Bilateral NSF/BIO-BBSRC Synthesis of Microcompartments in Plants for Enhanced Carbon Fixation

Lead Research Organisation: Lancaster University
Department Name: Lancaster Environment Centre

Abstract

Global demand for food and fuel is steadily increasing, while gains in yield of many major food crops through traditional breeding have leveled off in recent years. The natural variation that has been the source of substantial crop improvement is becoming exhausted, so that new efforts, including input from synthetic biology will be needed to improve photosynthetic efficiency. More than 90% of biomass is derived directly from photosynthetic products. The properties of the carbon-fixing enzyme Rubisco (ribulose-1:5-bisphosphate carboxylase/oxygenase) limit the efficiency of photosynthesis in land plants. Rubisco can catalyze the combination of RuBP (ribulose-1,5-bisphophate) with CO2, but also can catalyze the reaction of RuBP with oxygen, leading to photorespiration, a process in which previously fixed CO2 is lost. Cyanobacteria and some land plants have evolved to deal with an increase of oxygen in the atmosphere by developing mechanisms that concentrate CO2 near Rubisco. However, many of the globally important crop plants lack this ability; instead, they utilise Rubisco enzymes that have higher CO2 affinity but are slower than Rubisco enzymes in plants with carbon-concentrating mechanisms such as maize. Consequently, these plants must devote considerable amounts of protein, and thereby, nitrogen, to allow Rubisco to carry out adequate amounts of carbon fixation, reducing yield and biomass production.

One of the outstanding millenial goals is to find ways to improve photosynthetic yields for enhanced biomass production. Replacing endogenous Rubisco with a faster enzyme with less CO2 specificity, along with a carbon concentrating mechanism, is one way to significantly improve CO2 fixation, according to published computational models. We propose to undertake work to this end. We will install a novel cyanobacterial-based carbon-concentrating mechanism in a land plant chloroplast and provide the necessary molecular machinery to facilitate its operation. Regulatory modules will be produced to express components of the cyanobacterial carboxysome from the chloroplast genome and chloroplast membrane-targeted bicarbonate pumps from the nuclear genome. As a proof of principle, this work will be carried out in tobacco, a species in which chloroplast transformants can be most rapidly obtained. We have already established the ground work for this engineering feat, demonstrating that several of the components can be introduced; for example, to generate novel microcompartments within the tobacco chloroplast.

We expect the knowledge gained from the project will inform subsequent efforts to enhance photosynthesis in other species, for example by introducing synthetic microcompartments into species such as soybean, in which technology is already available for chloroplast and nuclear transformation. Knowledge will be gained through biochemical and microscopic studies, for example about the effect of stoichiometry of cyanobacterial proteins on microcompartment size, morphology, and function in carbon concentration and photosynthesis. We will examine features of the gene regulatory sequences on the synthetic chloroplast operons needed to express proteins in the amounts and ratios needed for assembly of microcompartments. We expect the findings to be valuable also for future projects and additions of other capabilities to plants by synthesis of artificial microcompartments.

Technical Summary

Experimental data and modeling indicate that increasing photosynthesis increases crop yields, provided that other constraints do not become limiting. One approach to enhancing the efficiency of photosynthesis is to introduce carbon-concentrating mechanism (CCM), which increases the CO2 substrate available for assimilation by Rubisco (ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase) and suppresses a competing reaction with O2. The oxygenation of RuBP is also catalysed by Rubisco and initiates photorespiration, with the consequent loss of fixed carbon, consumption of energy and release of nitrogen. CCMs limit photorespiration by altering the proportion of the two gaseous substrates, CO2 and O2, in the vicinity of Rubisco. While CCMs have evolved in a variety of organisms, including certain crop species such as maize, they are lacking in many globally important, so-called C3 crop species such as soybean and wheat. We propose to apply the methods of synthetic biology to engineer a CCM derived from cyanobacteria into the chloroplast in a model C3 plant, tobacco, with potential for future application to crop plants. The cyanobacterial Beta-carboxysome will be constructed in the tobacco chloroplast through expression of component proteins from the chloroplast genome. We will optimize chloroplast vectors for modular cloning that will allow rapid design of new operons to introduce carboxysomal proteins in the proper ratio for assembly of the microcompartment. Our efforts to adjust protein levels will be guided by assays of transgene expression, electron and fluorescence microscopy, and immunolocalization. Incorporating the complete system into chloroplasts will also require introduction of a nuclear-encoded bicarbonate transporter and genetic removal of carbonic anhydrase from the chloroplast stroma. Promising transgenic lines will be assayed for carbon assimilation, stomatal conductance, Rubisco amount and activity, and growth rate and biomass accumulation.

