# Advances in the Biosynthesis and Molecular Evolution of Steroidal Saponins in Plants
## 7 Conclusions and Perspectives  
In this review, we systematically introduced the genes involved in the biosynthesis of steroidal saponins and their functions in plants. The key biosynthetic steps mainly focus on the biosynthesis of cholesterol and the hydroxylation, oxidation, and glycosylation of its side chains. The biosynthetic pathway of cholesterol has been thoroughly studied, but the specific catalytic step (i.e., the modification of the cholesterol C22 position) of diosogenin from cholesterol is still controversial. In addition, the production of different steroidal saponins by diosgenin under the catalysis of CYP450 and UGT enzymes is still under-studied. Therefore, it is necessary to further explore how diosgenin can synthesize steroidal saponins through glycosylation and other modifications by CYP450 and UGT. Moreover, there have been few in-depth studies on the regulation mechanism of steroidal saponin biosynthesis, and the identification of these various factors involved in the regulation of biosynthesis, including exogenous hormones, and transcription factors, can be used to enhance the biosynthesis of steroidal saponins in plants.  
Due to the complex genetic background and metabolic composition of plants containing steroidal saponins, it is difficult to obtain plant calluses that can be used for transgenic verification, which is the greatest difficulty in the functional verification of genes related to steroidal saponin biosynthesis in plants. In addition to improving the content of steroidal saponins in plants using transgenic technology, it is also possible to use corresponding substrates in microbial systems (such as yeast) for the exogenous synthesis of steroidal saponins through synthetic biology methods. This strategy can also be used for the screening and identification of key genes for steroidal saponin biosynthesis. Therefore, the studies on steroidal saponin biosynthesis in the engineering bacteria system can provide important clues for screening and identifying new candidate genes that may also be applied to the industrial production of drugs.  
We also reviewed the functions and distribution of steroidal saponins in plants and discussed the molecular evolutionary mechanism of steroidal saponin biosynthesis. These research advances can not only provide a reference for studying how steroidal saponinsdistribute and evolve in plants, but can also provide clues for exploring and mining new steroidal saponin resource plants.  
In our opinion, without breaking through the technical problem of using transgenic plants to verify the function of pathway genes, the most effective research strategy to explore the biosynthesis and regulation mechanism of steroidal saponins is the use of microorganisms. At the same time, understanding the molecular evolution mechanism of steroidal saponins can also provide a reference for other specialized plant metabolites.  
The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/ijms24032620/s1](https://www.mdpi.com/article/10.3390/ijms24032620/s1)]([https://www.mdpi.com/article/10.3390/ijms24032620/s1](https://www.mdpi.com/article/10.3390/ijms24032620/s1)). References [141; 142; 143; 144; 145; 146; 147; 148; 149; 150; 151; 152; 153; 154] are cited in the supplementary materials.  
Conceptualization, J.L. and Y.L.; writing--original draft preparation, Y.L., H.Y. and Z.L.; writing--review and editing, Y.L., H.Y., Z.L. and S.L.; supervision, J.L. All authors have read and agreed to the published version of the manuscript.  
This work was supported by grants from the National Natural Science Foundation of China (30370152, 31270345, and 31470388, all to J.L.), and the project of Academician Expert Workstation Project of Yunnan Province (202005 AF150019 to J.L.).  
Not applicable.  
The authors declare no conflict of interest.