At the beginning of our project design, we surveyed the public's understanding of plant synthetic biology and their acceptance of its products, aiming to gauge their knowledge, attitudes, and concerns.

Fig.1 Survey on the public's understanding of plant synthetic biology and product acceptance

Over one month, we received 312 valid responses. We analyzed the responses from the following three aspects:

1.The public's understanding of plant synthetic biology

35.51% of respondents had never heard of "plant synthetic biology," 39.88% had heard of it but were unfamiliar with it, 17.76% had some knowledge, and 6.85% were very knowledgeable. It shows that most people have little or no understanding of "plant synthetic biology." We should carry out related science popularization activities to raise public awareness, increase its visibility, and enhance understanding.

Regarding the application fields of plant synthetic biology, agriculture was considered the most useful, with 93.77% of respondents selecting it. It indicates that people generally believe plant synthetic biology can help improve crop yields and disease resistance, positively impacting agriculture.

The second most recognized field was healthcare, accounting for 82.87%. It shows that people are optimistic about the potential of plant synthetic biology in drug production, expecting it to bring innovation and improvements to the medical industry.

2.The public's attitudes toward plant synthetic biology products

When asked whether they support the continued research and development of plant synthetic biology technologies for producing natural metabolites, most respondents (60.75%) highly supported further research and development. 31.15% were likely to support it. Only a small number of respondents expressed uncertainty (6.85%), possible opposition (0.62%), or complete opposition (0.62%).

More than 80% of respondents indicated they would likely or be willing to purchase medicines, essential oils, or fragrances produced using plant synthetic biology technology. Fewer than 4% of respondents said they would likely not purchase such products. Meanwhile, 14.95% of respondents were uncertain, suggesting that further information or education may be needed to increase their trust. During commercialization, it will be essential to provide more information about plant synthetic biology technologies, such as their benefits, safety, and environmental friendliness, to improve the acceptance rate among the uncertain group.

3.The issues the public concerned about regarding such products

Several factors influence the public's acceptance of plant natural metabolites produced by synthetic biology, with health and safety being the most significant, accounting for 83.49%. It indicates that people prioritize health and safety when choosing products. Product price is another factor, with 51.71% of respondents listing it as a consideration. It suggests that competitive pricing is also essential when promoting plant natural metabolites produced through synthetic biology.

Exploration of Application Models

Prof. Yongfeng Guo

To enable VersaTobacco to produce large-scale plant natural metabolites, we must overcome significant challenges, including mass cultivation, infiltration, and plant sampling.

We learned from Prof. Yongfeng Guo that Agrobacterium is injected using a syringe for transient transformation in ordinary biological laboratories. There are successful cases of industrialized transient expression abroad. For example, Medicago's COVID-19 vaccine utilized transient expression in N. benthamiana.

Professor Guo mentioned that in China, the regulation of genetically modified organisms primarily focuses on field planting. For production materials, soilless cultivation in laboratory-scale factories offers more uniformity between batches and individuals, which is beneficial for ensuring the stability of the final product. Additionally, large-scale production cannot rely on syringe injection for Agrobacterium infiltration. A standard factory method uses vacuum pressure to draw the Agrobacterium suspension into the intercellular spaces. This method is very effective for N. benthamiana, as its tissues are relatively loose but much more costly than field planting.

If we can construct highly effective tobacco materials that produce valuable compounds, Professor Guo suggested collaborating with enterprises or applying for patents to facilitate the transformation of our results. However, this would undoubtedly present significant challenges.Based on Professor Guo's description, we researched similar application precedents[2, 3] and initially determined our application model.

We aim to cultivate a large quantity of N. benthamiana using artificially lit and temperature-controlled growth facilities, utilizing soilless media. By exploring suitable fermentation protocols in industrial fermenters, we intend to produce large quantities for injection into Agrobacterium. The N. benthamiana plants used for infiltration experiments were grown for 5 weeks. For infiltration, we placed the flowerpots in a small basin with a barrier and inverted the small basin into the Agrobacterium suspension. In this case, the fixed flowerpots were inverted into a stainless steel tank, allowing all N. benthamiana leaves to be immersed in a suspension of A. tumefaciens. Then, agroinfiltration was carried out using a vacuum infiltration device. Upon starting the device, the vacuum chamber's pressure dropped to a particular value, drawing out the air from the intercellular spaces and allowing the suspension to enter the leaf cells. Finally, the plants were cultured in a dark chamber for 24 hours before being returned to the greenhouse for another 4 days until they were ready to be harvested. The infiltrated leaves were then subjected to the subsequent extraction, purification, and analysis steps(Fig.2).

Fig.2 A diagram of the large-scale production process of VersaTobacco in industrial applications

Cost and Risk Assessment

Fig.3 SCU-China team members meeting with Mr.Xie from Aktin Chemicals, Inc.

To understand the industry perspective, we had a meeting with Mr. Xie, Chairman of Aktin Chemicals, Inc. Aktin Chemicals, Inc. is dedicated to improving people's lives by developing and manufacturing quality bioactive ingredients and molecules, as well as providing CMO/CDMO solutions for global nutraceutical, cosmetic, and pharmaceutical industries.

He acknowledged the scientific significance of our project but expressed concerns about its commercial value. He pointed out that for our project to achieve industrialization, we need to overcome at least four cost issues: the cost of N. benthamiana, the cost of fermentation, the cost of the infiltration process, and the cost of product extraction. Firstly, it is crucial to significantly reduce the cost of industrial transformation, ideally achieving high yields with a single spray. Secondly, we need to lower the cultivation costs of N. benthamiana (such as greenhouse cultivation costs). Furthermore, the yield of the products in N. benthamiana must be increased to at least 10% of the total fresh weight for companies to justify mass production.

