Stevioside is a natural compound synthesized in Stevia. The steviol glycosides in the leaves of Stevia rebaudiana are a mixture of different structural molecules, mainly including steviol glycosides, rebaudioside A, rebaudioside D, etc. The sweetness of Stevia glycosides is 250-350 times that of sucrose, which can replace sucrose as a new generation of zero-calorie natural sugar source.

Stevia glycosides have the characteristics of good safety and high sweetness and have broad application prospects in the food industry. Nowadays, high sugar intake in daily diet is one of the factors causing obesity. Cardiovascular diseases such as hypertension, hyperglycemia, and diabetes caused by obesity are a major public health crisis affecting global human health. Stevia glycosides are added as food additives to reduce sucrose intake and assist in controlling obesity. Clinical experiments have shown that stevia glycosides can not only be used as food additives but also have certain health and medical effects. It has certain healthcare functions for anti-diabetes, anti-cardiac fibrosis, anti-fatty liver, anti-inflammatory, antibacterial, anti-tumor and other diseases.

Due to its excellent characteristics, steviol glycosides have a wide demand in the market (as of 2016, the consumption population has exceeded 4 billion people), and the future trend is predicted to have a good growth rate (it is expected that the market value of steviol glycosides will grow at a compound annual growth rate of 8.0% by 2028, and the market size is expected to reach 1.14 billion US dollars). It is a very promising natural compound. Therefore, exploring production methods with high economic value and environmental friendliness is the goal of modern industrial production.*(李亚桐）

Up to now, the preparation methods of stevia glycosides are mainly divided into traditional plant-based stevia glycoside extraction methods, including hot water extraction, solvent extraction, impregnation, macroporous resin adsorption, and modern innovative microwave-assisted extraction, ultrasonic-assisted extraction, and rapid solid-liquid dynamic extraction. However, the above innovative extraction methods are still in the laboratory research stage and have not been applied in actual industrial production. At present, the use of microbial heterologous synthesis of stevia glycosides is a good preparation method choice, with high economic value and environmental friendliness, which can effectively reduce the carbon footprint in traditional production methods.

Therefore, we plan to explore new pathways for the synthesis of stevia glycosides in our project and further efficiently utilize microbial heterologous synthesis of stevia glycosides. To improve the biosynthesis efficiency of steviol glycosides, we plan to design a heterologous synthesis pathway in Escherichia coli to synthesize steviol glycosides through basic nutrients such as glucose. To increase production yield, we overexpression several genes to enhance the precursor supply.

Current methods of synthesis of steviol glycosides

Our solutions - genetic pathway, synthetic pathway *(WHO).

Steviol glycosides are obtained from Stevia rebaudiana leaves and are now frequently used in the food industry as natural low-calorie sweeteners. Current ways to synthesize steviol glycosides include polyploidy induction and micropropagation with elicitors.

Polyploidy breeding can be an auspicious approach to enhance steviol glycoside yields in Stevia rebaudiana. Polyploidy is known as a major mechanism of adaptation in the evolution of plants. Polyploidy plants can arise in nature by several mechanisms. They are large and healthy in general. Through experimenting, germinating seeds treated with 0.05% colchicine for 48 hours or 0.1% colchicine for 24 hours had been proven to be polyploidy induced with a higher number of secondary branches, more leaf thickness, and a higher steviol glycoside content in the leaf. Thus, the method of polyploid induction is effective in increasing the production of steviol glycoside.

Elicitation is a common treatment used in vitro cultures for obtaining secondary metabolites (small organic molecules produced by an organism that is not required for their development and growth) by using different elicitors. Through the use of the elicitors, the production of steviol glycoside can be significantly enhanced. Elicitors are divided into two categories: physical and chemical. Common physical elicitors will include light and temperature. For light, photoperiods are essential to induce vegetative growth that can increase leaf biomass, while light intensity is an important factor affecting plant growth and the production of secondary metabolites. Temperature plays a role as an abiotic stress factor, which also influences plant growth and secondary metabolites' accumulation. Chemical elicitors can include plant growth regulators(Me-JA), complex organic extracts(coconut water and yeast extracts), and amino acids(glutamic acid and proline) that enhance callus growth, steviol content, and plant growth.

There are many more methods that are currently used to escalate the production of steviol glycoside. However, the demand for steviol glycoside is still growing as companies are using it as a sweetener in food and beverages. Growing demand is a result of the increasing awareness of Stevia and the consumers' demand for healthier products. Therefore, we actively looked for new pathways in this project to make the synthesis of steviol glycoside more efficient.

We selected E. coli BL21(DE3) as the host for the heterologous synthesis of steviol glycosides. We incorporated the steviol glycosides biosynthetic pathway (KS_Sr, CDPS_Sr, CPR_Sr,GGPPS, KO,17αtr29CYP714A2,UGT74G1, UGT76G1, UGT85C2, EUGT11M(F379A) ) into BL21(DE3) to produce rebaudioside A , D , and M(reb A, reb D， reb M) as the final product. Besides, we introduce the isopentenol utilization pathway (IUP) to enhance the isoprenoid precursor supply (Chatzivasileiou et al., 2018b).

We divided the project into three modules: the isoprenoid (DMAPP/IPP) module, the steviol module, and the glycosyltransferase module. We synthesized the genes for each module and assembled them using a method like NEB BioBrick assembly. These modules were then inserted into vectors pACYCduet-1, pET21a, and pET28a. Under induction with IPTG, we expect to detect the rebaudiosides products.

Each module is controlled by the T7 promoter for transcription initiation and expressed in the presence of IPTG induction. The genes within the modules contain independent RBSs. After fermentation in the shake flask, Reb A, Reb D, and Reb M were detected.

https://www.who.int/news-room/fact-sheets/detail/diabetes

https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight

https://d.docs.live.net/54d98c19ea0fce07/Desktop/igem/文献/甜菊糖苷的应用及生物合成研究进展_李亚桐.pdf

https://pubs.acs.org/doi/10.1021/acs.jafc.3c01753

https://www.mdpi.com/2076-3417/12/17/8640

https://patents.google.com/patent/WO2018071744A1/en

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543730/

www.steviashantanu.com

https://www.cbi.eu/market-information/natural-food-additives/stevia/market-potential

Wang et al. 2016 -

Wang, J., Li, S., Xiong, Z. et al. Pathway mining-based integration of critical enzyme parts for de novo biosynthesis of steviol glycosides sweetener in Escherichia coli. Cell Res 26, 258–261 (2016). https://doi.org/10.1038/cr.2015.111

Chatzivasileiou, A. O., Ward, V. C., Edgar, S., & Stephanopoulos, G. (2018). Two-step pathway for isoprenoid synthesis. Proceedings of the National Academy of Sciences of the United States of America, 116(2), 506–511. https://doi.org/10.1073/pnas.1812935116

Prakash, I., Markosyan, A. A., & Bunders, C. (2014). Development of next generation Stevia Sweetener: Rebaudioside M. Foods, 3(1), 162–175. https://doi.org/10.3390/foods3010162

Xu, Y., Wang, X., Zhang, C., Zhou, X., Xu, X., Han, L., Lv, X., Liu, Y., Liu, S., Li, J., Du, G., Chen, J., Ledesma-Amaro, R., & Liu, L. (2022b). De novo biosynthesis of rubusoside and rebaudiosides in engineered yeasts. Nature Communications, 13(1). https://doi.org/10.1038/s41467-022-30826-2