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等人)。2015年)。人们正在研究作物生长的许多方面以提高表现，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等人。这个项目的目标是：1.格里菲斯属植物的基因组特征。对念珠藻的基因组进行测序将使人们能够发现伴侣蛋白和相关因子，从而能够将功能性的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相关蛋白的搜索。鉴定伴侣对于开发“红色”RuBiscos提供的改进的动力学是必要的。一旦我们对鹰嘴兽的基因组进行了测序，我们将使用基于同源性的方法，结合我们小组之前收集的RNAseq数据(未发表)来确定伴侣候选。根据Aigner等人建立的技术，候选基因将与GmRubisco在缺乏天然Rubisco和Rubisco生物发生蛋白的莱茵哈迪氏杆菌菌株中共表达，并在大肠杆菌中共表达。(2018)。能够成功折叠和组装Rubisco的蛋白质将被进一步研究。在我们的研究中发现的任何伴侣都将在大肠杆菌中进行重组表达和纯化，用于结构研究，既作为单独的蛋白质，也作为单独的Rubisco亚基和L8S8 Rubisco的复合体。将使用X射线结晶学和低温电子显微镜进行这些研究。设计一种复杂的克隆系统，利用多种“红色”和“绿色”RuBisco物种的序列产生Rubisco嵌合体。将设计一种克隆系统，利用细分的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。我们将使用克隆策略的组合，包括金门、启动-停止和吉布森组装，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。成功将使我们能够鉴定出莱茵哈迪尔乳杆菌伴侣蛋白相互作用的必要基序，并有可能从一些未被研究的物种中表达出没有物种特异性伴侣的“红色”RuBiscos。Rubisco异源表达条件的优化本实验室将对Rubisco突变株的表达和研究进行优化，并对Rubisco的重组表达转化、培养技术和菌种文库维护进行优化。高通量Rubisco定量和动力学筛选的开发。该领域的一个主要补充将是开发突变Rubisco表达和功能的高通量筛查。由于折叠差异，突变体将在不同的水平上表达，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异体具有改善的性质和良好的表达将是无价的。我们希望使用一种不依赖14C的屏幕，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Griffithsia monilis we will use a homology-based approach, combined with RNAseq data previously gathered by our group (unpublished), to identify chaperone candidates.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.