Regarding selecting target products for production, he mentioned that resveratrol and crocin could serve as chassis validation products. However, the target products for commercial production must consider market demand and optimal methods. He also highlighted the importance of innovation at the gene-editing level, indicating that the core work should focus on gene design and improvement before returning to the downstream experimental components to mitigate risks.

Afterward, we gathered the relevant data from Aktin Chemicals, Inc for commercial modelling. For related results, please refer to the Market Promising Analysis

Prevention of Agrobacterium and plasmid residues

Mr. Zhang emphasized that since we are using Agrobacterium for transformation, we need to pay attention to potential residues of bacterial liquid and plasmid in the leaves. He identified several factors to avoid such residues:

• Bacterial strain toxicity: Given that we are using the commercial Agrobacterium strain GV3101, the strain poses minimal risk.
• Plant washing: After sampling, the leaves can be washed multiple times to remove surface Agrobacterium, reducing the amount that may enter during subsequent extraction processes.

Upon learning that we will still need to conduct plant and product purification after leaf sampling, Mr. Zhang stated that it is possible to almost completely avoid any residues with a well-designed extraction process. During metabolite extraction and purification, various organic solvents are used, and multiple chromatographic techniques (such as affinity chromatography, ion exchange chromatography, and gel filtration) are employed. These not only enhance purity but also effectively remove Agrobacterium and vector residues.

However, the actual procedures and regulatory standards to be followed depend on specific circumstances and requirements. According to the Guidelines for the Safety Evaluation of Gene-Edited Plants for Agricultural Use (Trial) issued by China's Ministry of Agriculture and Rural Affairs, a comprehensive evaluation of the absence of Agrobacterium and plasmid residues, requires analyzing residual vector sequences, including analyzing the backbone sequence, main components, and providing details on test methods, data quality, analysis methods, and conclusions. Additionally, assessing and providing evidence for the gene-editing off-target effects is essential.

Prevention of the release of gene-edited plant seeds into the natural environment

After using CRISPR/Cas9 to knock down chlorogenic acid levels, it is theoretically expected that lignin will accumulate, leading to higher biomass accumulation in the gene-edited plants compared to wild types. Although the target gene knockout does not confer any genetic preference or ecological niche advantage, passing environmental and food safety assessments will require adhering to the guidelines set by the Ministry of Agriculture and Rural Affairs of China in the Safety Evaluation Guidelines for Gene-Edited Plants for Agricultural Use (Trial). These guidelines mandate the assessment of the target traits across different generations, requiring at least three generations of experimental data. If the target traits do not increase environmental safety risks, data or analysis proving this must be provided. Similarly, if the traits do not increase food safety risks, corresponding data or analysis should be submitted. Following this, further trials, including intermediary tests, are necessary, with a comprehensive experimental report to be provided.

All these evaluations are contingent on the success of initial lab tests. Lab test success criteria include the following:

1. Stable yields and reliable product quality;
2. Defined operational conditions, and established analysis methods for products, intermediates, and raw materials;
3. Corrosion resistance tests on specific equipment and pipelines have been conducted, and the necessary general equipment is available;
4. A material balance has been performed, and preliminary methods for handling industrial "three wastes" (waste gas, wastewater, and solid waste) have been proposed;
5. Specifications and consumption levels of raw materials have been established;
6. Safety production requirements have been outlined.

We have considered the above aspects to the best of our ability. Regarding point 2, we have established analysis methods for the raw materials in the wet laboratory section. For point 4, since we are developing a universal chassis with various target compounds, we cannot determine specific analysis methods. However, we consulted Mr. Zhang about the general framework for the analysis methods, which primarily involved solvent extraction and macroporous resin separation. After separation, recovery is performed, and crystallization methods are designed based on the solvent properties (this involves experimenting with different solvents and reviewing relevant literature to design experiments). The general process cycle takes approximately 5 to 7 days. Concerning waste management, Mr. Zhang indicated that once the extraction method is finalized, waste handling can be coordinated with specialized companies authorized by the government to design the collection, transportation, and treatment methods for the waste liquids.

For point 5, using the production of resveratrol from VersaTobacco as an example, we obtained relevant production data from Mr. Xie, Chairman of Aktin Chemicals, Inc. We conducted a preliminary material balance and performed an ROI Analysis and Risk-Return Analysis in the modelling section.

Future Possibilities

During our discussions, stakeholders presented us with many brilliant ideas to help explore further possibilities for VersaTobacco. Unfortunately, we cannot complete this during the current season due to time constraints. We have compiled the experts' suggestions here to provide iGEM teams with different perspectives for consideration.

Dr. Jinquan Huang

Dr. Huang is a researcher at the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, focusing on the extracellular biosynthesis and regulation of plant natural products. He provided several improvement strategies for enhancing plant metabolite yields from various angles, including vector construction and plant biomass.

While the transient transformation of N. benthamiana allows each gene to be placed on different plasmids, mixing and injecting Agrobacterium containing different plasmids does not guarantee that all genes will be introduced into the same cell. Some cells within a leaf may receive all genes, while others may only receive one. This discrepancy could significantly affect production efficiency. Dr. Huang suggested we add linkers between gene sequences to express the genes in a polycistronic format.

The advantage of N. benthamiana lies in its ability to produce target metabolites within 1 to 2 weeks through transient transformation; however, its biomass is significantly smaller than that of conventional tobacco. Dr. Huang recommended that we find ways to increase the biomass of N. benthamiana, potentially through hormone application or further modification of the chassis.

To enhance the content of a specific class of compounds, we could consider overexpressing related transcription factors. For instance, if we aim to increase flavonoid content, we could explore introducing AtMYB12 into tobacco[4].