Team:Lambert GA/Iron
INSPIRATION
During the spring, Dr. Todd Guerdat, an Agricultural Engineer with the Natural Resources Conservation Service at the USDA, aided in directing Lambert iGEM's project away from phosphate and nitrate synthesis and towards biosensing the Pho and Nar pathways as well as finding a method to solubilize iron, as production of phosphate and nitrate ions already occurs efficiently in fish. However, according to Dr. Guerdat, there is no method to extract and reduce iron (another essential nutrient) from fish excrement (sludge) and solubilize it to allow plants to utilize it within an aquaponic system. Further research informed the team that up to 85% of iron is in an insoluble form in the fish sludge.
RECOMBINANT CYBRD1
Based on discussion with Dr. Guerdat and further literature review, Lambert iGEM decided to pursue a synthetic biology approach in order to effectively solubilize the insoluble iron (Ferric or Fe3+) to a soluble form (Ferrous or Fe2+), allowing plants to uptake iron more easily.
The CYBRD1 gene is a member of the cytochrome b(561) family that encodes an iron-regulated protein (Duodenal Cytochrome B). This gene is commonly found in primates including humans. It is expressed on the duodenum (small intestine) membrane and has ferric reductase activity. Additionally, the Duodenal Cytochrome B protein plays a physiological role in dietary iron absorption [1].
Figure 1. Diagram of CYBRD1 Protein
The Duodenal Cytochrome B protein acts as a ferric reductase enzyme by reducing ferric iron to ferrous iron in the presence of the electron donor ascorbate [2]. Lambert iGEM decided to create recombinant cytochrome B protein in E. coli.
This led the team to a publication, High-yield production, purification and characterization of functional human duodenal cytochrome b in an Escherichia coli system, where a team of University of Texas Health Science Center researchers were able to encode a cytochrome gene and receive protein yields in E. coli [3]. Their paper acted as a proof of concept for Lambert iGEM's potential approach, so the team reached out to one of the authors, Dr. Richard Kulmacz, to further inquire about their experimental procedure and the gene sequence used. Dr. Kulmacz suggested that addition of ascorbate or another reducing agent would be capable of solubilizing iron even in the absence of an electron transport protein such as Duodenal Cytochrome B. This conversation led the team to re-evaluate the initial approach to encode the CYBRD1 gene in E. coli.
To receive further feedback, Lambert iGEM pitched the idea of using the CYBRD1 gene to Dr. Styczynski from the Georgia Institute of Technology, Dr. Ichiro Matsumura from Emory University, and Dr. Sammy Bell from Boehringer Ingelheim. Dr. Bell claimed there needed to be a strong justification to choose the synthetic biology approach over chemical reduction because the CYBRD pathway was labor intensive. Further research led the team to the phenanthroline method, which was significantly more efficient and effective at reducing ferrous iron than the synthetic pathway. Thus, Lambert iGEM decided not to pursue the CYBRD gene idea.
After the initial CYBRD pathway was rejected, the team reconvened with Dr. Guerdat and asked about the prevalence of iron deficiency in aquaponic systems. He told Lambert iGEM that rather than looking into microorganisms that can supplement iron uptake, the team should look into plant physiology barriers.
PLANT PHYSIOLOGY
Lambert iGEM began researching plant physiology of iron uptake systems and tried to find issues with the natural system, especially natural ferric to ferrous conversion. In general, plants can uptake both ferric and ferrous by reducing ferric iron into ferrous iron. Before crossing the plasma membrane through Iron-Regulated Transporter 1 (IRT1), Ferric Reduction Oxidase 2 (FRO2) at the plasma membrane transports an electron from cytosolic NADH to apoplastic Fe3, reducing ferric iron [4]. Despite the plants’ ability to uptake both soluble and insoluble iron, plant iron deficiency still occurs, and one of the biggest reasons for that is iron and manganese interference [5]. Typically, iron and other metals are uptaken by IRT1; However, IRT1 permeates other metals that are not iron and has low selectivity for iron. Thus, even when the iron exists, it cannot be sufficiently uptaken by the plants [6].
