Why plant?

As a widely recognized medicinal herb, Danshen (derived from the root of Salvia miltiorrhiza) has its medicinal components’ effectiveness widely acknowledged, leading to a significant demand in the market. However, traditional methods of cultivation and extraction have limitations, making it particularly important to find more efficient production methods. When propagated from seeds, Salvia miltiorrhiza typically requires 2 to 3 years to reach harvest maturity. By using rootstocks (or rhizomes), the cultivation cycle can be shortened to 1 to 1.5 years, allowing for harvesting at that time. Du ring cultivation, a large amount of land is needed, and atten tion must be paid to irrigation, fertilization, and pest control, which consume substantial resources. Therefore, employing synthetic biology to produce the active components of Danshen is a promising strategy. This method reduces dependence on land and water resources, simplifies the cultivation and extraction process, thus enhancing production efficiency and sustainability.

Research has found that the biosynthesis of salvianolic acid in Salvia miltiorrhiza involves two parallel pathways, which branch off from the phenylalanine and tyrosine metabolic pathways, respectively. Specifically, 4-coumaroyl-CoA, produced through the phenylalanine pathway, acts as the acyl donor, while 4-hydroxyphenyllactic acid or danshensu, produced through the tyrosine pathway, serves as the acyl acceptor. Rosmarinic acid is synthesized under the catalysis of rosmarinic acid synthase (RAS) and cytochrome P450 enzymes of the CYP98A family. In the early stages of our project, we conducted in-depth research on various host cells that could potentially be used to reconstruct the biosynthetic pathway of salvianolic acids, comparing their advantages and disadvantages. N. benthamiana, a tobacco plant, has significant advantages compared to yeast and E. coli. First, N. benthamiana has the phenylalanine pathway, which can efficiently produce 4-coumaroyl-CoA. 4-Coumaroyl-CoA is expensive and difficult to produce on a large scale. Second, N. benthamiana demonstrates high efficiency in expressing heterologous proteins, and the gene transformation technology is relatively advanced. In contrast, the transformation technology for transgenic danshen is complex, with a longer subsequent cultivation cycle and higher management costs. Additionally, as a model plant, N. benthamiana has a short growth cycle and produces many leaves, which is conducive to increasing the yield of the target product. Finally, the safety of N. benthamiana as a production platform has been confirmed. Based on these considerations, our project has decided to produce rosmarinic acid by reconstructing the tyrosine pathway in N. benthamiana.

Creating new production systems in plants

We devised two different biosynthetic pathways for rosmarinic acid based on available studies (see Engineering section for details). N. benthamiana was then grown and the 4-week-old was selected to carry out our work.

Tobacco Planting and Transplanting:

Initiate germination in a controlled environment maintained at 23°C with adequate lighting for a period of 7-10 days. Once the tobacco seedlings develop at least four leaves, they are ready for transplanting. To minimize root disturbance, carefully lift the seedlings along with the soil clump. Prepare the planting holes in the new soil mix using your finger, and plant 2-3 seedlings per container. Handle the seedlings delicately to prevent damage.

Figure 1. Photo of freshly sprouted (left) and 4-week-old (right) N. benthamiana.

Agrobacterium Solution Preparation:

The Agrobacterium culture should be grown overnight and its optical density (OD600=0.5-0.8) measured before use. Ensure the culture is at the correct concentration, which is critical for achieving efficient and successful transformation. Precise control over the concentration is essential for optimal results.

Figure 2 Determination of Agrobacterium concentration.

Agrobacterium Infection:

Conduct Agrobacterium-mediated transformation when the tobacco seedlings are large enough that their leaves begin to touch. Before infection, expose the seedlings to white fluorescent light for one hour to facilitate stomatal opening, which aids in Agrobacterium uptake. Select seedlings with three to four fully expanded leaves for the procedure. Press the leaf surface with a syringe (without a needle) while supporting the leaf tip with your index finger to create a microenvironment for Agrobacterium penetration. Exercise caution to prevent leaf injury and take safety measures to avoid splashing of the bacterial suspension. During the injection process, due to our lack of experience, we encountered numerous challenges. We have summarized these issues and documented the key considerations and solutions in detail. You can find this information in our contribution section. We hope that these summaries can assist other iGEM teams in conducting similar experiments.

For our project, in order to enhance the efficiency of product production, we have refined our approach, particularly focusing on the plant cultivation methods and the sequence of injection for the bacterial suspension and the substrate.

1.Post-Infestation Plant Culture: Following infestation, the plants were initially cultured in darkness for 24 hours. This dark culture condition slows down the metabolism of plant cells, facilitating more effective invasion by Agrobacterium. Subsequently, the plants are transitioned to light culture to ensure the robust expression of exogenous genes.

2.Substrate Injection: Rather than injecting the substrate and strain simultaneously, we opted to inject the strain first. Once the exogenous gene achieves stable expression, the substrate is then introduced, ensuring a more efficient reaction process.

Figure 3 Process of plant cultivation after infection

Optimization of active ingredient production in Salvia miltiorrhiza with improved plants

Danshen, an important traditional Chinese medicine, is well-known for its clear active components. However, it has a long growth cycle and faces challenges with continuous cropping, leading to frequent shortages in meeting market demand. Plant scientists have been working hard to address these issues by modifying model plants, which has great potential for solving challenges faced by humans.

Our project is a beneficial attempt to use tobacco as the production host organism, optimizing the experimental methods for the biosynthesis of foreign proteins in plants. By using LC-MS/MS technology, we have successfully detected the target product, proving the feasibility of our strategy. Of course, we will continue to explore methods to increase production using plants as the host organism.