侣对于利用“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），来确定伴侣候选物。候选基因将与GmRubisco在缺乏天然Rubisco和Rubisco生物发生蛋白的C. reinhardtii菌株中共表达，并按照Aigner等人（2018）建立的技术在大肠杆菌中共表达。使Rubisco成功折叠和组装的蛋白质将被进一步研究。新型“红色”伴侣的结构特征。在我们的研究中发现的任何伴侣都将在大肠杆菌中重组表达和纯化，以进行结构研究，无论是作为单个蛋白质还是与单个Rubisco亚基和L8S8 Rubisco复合物。x射线晶体学和低温电镜将用于这些研究。设计一个复杂的克隆系统，利用来自多个物种的“红色”和“绿色”Rubisco的序列产生Rubisco嵌合体。克隆系统将被设计成能够使用细分的rbcL和rbcS基因组装Rubisco序列的多次迭代，允许环和螺旋在物种之间互换。我们将使用包括Golden-Gate， Start-Stop和Gibson组装在内的克隆策略组合，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序列库，以允许跨物种的“拾取和混合”组装。成功将允许识别必要的基序为C. reinhardtii伴侣相互作用和潜在的表达“红色”Rubiscos从一些未被充分研究的物种没有物种特异性伴侣。Rubisco在莱茵花中异源表达的优化。Rubisco突变体表达和研究的主要基础将是C. reinhardtii；我们的实验室将优化Rubisco重组表达的转化，以及培养技术和菌株库的维护。Rubisco定量和动力学高通量筛选的开发。该领域的一个主要补充将是开发高通量筛选突变Rubisco表达和功能。由于折叠差异，突变体的表达水平不同，不可避免地存在动力学差异；一个高通量的工具对于快速监测哪些变体具有改进的特性和良好的表达是非常宝贵的。我们希望使用一种不依赖于14C的筛选，因此我们将探索荧光抗体和下游3-PGA反应的潜力。

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Griffithsia monilis we will use a homology-based approach, combined with RNAseq data previously gathered by our group (unpublished), to identify chaperone candidates.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.