Team:Lethbridge HS/Contribution




Bronze Contribution

For our part contribution, we decided to pick three different Registry Parts we were interested in learning more about. Our summaries of the recent literature surrounding these parts are shown below as well as the original part page.

Rose Odor Generator Device (Geranoil) (BBa_K727007), AUC Turkey 2012



Apart from being a component in rose and other aromatic oils, Geraniol has many pharmacological properties. According to a study using a streptozotocin induced diabetic model in rats, the administration of GE for a period of 45 days was seen to have decreased the levels of hemoglobin HbA1C and plasma glucose; this resulted in a restoration of glucose homeostasis [1]. Geraniol is also seen to alleviate other diabetes based health problems, such as having a protective effect on diabetic neuropathy [2], attenuating diabetes-induced cardiac functional disorder [3], and improving heightened vasoconstriction caused by diabetes [4]. In general, Geraniol has been seen to have a plethora of pharmacological benefits, and is believed, due to mounting evidence in the last several decades, to be a botanical compound that does not have any harmful effects [5].


Additionally, studies of the compound demonstrate fungicidal and antilarval properties, suggesting GE’s potential in the biocide industry. The antifungal effects of geraniol may be due to the ability of this monoterpene to travel across the cell membranes of phytopathogenic fungi and, in doing so, interacting with various membrane proteins. This interaction can result in the hyphae, an aspect of the fungi’s mycelium [6] and the plasma membrane[7]. Despite these considerable benefits of GE, it is seen to have low economic value, as it can only be extracted at very low concentrations from plants [8]. Most microorganisms, with the exception of some Saccharomyces cerevisiae strains [9], are unable to synthesize monoterpenes, and cannot synthesize GE naturally, due to the lack of a geranyl diphosphate synthase (GPPS). However, a 2016 paper [8] attempted to increase the yield of geraniol production, by engineering E.coli and utilizing the biotransformation from geraniol acetate to geraniol.

We chose to update this part, because many of the responses from the survey we conducted as part of our human practices, stated that a barrier to composting for them was the presence of pests, or the “ick” factor. To counter this problem, it’s valuable to look into certain aromatic compounds, so that in the future, we can continue our project to make composting better and more accessible for more people.



Pectinase ( BBa_K2377003), Lanzhou 2017



Recent literature has shown that pectinases can be produced in large scale batches using organisms such as Aspergillus niger using an optimal growth media [10]. However most of these pectinases produced for commercial purposes are active only at about 50˚C [11]. Some pectinases found in plant leaf tissue can provide more stable products but there are still limitations to thermostable options [12]. These industrial pectinase enzymes are commonly purified using ammonium salt precipitation, gel filtration chromatography and ion exchange chromatography [13].


The pectinase represented from this part is a pectinase enzyme from Alkalihalobacillus halodurans or also known as Bacillus halodurans. Since the making of this part, there has been only one publication on this particular pectinase, published in 2013. Mei et al., [14] were able to clone, purify by affinity chromatography, and test the biochemical properties of this enzyme. The M29 strain of A. halodurans is an aerobic alkaliphilic bacterium that can handle temperatures up to 65˚C. The identified 39 kDa pectinase was cloned into E. coli for purification and characterization. This protein was determined to have optimal activity at pH 10 and a temperature of 80˚C. It was also determined that calcium was a necessary cofactor for enzymatic activity

We chose to look into this part because it is another pectinase that has been used in other iGEM projects, which intrigued us. Furthermore, as this protein is thermophilic it provides some insight into other purification methods and assays that we may want to use for our own thermophilic variants.



Lumazine SynthaseBBa_K249002 ), Lethbridge 2009




The last part we wanted to contribute to was the BBa_K249002, which describes lumazine synthase. Although this part has nothing to do with our project, we found it to be a very interesting protein, especially with what it has been recently used for in the literature. As this protein has been engineered to gain different electrostatic properties, it much like the engineering we are doing with the pectinase to gain thermostability


Wild type Lumazine synthase from Aquifex aeolicus naturally forms a 60 subunit 13 nm diameter capsid [15]. It naturally encapsulates riboflavin synthetase; both proteins take part in the production of riboflavin or vitamin B12. As the inside layer of the lumazine synthase capsid is negatively charged, the encapsulation of a positively charged capsid can easily be done. However, with the use of protein engineering, two Lumazine synthase proteins have been made called AALS-neg and AALS-13 where the first is a more negatively charged variant and the second is an optimized version of AALS-neg [16] These two variants have been used to encapsulate a Green fluorescent protein that has a poly-arginine tag. This tag provides a positive charged end that can interact with the inside of the capsid.

Besides the engineering of the protein to encapsulate novel cargo’s, Lumazine synthase has been also used for the delivery of antigens in vaccine development [17]. Typically, the antigens are displayed on the surface of the formed Lumazine synthase capsid, which improves the activation of the immune system and the overall positive response to the vaccine.



