Implementation
Communities throughout the world struggle to meet daily nutritional needs. One solution is to improve crop yield by optimizing the process of photosynthesis, the use of light energy to form sugar molecules. Learn more about that here!
Team:MiamiU OH/Plant
Plant Synthetic Biology
Award Nominee
Special thanks to the hard work put in by the Miami University iGEM, the CROP project was nominated for Best Plant Synthetic Biology Team in the 2021 iGEM competition. Learn more about the award and other nominees at jamboree.igem.org/results/specials/sustainable_development.
The Problem
As the population continues to rise, the need for greater crop yield is an issue of increasing concern. This issue is compounded by rising need in various sectors; not only is crop biofuel demand increasing as carbon emissions rise, but agricultural production is predicted to require drastic increases by 2050 to fulfill nutrition requirements of the growing population (1). Rising needs conflict with sustainability goals to reduce farmland (2). These conditions strain the agricultural industry, whose best solution is increasing crop production efficiency at the level of individual plants. One issue inhibiting greater production efficiency is the inefficiency of photosynthesis, the primary energy source for plants. Several studies suggest that photosynthesis only converts a maximum of 4.6% of solar energy into energy stored in biomass (3).
Although many aspects of photosynthesis do not efficiently convert energy, our team focused on the regenerative segment of the Calvin-Benson-Bassham (CBB) cycle. The CBB cycle reactions convert carbon dioxide into organic carbon. The regenerative segment of the cycle recycles portions of the carbon compounds into ribulose-1,5-bisphosphate (RuBP) to allow continuous cycle activity. The major inefficiency of this portion stems from the intermediates being consumed in other metabolic processes. Our team selected this portion of the cycle as our target for redesign due to a clear solution; if enzymes could be introduced to circumvent some steps, thereby reducing the number of intermediates available to leave the cycle, RuBP regeneration efficiency could be increased. By increasing RuBP regeneration, growth rate could be improved by creating a more continuous and efficient photosynthetic cycle.
Our Project
To model plant metabolomics, our group used cyanobacterial strain Synechococcus elongatus PCC 7942 as our chassis. Photosynthesis, especially the CBB cycle, is well conserved across photosynthetic organisms. Cyanobacteria make an excellent model organism for crop plants, particularly due to the ease of genetic manipulation compared to eukaryotic organisms (4).
Figure 1. A comparison of the native pathway and the modified pathway. More steps lead to more intermediates that can be used on other metabolic pathways. Created with BioRender.com Potential metabolic redesigns for RuBP regeneration were thoroughly investigated via computational modeling. Two potential pathways were explored: the transaldolase pathway and the glycolaldehyde pathway. Between these pathways, our team proceeded to test the transaldolase pathway in vivo. The fully redesigned pathway requires the deletion of glpX, which encodes SBPase, and the overexpression of tal, encoding transaldolase. SBPase dephosphorylates sedoheptulose-1,7-bisphosphate (SBP) to sedoheptulose-7-phosphate (S7P) and is present in the native CBB cycle (Figure 1). By knocking out SBPase and overexpressing tal, which catalyzes the reversible conversion of erythrose-4-phosphate (E4P) and fructose-6-phosphate (F6P) to S7P and glyceraldehyde-3-phosphate (G3P), the redesigned pathway reduces the number of intermediates that can be consumed in other reactions (Figure 1). We additionally investigated the effect of fructose-1,6-bisphosphatase (fbp) overexpression, finding that it could help drive our pathway in conjunction with tal overexpression.
SBPase is an essential protein, and therefore cannot be deleted before giving its relevant pathway an alternative. To address this issue, our team first induced the overexpression of transaldolase to allow cell survival of SBPase deletion.
Essentially, this reduction of the number of intermediates in the regenerative segment of the CBB cycle allows fewer metabolic processes to slow down the regeneration of RuBP by skipping two steps of the CBB cycle.