Planned Impact

This proposal is for fundamental research to develop new conceptual approaches relevant to ideas emerging within the international plant, systems and synthetic biology communities. The research to develop a new micro-compartment will stimulate thinking around strategies for modelling and for applications of synthetic biology in plants, especially in relation to photosynthesis, and it should strengthen methodologies relevant at many levels from cell to crop engineering. Thus, the research is expected to benefit fundamental researchers and, in the longer-term, agriculture and industry, through conceptual developments and approaches to not only improve carbon capture by plants but also for other applications (e.g. packaging bioactive molecules). The research will feed into higher education training programmes through capacity building at the postgraduate and postdoctoral levels. Additional impact is proposed through public displays and the development of teaching resources building on the background work for this proposal. Finally, the research will help guide future efforts in applications to agricultural/industrial systems. The applicants have established links with industrial/technology transfer partners and research institutes to take advantage of these developments. Further details of these, and additional impacts will be found in Part 1 of the Case for Support and in the attached Pathways to Impact document.

Publications

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Hanson MR (2016) Towards engineering carboxysomes into C3 plants. in The Plant journal : for cell and molecular biology

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Lin MT (2021) A procedure to introduce point mutations into the Rubisco large subunit gene in wild-type plants. in The Plant journal : for cell and molecular biology

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Zhu XG (2020) A wish list for synthetic biology in photosynthesis research. in Journal of experimental botany

 
Description Recently we completed analysis of transgenic lines which express a mixture of tobacco and cyanobacterial Rubisco, with and without a cyanobacterial aggregation factor. This work was recently published (Orr et al. 2020, Plant Physiology) and highlights the unexpected ability of the Rubisco small subunits from tobacco to assemble into complexes with the cyanobacterial Rubisco large subunit, and where an aggregation factor was also introduced, to also form large aggregates of Rubisco. The hybrid composition of these Rubisco enzymes had a major impact of growth and photosynthesis.

In June 2019 Lancaster based postdoc Douglas Orr attended the Gordon Research Seminar on CO2 assimilation, where he presented current findings and was able to take the chance to share thoughts and discuss work with leading researchers in photosynthesis. In January 2020 both Prof Maureen Hanson (project co-leader at Cornell University) and postdoc Douglas Orr were invited to speak about the project at the Plant and Animal Genome conference, where they presented the latest findings of the project, and were able to take part in discussions with other leaders in the field during an 'Organellar Genetics' session. This was also an invaluable opportunity for Prof Hanson and Dr Orr to discuss the project in person and plan future project activities. These discussions have already led to very interesting ideas about how to take forward the work with newer transgenic lines.

The analysis of newer transgenic lines from our collaborators is ongoing. These lines are expressing larger numbers of carboxysome components and are being analysed to determine if they are correctly producing all the introduced components, and whether this results in an assembled structure. We have so far had some interesting results that are inspiring us to evolve our analysis to decipher the structural composition of Rubisco aggregates, and their impact on carbon fixation biochemistry, plant growth and development.
Exploitation Route The understanding of how different compoments alter expression may be exploited more widely in synthetic biology projects
Sectors Agriculture, Food and Drink,Manufacturing, including Industrial Biotechology

 
Title Transient expression system using rice mesophyll protoplasts 
Description We have developed a high-throughput transient expression system using rice mesophyll protoplasts for assessing the efficacy of expression cassettes prior to stable plant transformation. This, coupled to our extensive library of DNA parts and the highly efficient Golden Gate cloning system, is allowing us to screen large numbers of single and multigene expression cassettes. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact This method coupled to our extensive library of DNA parts and the highly efficient Golden Gate cloning system, is allowing us to screen large numbers of single and multigene expression cassettes and identify useful promoters. 
 