References


  1. Babukumar, S., Vinothkumar, V., Sankaranarayanan, C., & Srinivasan, S. (2017). Geraniol, a natural monoterpene, ameliorates hyperglycemia by attenuating the key enzymes of carbohydrate metabolism in streptozotocin-induced diabetic rats. Pharmaceutical Biology, 55(1), 1442-1449. doi:10.1080/13880209.2017.1301494
  2. Babukumar, S., Vinothkumar, V., Sankaranarayanan, C., & Srinivasan, S. (2017). Geraniol, a natural monoterpene, ameliorates hyperglycemia by attenuating the key enzymes of carbohydrate metabolism in streptozotocin-induced diabetic rats. Pharmaceutical Biology, 55(1), 1442-1449. doi:10.1080/13880209.2017.1301494
  3. El-Bassossy, H. M., Ghaleb, H., Elberry, A. A., Balamash, K. S., Ghareib, S. A., Azhar, A., & Banjar, Z. (2017). Geraniol alleviates diabetic cardiac complications: Effect on cardiac ischemia and oxidative stress. Biomedicine & Pharmacotherapy, 88, 1025-1030. doi:10.1016/j.biopha.2017.01.131
  4. El-Bassossy, H. M., Elberry, A. A., & Ghareib, S. A. (2016). Geraniol improves the impaired vascular reactivity in diabetes and metabolic syndrome through calcium channel blocking effect. Journal of Diabetes and Its Complications, 30(6), 1008-1016. doi:10.1016/j.jdiacomp.2016.04.006
  5. Lei, Y., Fu, P., Jun, X., & Cheng, P. (2018). Pharmacological Properties of Geraniol – A Review. Planta Medica, 85(01), 48-55. doi:10.1055/a-0750-6907
  6. Kaur, G., Ganjewala, D., Bist, V., & Verma, P. C. (2019). Antifungal and larvicidal activities of two acyclic monoterpenes; citral and geraniol against phytopathogenic fungi and insects. Archives of Phytopathology and Plant Protection, 52(5-6), 458-469. doi:10.1080/03235408.2019.1651579
  7. Hyphae - Production, Structure, Morphology, Types. (n.d.). Retrieved October 26, 2020, from https://www.microscopemaster.com/hyphae.html
  8. Liu, W., Xu, X., Zhang, R., Cheng, T., Cao, Y., Li, X., Guo, J., Liu, H., & Xian, M. (2016). Engineering Escherichia coli for high-yield geraniol production with biotransformation of geranyl acetate to geraniol under fed-batch culture. Biotechnology for biofuels, 9, 58. https://doi.org/10.1186/s13068-016-0466-5
  9. Carrau, F. M., Medina, K., Boido, E., Farina, L., Gaggero, C., Dellacassa, E., Versini, G., & Henschke, P. A. (2005). De novo synthesis of monoterpenes by Saccharomyces cerevisiae wine yeasts. FEMS microbiology letters, 243(1), 107–115. https://doi.org/10.1016/j.femsle.2004.11.050
  10. Enshasy, G., Elsayed, E., Suhaimi, N., Malek, R., Esawy, M. (2018) Bioprocess optimization for pectinase production using Aspergillus niger in a submerged cultivation system. BMC biotechnology. 18(71), https://doi.org/10.1186/s12896-018-0481-7
  11. Carrasco, M., Rozas, J,. Alcaino, J., Cifuentas, V., Baezo, M. (2019)Pectinase secreted by psychrotolerant fungi: identification, molecular characterization and heterologous expression of a cold-active polygalacturonase from Tetracladium sp. 18 (45), https://doi.org/10.1186/s12934-019-1092-2
  12. Daniel, H., Ribeiro, T., Lin, S., Saha, P., McMichael, C., Chowdhary, R., Agarwal, A. (2019) Validation of leaf and microbial pectinases: Commercial launching of a new platform technology. . 17(6), https://doi.org/10.1111/pbi.13119
  13. Javed, R., Nawaz, A., Munir, M., Manif, M., Mukhtar, H., Haw, U., Abdullah, R. (2018) Extraction, purification and industrial applications of pectinase: A review. Journal of Biotechnology & Bioresearch.1(1). JBB.000503.2018
  14. Mei, Y., Chen, Y., Zhai, R., and Liu, Y. (2013) Cloning, purification and biochemical properties of a thermostable pectinase from Bacillus halodurans M29. Journal of Molecular Catalysis B: Enzymatic. 94, 77-81. https://doi.org/10.1016/j.molcatb.2013.05.004
  15. Azuma, Y., Edwardson, T., and Hilvert, D. (2018) Tailoring lumazine synthase assemblies for bionanotechnology. Chemical society Reviews 47, 3543. DOI: 10.1039/cBcs00154e
  16. Worsdorfer, B., Woycechowsky, K., and Hilvert, D. (2011) Directed Evolution of a protein container. Science. 331(6017), 589-592
  17. Sciutto, E., Toledo, A., Cruz, C., Rosas, G., Meneses, G., Laplagne, D., Ainciart, N., Cervantes, J,. Fragoso, G., Goldbaum, F. (2004) Brucella spp. Lumazine synthase: a novel antigen delivery system. Vaccine 23(21), 2784-2790