A Structural and Functional Investigation of "red" Rubisco
Lead Research Organisation:
Newcastle University
Department Name: Sch of Natural & Environmental Sciences
Abstract
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Griffithsia monilis we will use a homology-based approach, combined with RNAseq data previously gathered by our group (unpublished), to identify chaperone candidates.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.
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.