Description Cornell University 
Organisation Cornell University
Department Department of Molecular Biology and Genetics
Country United States 
Sector Academic/University 
PI Contribution Led the development of grant proposals building on our previous collaborative research work
Collaborator Contribution Assisted in the development of this grant proposal building on previous collaborative research work
Impact Multiple publications listed under awarded grants. Numerous talks at International events.
Start Year 2012
 
Description University of Glasgow 
Organisation University of Glasgow
Country United Kingdom 
Sector Academic/University 
PI Contribution Led the development of grant proposals
Collaborator Contribution Assisted in the development of the research grant proposal
Impact Awarded research grant
Start Year 2015
 
Description Food, Health and Environmental Security 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact New breeding approaches to increase the yield & quality of crops. Palma de Mallorca, February 20 - 22nd, 2018
Year(s) Of Engagement Activity 2018
 
Description 2nd Agriculture and Climate Change Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 2nd Agriculture and Climate Change Conference
Year(s) Of Engagement Activity 2017
 
Description 3rd Synthetic Biology Congress, 20th -21st October, 2016, London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited presentation 'Synthesis of Microcompartments in Plants for Enhanced Carbon Fixation' at the 3rd Synthetic Biology Congress, 2016, London
Year(s) Of Engagement Activity 2016
 
Description CCM9 Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation at the IXth International Symposium on Inorganic Carbon Utilization by Aquatic Photosynthetic Organisms held in Cambridge.
Year(s) Of Engagement Activity 2016,2017
 
Description DEFRA University Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact This workshop brought together a group of universities with Defra policy teams / evidence specialists to explore the opportunities available through academic partnerships (including fellowships, studentships and other grant processes) and the benefits of engaging with external academic experts through developing contacts and networks.
Year(s) Of Engagement Activity 2018
 
Description Invited lecture 'Improving Rubisco' at Enhancing Photosynthesis in crop plants: Targets for improvement' at the Royal Society London December 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited lecture 'Improving Rubisco' at Enhancing Photosynthesis in crop plants: Targets for improvement' at the Royal Society London December 2016
Year(s) Of Engagement Activity 2016
 
Description Keynote for Keys Symposia 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Keynote presentation at Keygene Symposia
Year(s) Of Engagement Activity 2017
 
Description Newton Sustainable Rice Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Increasing photosynthesis and yield of rice through exploitation of a cyanobacterial CO2 concentrating mechanism
Year(s) Of Engagement Activity 2017
 
Description Waitrose Agronomy Group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Plant breeding and genetics for enhanced yield
Year(s) Of Engagement Activity 2017
 
Description World Grain Forum 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Keynote lecture and breakfast discussion with minister of agriculture at the World Grain forum in Sochi, Russia
Year(s) Of Engagement Activity 2016
 
Description World Life Science Conference in Plant and Environment session, Beijing 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Keynote lecture 'Improving Photosynthesis the engine of Life to Increase Crop Yields' in Plant and Environment session at the World Life Science Conference, Beijing 2016
Year(s) Of Engagement Activity 2016
 
Description • 'Discovery and creation of genetic variation to enhance the yield potential of wheat' Keynote lecture at 9th International Wheat Conference IWC Sydney, Australia September 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited talk 'Discovery and creation of genetic variation to enhance the yield potential of wheat' Keynote lecture at 9th International Wheat Conference IWC Sydney, Australia September 2015
Year(s) Of Engagement Activity 2015
 
Description • 'Plenary Lecture at the 1st International Workshop on Food and Health Security, Lima Peru October 2016. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Plenary talk on 'Discovery and creation of genetic variation to increase crop performance for current and future environments' at the 1st International Workshop on Food and Health Security, Lima Peru October 2016.
Year(s) Of Engagement Activity 2016