The Problem
Pueraria lobata var mirifica (P. mirifica) is a plant highly valued for containing important phytoestrogens: puerarin, daidzein, and genistein.[5] These phytoestrogens (class: isoflavonoids) possess essential antioxidative and neuroprotective properties, making P. mirifica a valuable plant used in supplements. However, the plant’s tuberous roots have been heavily exploited for commercial purposes, putting the species at risk for eventual extinction.
[2]Current harvesting practices in producing P. mirifica supplements result in significant physical disruptions to the surrounding soil and natural habitat, leading to erosion, degradation of soil structure, and potential damage to local ecosystems – they are unsustainable.[5] Moreover, P. mirifica’s long maturation period of 3 years makes its continual production more difficult to uphold, jeopardizing this herbal medicine’s availability.[4]
Our Plant Chassis
The plant chassis used is Nicotiana benthamiana. This is a key model plant that has been used in plant synthetic biology for a long time due to its rapid growth time and ease of genetic manipulation. More specifically, N. benthamiana is very prone to Agroinfiltration, with chances of successful agroinfiltration in N. benthamiana being higher than in other plants. Especially for this competition, where we needed a plant chassis that would be easily accessible and not take too long to grow, N. benthamiana stood out with its maturation period of only 2 months, compared to the 3 years it takes to grow P. mirifica.[2][3]
A more suitable plant chassis for our project would be Glycine max (G. max). Since G. max is also a leguminous plant like P. mirifica, with a longer but still short maturation period, it would be suitable for the relocation of our enzymes required. This is because G. max goes further in the isoflavonoid synthetic pathway, so fewer enzymes would need to be relocated. G. max still does not produce puerarin, daidzein, and genistein, so it is not used as a herbal supplement.[1] Moreover, G. max is widely grown in Thailand, as opposed to P. mirifica, whose growing conditions do not suit the tropical climate. If we had been given more time, this chassis would have been experimented on too.
Our Project
The Thailand-RIS team addressed our issue by developing a more sustainable and less resource-intensive way to produce isoflavonoids – by relocating the enzymes responsible for the production of the important isoflavonoids in P. mirifica to N. benthamiana.
We wanted to create a genetically modified N. benthamiana that would express: chalcone synthase (PmCHS), chalcone reductase (PmCHR), chalcone isomerase (PmCHI), isoflavone synthase (PmIFS), 2-hydroxyisoflavanone (PmHID), and C-glycosyltransferase (PmC-UGT) to complete the isoflavonoid pathway. To prove their concept, we agro-infiltrated N. benthamiana and managed to find proof of the production of genistein, daidzein, and puerarin in N. benthamiana, which naturally does not produce these.
We also thought of possible safety systems to prevent the spread of this plant. One of these was a kill switch, which would prevent the growth of the plant when exposed to red light. Unfortunately, this composite part was not able to be tested in the lab environment; however, if this project were to be developed further, future prospects would include testing of this kill switch to see if it is a viable part.
Conclusion
In our iGEM project, we focused on addressing the challenges in growing P. mirifica. By relocating the key enzymes responsible for isoflavonoid production to N. benthamiana, we aimed to create a genetically modified plant capable of synthesizing the same valuable compounds of daidzein, genistein, and puerarin, though puerarin was produced in minimal amounts. Our approach focused on minimizing ecological disruption and resource consumption. Our successful agroinfiltration provided proof that N. benthamiana could be used as an alternative to P. mirifica to produce valuable herbal supplements.
Despite our accomplishments, resource constraints prevented the full testing of the red light kill switch system designed to limit the spread of our product plant. Time constraints also limited testing in what we believed to be a more suitable alternative plant chassis, Glycine max
Our exploration of this solution to more sustainably produce a Thai traditional herb through synthetic biology represents a breakthrough in addressing unease in Thailand regarding biomanufacturing through genetically modified plants. Currently, synthetic biology is not widespread in Southeast Asia, making it difficult to find resources necessary for projects like this. However, we hope that our findings will inspire future teams and researchers to further explore and experiment with plant synthetic biology.