When researching plant physiology, the team found mutations on the FRO2 gene, which is the ferric reduction oxidase within certain species of plants. This gene is a vital piece of the iron uptake pathway in plants as it encodes for an enzyme embedded in the membrane of a cell that reduces Fe into a bioavailable form. This mutation was known to cause growth defects within plants when grown under -Fe conditions. Lambert iGEM attempted to find a synthetic biology solution to this mutation in order to improve iron uptake within the cell rather than converting ferric to ferrous iron outside the cell, as this would still reach the goal of improving iron concentrations within the plant. However, with further research, multiple pieces of literature directed the team to the conclusion that there was simply not enough research done on the genetic components and position of this certain mutation, and therefore it would be strenuous to continue attempting to improve this pathway [7].
CONCLUSION
While looking into other aspects to improve, Lambert iGEM looked into the mineralization process, a process where chemicals in organic matter decompose or oxidize in order to be readily absorbed by plants [8]. However, Dr. Guerdat suggested that there were no parts of this naturally occurring process with the potential to be improved by synthetic biology, so the team continued discussion with other stakeholders to identify problems in aquaponics. While having multiple discussions with different members of the community and professors, the need for biosensors for iron arose as a possible approach that the team could take. After researching iron biosensors, however, Lambert iGEM discovered that there were many existing variations. Due to the lack of novelty, the idea was not further explored.
REFERENCES
[1] LanLane, D. J., Bae, D. H., Merlot, A. M., Sahni, S., & Richardson, D. R. (2015). Duodenal cytochrome b (DCYTB) in iron metabolism: an update on function and regulation. Nutrients, 7(4), 2274-2296. doi: 10.3390/nu7042274
[2] Ganasen, M., Togashi, H., Takeda, H., Asakura, H., Tosha, T., Yamashita, K., ... & Hamza, I. (2018). Structural basis for promotion of duodenal iron absorption by enteric ferric reductase with ascorbate. Communications biology, 1(1), 1-12. Retrieved from https://www.nature.com/articles/s42003-018-0121-8
[3] Liu, W., Wu, G., Tsai, A. L., & Kulmacz, R. J. (2011). High-yield production, purification and characterization of functional human duodenal cytochrome b in an Escherichia coli system. Protein expression and purification, 79(1), 115-121.Retrieved from https://www.sciencedirect.com/science/article/abs/pii/S1046592811000817
[4] Connorton, J. M., Balk, J., & Rodríguez-Celma, J. (2017). Iron homeostasis in plants–a brief overview. Metallomics, 9(7), 813-823. Retrieved from https://pubs.rsc.org/en/content/articlehtml/2017/mt/c7mt00136c
[5] Somers, I. I., & Shive, J. W. (1942). The iron-manganese relation in plant metabolism. Plant Physiology, 17(4), 582. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC438057/pdf/plntphys00290-0076.pdf
[6] Eroglu, S., Meier, B., von Wirén, N., & Peiter, E. (2016). The vacuolar manganese transporter MTP8 determines tolerance to iron deficiency-induced chlorosis in Arabidopsis. Plant physiology, 170(2), 1030-1045. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4734556/
[7] Satbhai, S. B., Setzer, C., Freynschlag, F., Slovak, R., Kerdaffrec, E., & Busch, W. (2017). Natural allelic variation of FRO2 modulates Arabidopsis root growth under iron deficiency. Nature communications, 8(1), 1-10. Retrieved from https://www.nature.com/articles/ncomms15603
[8] Konhauser, K. O. (1998). Diversity of bacterial iron mineralization. Earth-Science Reviews, 43(3-4), 91-121. Retrieved from https://www.eas.ualberta.ca/konhauser/Reprints/EarthScienceReview-KOK%281998%29.pdf