Our Entry: Best Plant Synthetic Biology

Read about why we chose to build our project using a plant chassis and what we achieved.

Leveraging Plants

Over 50 years ago, as scientists gained more knowledge about genetics and basics of molecular biology, recombinant DNA technologies were developed leading to the establishment of Escherichia coli as a bio factory for low-cost production of peptides and proteins of interest ¹ . Just a few years later, recombinant human insulin produced by E. Coli was approved for treatment by the FDA, thus opening the door to the market for other protein-based recombinant pharmaceuticals ² . The number rose steadily in the following decades and reached 151 licensed drugs by FDA and/or by EMA, thus showing the great potential this technology has ³ . Yet with the ever-growing problem of climate change, we should strive to minimalize the greenhouse gas emissions in any technology we use nowadays. Bioreactors requiring heating and constant surplus of growth medium for the bacteria are no exception and it’s no wonder that in past years, many researchers explored the option of using microalgae as an alternative organism for production of recombinant proteins ⁴ ⁵ . Here however, designing a bioreactor with desired properties, mainly such as optimal light penetration to improve yields, has proven to be a challenge of its own ⁶ . We are convinced, that this problem could be bypassed by omitting the need for bioreactors completely and using higher plants, such as Nicotiana benthamiana , for the production of desired recombinant proteins. This approach was criticized in the past for the long periods required to grow transgenic plant, however this can be nowadays bypassed by utilizing the Agrobacterium tumefaciens mediated transient expression ⁷ ⁸ . To demonstrate the capabilities of this system, we chose to tackle another rising problem of this century, the antimicrobial resistance.

Discovery of antibiotics caused a revolution in medicine and soon, many previously lethal infections became easily treatable. However, as years passed and science knowledge expanded, it became obvious that microorganisms can develop resistance against these substances rather easily and quickly, thus making infections believed to be curable deadly once again, even though new antibiotics kept being developed. To battle this issue, more and more new antibiotics were developed. However, with the costs of research rising exponentially and legal framework halting use of these antibiotics to limit the development of resistance, pharmaceutical companies aren’t interested in following this research path ⁹ . Yet the issue keeps getting bigger and recently, the WHO adopted a global action plan to combat antimicrobial resistance and later proceeded to declare it one of the 10 biggest threats to humanity ¹⁰ ¹¹ . Therefore, the race for investigation of alternative approaches is on.

Traditionally, many cultures used a plethora of plant-based remedies to treat all sorts of symptoms. Our project revolves around plant-originated molecules as well. These are called cyclotides, a class of antimicrobial peptides (AMPs) known for their high stability achieved via disulfide bonds in a so called cyclic cystine knot motif (CCK) ¹² . Researchers have considered AMPs to be one of the promising alternatives to antibiotics, however their therapeutic application is so far is hindered by either missing specificity against the pathogenic bacteria, resulting in hemolytic or cytotoxic effect on the host cells, as well as lack of stability under physiological conditions. Inspired by the work of Prof. Craik from the University of Queensland, we developed a strategy based on using the cyclic backbone of MCoTI-II. This cyclotide possesses no hemolytic or cytotoxic effect compared to most cyclotides and is therefore a well-suited candidate for grafting of promising but unstable AMPs in order to enhance their stability ¹³ .

Our approach consists of multiple steps , beginning with modelling to predict, if the AMPs of our interest will keep its antimicrobial properties when grafted into the cyclic backbone. This enables us to save resources in the next step, cloning. Here, we developed a 3in1 binary vector for Agrobacterium tumefaciens mediated transient expression in N. benthamiana . Using Golden Gate Assembly, any gene encoding an AMP of interest can be cloned into this plasmid in a single cloning step, resulting in the production of the desired recombinant peptide in plant cells within three to five days upon the leaf infiltration, thus bypassing the long waiting times required with traditional transgenic plants. Next, we propose an extraction and purification protocol, that enabled us to isolate the recombinant cyclic peptides for further testing of desired antimicrobial properties. Last but not least, if all expectations are met, we hypothesize about how industrial scaled production could be achieved using transgenic plants.

Overall, we are hoping to do our part in combating the antimicrobial resistance, but also to show that transient expression in higher plants is a promising method with great potential. In order to highlight the promises this method holds, we visited a local radio where we discussed this method among other things. We also interviewed a guest specializing in plant molecular science in our podcast and did a series of educational posts on our Instagram account throughout the project. In addition, we visited a company specializing on the production of pharmaceutical products to learn about applicability of in planta production on a large, industrial scale. Additionally, we organized a panel discussion with experts from the field, targeting high-school student as prospective future scientists, to inspire them to use in planta production in their future research.