Our project also significantly focused on educational and dialogue-based outreach in order to give our community a scientifically accurate foundation of GMO knowledge, detailed on our Education page. We targeted two age groups: elementary school children and older adults. For the elementary students we created informational resources and interactive games that educators could incorporate into their lesson plans, and we distributed those to local schools and a museum. For our outreach to older adults, we held a presentation and conversation at our local adult care home. Both of these efforts focused on building knowledge for the creation of GMOs. If our community has an accurate understanding of the science of GMOs, then discussions about projects like ours can go further, and we can create a more educated community.
Our educational components also expanded to photosynthesis as a general process. We submitted a manuscript on the light to dark transition in photosynthesis for the Frontiers for Young Minds journal, linked on the Education page, describing research accomplished using genetic modification and providing open access material on photosynthetic complexity.
Our Accomplishments
Our team was able to successfully model our alternative pathway and generate predictive results of how these redesigned pathways would affect cell physiology and biomass generation, detailed on our Computational Modeling page.
Figure 2. Growth curve for tal, fbp, and tal-fbp overexpression cells. Our team also expanded the iGEM Part’s Registry in terms of cyanobacterial parts, submitting 40 parts described on our Contributions and Parts pages, incorporated into our 4 plasmids. We were able to achieve successful transformation with all our plasmids in our photosynthetic chassis organism. Due to the multiple genome copies and time restraints of the competition, however, we have not yet achieved total segregation for transformants.
In wet lab, we were able to successfully transform the overexpression genes into the cyanobacteria. Due to SBPase being previously established as an essential enzyme, we had doubts that we would be able to knock it out completely without first implementing an alternative pathway. However, there was evidence that it was knocked out of at least a few copies of the genome because the strains transformed with the pGEM-SBPase plasmid showed a resistance to antibiotics encoded in the plasmid.
Figure 3. A comparison of the wild type and overexpression mutant tal-fbp growth in the multicultivator. We did have colonies successfully transformed with overexpression genes for transaldolase, fructose-1,6-bisphosphatase (FBPase), and both genes. These results are further detailed in our Proof of Concept page. To provide a brief overview, the growth experiment results demonstrated that the simultaneous overexpression of FBPase and transaldolase was more effective for RuBP regeneration than the individual overexpression of either gene (Figure 2). This finding can be explained by the successful activation of our transaldolase pathway, the logic of which is detailed on our Proof of Concept page.
Even with overexpression of both tal and FBPase, however, there was less growth than the wild type culture (Figure 3). This is likely due to the presence of functional SBPase, as the SBPase and transaldolase pathways are actively competing for substrates and could limit growth. Therefore, we expect that our tal-fbp strains would exhibit growth comparable or even outcompeting that of the wildtype strains once undergoing full SBPase deletion.
References
1. Hunter MC, Smith R., Schipanski ME, Atwood LW, Mortensen DA. 2017. Agriculture in 2050: Recalibrating Targets for Sustainable Intensification. BioScience 67:386–391. (http://www.jstor.org/stable/90007777)
2. Balmford A, Green R and Phalan B. 2012. What conservationists need to know about farming. Proc. R. Soc. B. 279: 2714–2724 (https://doi.org/10.1098/rspb.2012.0515)
3. Zhu X-G, Long SP, Ort DR. 2008. What is the maximum efficiency with which photosynthesis can convert solar energy into biomass? Current Opinion in Biotechnology 19:153–159. (https://doi.org/10.1016/j.copbio.2008.02.004)
4. Jensen PE, Leister D. 2014. Cyanobacteria as an Experimental Platform for Modifying Bacterial and Plant Photosynthesis. Frontiers in Bioengineering and Biotechnology 2:7. (https://doi.org/10.3389/fbioe.2014.00007)
Explore Next
Proof of Concept
Explore the experiments conducted by our wet lab team and see the results of our genetic modification in cyanobacteria!
Contact Info
The main point of contact on the Miami University iGEM team is the principal investigator Xin Wang. Xin Wang can be inquired below. For additional contact information on the team, please refer to the Team page.
- xwang@miamioh.edu
- (513) 529-5427
- 212 Pearson Hall, 700 E. High St
Oxford, OH 45056
Special Thanks
This work was funded and made possible by the National Science Foundation!