14-PSIL MAGIC: a multi-tiered approach to gaining increased carbon

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

Abstract

In the Calvin-Benson cycle of plants, the enzyme RuBisCO fixes CO2 to produce two molecules of 3-phosphoglycerate. RuBisCO evolved ~3.6bn years ago in an atmosphere of high CO2 and low O2, with little need to discriminate between the two gases. In today's atmosphere RuBisCO fixes both CO2 and O2. The latter generates phosphoglycolate, which is retrieved by photorespiration but at an energy cost that represents a significant loss in photosynthetic efficiency. One method to reduce O2 fixation by RuBisCO is to raise the partial pressure of CO2. Carbon concentrating mechanisms (CCMs) have evolved multiple times to this end. For example, C4 photosynthesis uses phosphoenol-pyruvate carboxylase (PEPC), an enzyme that does not possess oxygenase activity, to fix HCO3- temporarily in C4 acids; cellular specialization allows release and concentration of CO2 for refixing by RuBisCO. As much as a 50% increase in yield might be realized in crops were O2 fixation by RuBisCO to be bypassed in a similar manner. Significant resources have already gone into engineering RuBisCO for increased CO2 selectivity and into introducing a single-celled version of C4 photosynthesis in rice, but a step change in photosynthetic efficiency has not yet been achieved.

Investigators from Universities in the US (John Golbeck (JG), Penn State; and Cheryl Kerfeld (CK), Michigan State) and the UK (Mike Blatt (MB), Glasgow; Nigel Burroughs (NB), Warwick; and Julian Hibberd (JH), Cambridge) participated in an NSF/BBSRC Ideas Laboratory in 2010, at which they proposed a novel strategy to address this problem, a proposal that has since matured to the level of technological implementation. They are now joined by Nick Smirnoff (NS, Exeter) and Manish Kumar (MK, Penn State), who bring additional and key expertise to the project. The research has two themes: a light driven ion pump, composed of halorhodopsin and an anion/HCO3- exchanger, AE1; and the use of artificial scaffolds for channelling CO2 to RuBisCO. A parallel goal is to re-engineer the light-driven ion pump to transport HCO3- directly and to absorb light energy not used by photosynthesis. These efforts are underpinned with mathematical modelling of CO2 delivery and assimilation to direct experimentation based around the following components.

Light-Driven Pump. Halorhodopsin (HR) is an integral membrane protein and consists of 7 transmembrane alpha-helices and a bound retinal. The retinal undergoes light-driven bond rotation between 13-cis and all-trans conformations to drive ion transport. HR transports other halides as well, and ion selectivity appears to be a localized feature of the pHR transport site. pHR is sufficiently promiscuous to make engineering a light-driven HCO3- pump a possibility.

Anion/Bicarbonate Exchanger: The erythrocyte Band3 protein (AE1) facilitates Cl-/HCO3- exchange across the membrane. It generates a high flux close to equilibrium and is largely insensitive to pH, making it well suited to engineering a HCO3- accumulating mechanism. Most promising for synthetic engineering, the AE1 transporter is functional in mammalian cell cultures, Xenopus oocytes, and yeast without adverse effects on homeostasis or growth. The modular structure of AE1, offers a realistic strategy for coupling HCO3- pumping coupled to pHR-driven Cl- transport.

Artificial Scaffolds: CO2 diffusion needs to be constrained locally for sufficient time to allow it to be fixed by RuBisCO. Substrate channelling is found in several natural systems, including plants. Efficiency gains arise from physical proximity and 'sponge'-like buffering that enables transfer of intermediates and minimizes runoff of substrates.

Technical Summary

We are building on progress that includes expression of pHR in E. coli, cyanobacteria and plants. These advances have put us in a strong position to deliver within the next few years. Mathematical modeling has validated the idea of using a light-driven ion pump for concentrating CO2; it now remains to assemble and express these pumps and validate function in chloroplasts. The idea to use scaffolds to concentrate CO2 at RuBisCO remains a goal, but our strategies have changed in light of new understanding of the interplay between diffusion and kinetics. We have successfully expressed scaffold proteins in cyanobacteria and plants, demonstrating that they can be both targeted to specific sites and that they function to recruit their respective substrates. Our mathematical models predict that the original idea of utilizing these constructs to enhance channeling of CO2 to RuBisCO will have negligible impact on CO2 assimilation. We need now to confirm this prediction in our cyanobacterial systems. A rethinking of the problem of concentrating CO2 at RuBisCO in C3 plants leads to development of a new approach. Our mathematical models highlight the poor CO2 capture probability of RuBisCO as a major constraint. Here, we propose designs to slow the diffusion rate of CO2 in the stroma and increase assimilation by introducing transient (stationary) binding sites near RuBisCO (a CO2 'sponge'), effectively enhancing the native characteristics recently identified in photosynthetic systems. We will use the cyanobacterial system to screen and optimize this approach and will use the scaffolds now proven in our hands to translate these to chloroplasts. Finally, we previously lacked the ability to quantify performance, ie. HCO3- concentration gains. This capability is now available through a lipid vesicle technique.

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 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 improving carbon capture by plants. 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 award builds on previous work towards a synthetic approach to improving photosynthesis in plants. The aim therefore is to provide an strategy for improving plant biomass production for agri-industrial applications and for food production in the face of global environmental change. To date, the main findings have application to academic research, but it is hoped that the knoweldge gained will have other applications in the near future. Major findings to date relate to in silico analysis to assess the limitations to incorporating C4 photosynthesis in C3 plants and the energetics required. These results suggest that permeation through the membrane barriers of the chloroplast and plasma membrane are key factors and are likely to determine the limits of C3-to-C4 conversion. They also suggest that estimates of membrane CO2 permeability are substantially in error based on past calculations.
Exploitation Route Guidance for future research directed to photosynthetic engineering
Sectors Agriculture, Food and Drink,Environment,Other

 
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