To make this process for them simpler, as well as for future iGEM team interested in similar projects, we wrote down a brief overview of steps necessary to successfully achieve the expression of your favorite peptide. We also listed all parts used in the process , including new parts we added to the registry, that are required for a successful cyclisation of your peptide of choice. This involves CtAEP1, a cyclizing asparaginyl endopeptidase required for the assembly of the CCK, Oak1 with a C-terminal c-myc-tag, a precursor that’s processed resulting into the cyclic backbone of MCoTI-II, as well as 3in1 Oak1 CtAEP sGFP binary vector. This composite part can be used by future teams to cyclize any peptide of interest via Golden Gate cloning, as all necessary parts for this process are obtained within the plasmid, including an expression control in form of a sGFP.

References

¹ Ahmad, N., Mehmood, M. A., & Malik, S. (2020). Recombinant Protein Production in Microalgae: Emerging Trends. Protein Pept Lett, 27(2), 105-110. doi:10.2174/0929866526666191014124855

² Human insulin receives FDA approval. FDA Drug Bull. 1982, 12: 18-19.

³ Redwan el, R. M. (2007). Cumulative updating of approved biopharmaceuticals. Hum Antibodies, 16(3-4), 137-158.

⁴ Gimpel, J. A., Hyun, J. S., Schoepp, N. G., & Mayfield, S. P. (2015). Production of recombinant proteins in microalgae at pilot greenhouse scale. Biotechnol Bioeng, 112(2), 339-345. doi:10.1002/bit.25357

⁵ Ahmad, N., Mehmood, M. A., & Malik, S. (2020). Recombinant Protein Production in Microalgae: Emerging Trends. Protein Pept Lett, 27(2), 105-110. doi:10.2174/0929866526666191014124855

⁶ Gimpel, J. A., Hyun, J. S., Schoepp, N. G., & Mayfield, S. P. (2015). Production of recombinant proteins in microalgae at pilot greenhouse scale. Biotechnol Bioeng, 112(2), 339-345. doi:10.1002/bit.25357

⁷ Rosales-Mendoza, S., Paz-Maldonado, L. M., & Soria-Guerra, R. E. (2012). Chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins: current status and perspectives. Plant Cell Rep, 31(3), 479-494. doi:10.1007/s00299-011-1186-8

⁸ Norkunas, K., Harding, R., Dale, J., & Dugdale, B. (2018). Improving agroinfiltration-based transient gene expression in Nicotiana benthamiana. Plant Methods, 14, 71. doi:10.1186/s13007-018-0343-2

⁹ Jit, M., Ng, D. H. L., Luangasanatip, N., Sandmann, F., Atkins, K. E., Robotham, J. V., & Pouwels, K. B. (2020). Quantifying the economic cost of antibiotic resistance and the impact of related interventions: rapid methodological review, conceptual framework and recommendations for future studies. BMC Med, 18(1), 38. doi:10.1186/s12916-020-1507-2

¹⁰ Mendelson, M., & Matsoso, M. P. (2015). The World Health Organization Global Action Plan for antimicrobial resistance. S Afr Med J, 105(5), 325. doi:10.7196/samj.9644

¹¹ Interagency Coordination Group on Antimicrobial Resistance (Ed.) (2019). NO TIME TO WAIT: SECURING THE FUTURE FROM DRUG-RESISTANT INFECTIONS.

¹² de Veer, S. J., Kan, M. W., & Craik, D. J. (2019). Cyclotides: From Structure to Function. Chem Rev, 119(24), 12375-12421. doi:10.1021/acs.chemrev.9b00402

¹³ Koehbach, J., Gani, J., Hilpert, K., & Craik, D. J. (2021). Comparison of a Short Linear Antimicrobial Peptide with Its Disulfide-Cyclized and Cyclotide-Grafted Variants against Clinically Relevant Pathogens. Microorganisms, 9(6). doi:10.3390/microorganisms9061249

About Us

We are the iGEM Team Tuebingen, a group of motivated students who are working on creating a fast screening platform for stabilized peptides. We are aiming to provide a system that gives everyone the ability to stabilize peptides such as antimicrobial peptides to create better medical agents.