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

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

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 Impact Pathways.

Publications

10 25 50
 
Description This project is in its early stages. Therefore, what developments there are arise from pre-application knowledge. These studies showed that it is possible to generate model plant species that express components necessary to build pyrenoid-like compartments. These compartments are normally found in algae and help enhance photosynthesis. The purpose of this project is to complete the construction of these compartments in order to enhance photosynthetic yields that will direct efforts towards enhanced crop biomass production.
Exploitation Route Assuming that the work is successful, the methods should be applicable to many horticultural and crop species and could be used to support agro-industry. In principle, therefore, the outcomes could aid in food production in the face of global environmental challenge.
Sectors Agriculture, Food and Drink,Energy,Environment,Manufacturing, including Industrial Biotechology,Other

 
Description Too early to say, so this is simply a placeholder.
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Energy,Environment
Impact Types Societal

 
Title 2in1 vector systems 
Description Synthetic biology vectors for transient and stable transformation with quantitative visual reporting on cell-by-cell basis 
Type Of Material Technology assay or reagent 
Year Produced 2009 
Provided To Others? Yes  
Impact Multiple publications from my own research group and over 100 research groups worldwide Vector system distributions to more than 500 research groups worldwide 
URL http://psrg.org.uk
 
Title EZ-Rhizo 
Description Computer software tool for quantitative measurement and analysis of root growth/development 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title Henry 
Description Software for electrophysiology and imaging data aquisition and analysis 
Type Of Material Technology assay or reagent 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title Multicistronic vector systems 
Description Synthetic biology vector systems for transient and stable transformation for expressing multiple, tagged proteins and for quantitative analysis of membrane traffic and transport 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and over 100 research groups worldwide Vector system distributions to more than 500 research groups worldwide 
URL http://psrg.org.uk
 
Title OnGuard 
Description Systems biology software for quantitative modelling of cellular transport and homeostasis 
Type Of Material Physiological assessment or outcome measure 
Year Produced 2012 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title SUS vector systems 
Description Synthetic biological vector systems for protein-protein interaction screening 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and over 100 research groups worldwide Vector system distributions to more than 500 research groups worldwide 
URL http://psrg.org.uk
 
Title Software tools for electrophysiology and imaging 
Description The laboratory continues to develop and refine software/hardware tools for data acquisition and analysis relevant to electrophysiology, single-cell imaging and analysis. These activities are long-standing and open-ended, and develop in line with the current research activities and needs of the laboratory. All software and related packages are made freely available to the research community through the laboratory website at psrg.org.uk 
Type Of Material Technology assay or reagent 
Provided To Others? Yes  
Impact The various software tools and packages have furthered the research activities of the laboratory since the 1990s and continue to provide key support and drivers for advancing much of current research. These tools and packages are disseminated, on average, to over 100 laboratories per year. 
URL http://psrg.org.uk
 
Title EZ-Rhizo 
Description Software for quantitative trait analysis and acquisition for root growth/development 
Type Of Material Database/Collection of data 
Year Produced 2010 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title Henry 
Description Software package for electrophysiology and imaging data acquisition and analysis 
Type Of Material Data handling & control 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title OnGuard 
Description Quantitative systems biology modelling of cellular transport and homeostasis 
Type Of Material Computer model/algorithm 
Year Produced 2012 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Title SDM-assist 
Description Software for molecular primer design that enables introduction of silent markers for molecular cloning 
Type Of Material Data analysis technique 
Year Produced 2013 
Provided To Others? Yes  
Impact Multiple publications from my own research group and research groups worldwide Online distribution has been accessed through the laboratory website with site views at a rate of >500 per month 
URL http://psrg.org.uk
 
Description PBL 
Organisation Plant Bioscience Limited Technology
Country United Kingdom 
Sector Private 
PI Contribution IPR on ABA receptor technology and ABA signalling
Collaborator Contribution Funding related to IPR on ABA receptor technology and ABA signalling
Impact Multiple outcomes in publications and industrial contacts
 
Description PSG 
Organisation POSCO - South Korea
Country Korea, Republic of 
Sector Private 
PI Contribution Base support for meetings and exchange of materials
Collaborator Contribution Base support for meetings and exchange of materials
Impact Base support for meetings and exchange of materials
 
Title Software tools and packages for electrophysiology and imaging 
Description The laboratory continues to develop and refine software/hardware tools for data acquisition and analysis relevant to electrophysiology, single-cell imaging and analysis. These activities are long-standing and open-ended, and develop in line with the current research activities and needs of the laboratory. All software and related packages are made freely available to the research community through the laboratory website at psrg.org.uk 
Type Of Technology Software 
Impact The various software tools and packages have furthered the research activities of the laboratory since the 1990s and continue to provide key support and drivers for advancing much of current research. These tools and packages are disseminated, on average, to over 100 laboratories per year. 
URL http://psrg.org.uk
 
Description International online services 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Prof. Blatt and members of his laboratory have contributed to various media events over the years, including online interview contributions (e.g. People behind the Science, a US-based media program)
Year(s) Of Engagement Activity Pre-2006,2006,2008,2011,2015,2016,2017,2018
 
Description Invited presentations 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I regularly speak to audiences, from small groups (5-20) to large audiences (>1000) in a variety of settings. In addition to teaching and extramural activities associated with the university, I also speak on invitation to national and international groups a number of times each year and in a variety of settings, academic as well as public. I also reach audiences through short video presentations mounted on the web, these primarily via my laboratory website and the ASPB websites. Anyone reading this entry is welcome to visit these sites to learn more.

The impacts arising from my presentations are varied. For example, a common consequence of my speaking in academic settings is to attract potential researchers to visit my laboratory and, frequently, to interest potential collaborators and students/postdocs to my research group. At scientific meetings, my talks often attract interest also from researchers interested in the various tools and materials that my research has produced, including the various vector systems and software packages that I
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk
 
Description Schools and displays 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact As these were multiple events, this question is not informative or useful. Participants varied from numbers in the tens to several thousands

Extensive training of participating laboratory members as well as broad scope reach to schools and communities, in the case of the GCC science days to the west of Scotland and in the case of the IFPD activities to audiences within and outside the UK
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk
 
Description Teaching Tools 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Schools
Results and Impact The PI has supported the editor in developing these tools since their inception in 2009 and has contributed to recent tools relating to membranes and transport education

The Tool received an international award in 2010 for excellence in education and has an acknowledged takeup worldwide in over 3000 institutions
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk
 
Description Teaching Tools 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Schools
Results and Impact The PI has supported the editor in developing these tools since their inception in 2009 and has contributed to recent tools relating to membranes and transport education

The Tool received an international award in 2010 for excellence in education and has an acknowledged takeup worldwide in over 3000 institutions
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014,2015,2016,2017,2018
URL http://psrg.org.uk