# Advances in the Biosynthesis and Molecular Evolution of Steroidal Saponins in Plants
## 2 Biosynthesis of Steroidal Saponins
### Modification of Cholesterol or Sitosterol Side Chain to Form Steroidal Saponins  
As steroidal saponins are generated by a series of complex catalytic reactions, such as the oxidation, hydroxylation, and glycosylation of cholesterol or the \(\beta\)-sitosterol molecular skeleton, the synthesis of steroidal saponins has been controversial at present [1; 19; 23] (Figure 2). Some candidate genes related to the biosynthesis of the steroidal saponin precursor diosogenin (such as _PpcyP90G4_, _PpcYP94D108_, _PpcYP94D109_, _PpcYP72A616_, _TfCYP90B50_, _TfCYP72A613_, and _TfCYP82J17_) were screened from _P. polyphylla_ and _Trigonella fonium graceum_ (fenugreek), and the metabolite changes in transgenic _Nicotiana benthamiana_ plants were verified by metabolomics [19]. Finally, the functions of two genes, _PpcYP90G4_ and _TfCYP90B50_, in plants were determined: they were involved in the formation of 22S-hydroxycholesterol by catalytic side chains of cholesterol [19]. The generated 22S-hydroxycholesterol was then catalyzed by CYPs and other enzymes to finally synthesize steroidal saponins [19; 22]. However, [PERSON] et al. showed that the _DzCYP90B71_ gene was involved in cholesterol C22-R oxidation and that the _DzCYP90G6_ gene was involved in catalytic C16 oxidation through the transcriptome sequencing and targeted metabolome analysis of _D. zingigherensis_[17]. They believed that the _CYP90_ gene should catalyze the formation of cholesterol, which should be 22R hydroxycholesterol, rather than 22S hydroxycholesterol [17; 19]. In general, diosogenin is generated from cholesterol through the catalysis of cholesterol C16/C22-monohydroylase (CYP90B/CYP94), cholesterol 26-hydroxylase (CYP2A), and other enzymes. The first step of the biosynthesis of steroidal saponins from diosogenin is the glycosylation of 3-O-sterol glycosyltransferase (S3 GT belongs to the UGT80 family), which transfers the glycosyl group to the 3-OH site of the diosogenin [24; 25]. At present, there have been few studies on the late modification of steroidal saponin aglycones, such as diosigenin. In addition to _UGT80_, [PERSON] et al. found that the _UGT73 CR1_ gene is involved in the later modification of steroidal saponins, such as the glycosylation of steroidal saponins, and has a higher affinity for diosegin than _UGT80_[25; 26]. In addition, furostanol glycoside 26-O-\(\beta\)-glucosidase (F26G) can not only catalyze the conversion of furostanol saponins to spirostanol saponins, but is also involved in catalyzing diosogenin to form steroidal saponins [20; 27]. B-sitosterol may also be catalyzed by a series of enzymes to form steroidal saponins, which needs further exploration. Although the current plant tissue culture technology is an effective and important means to explore and verify the biosynthesis of plant-specific metabolites [28; 29], it remains difficult to construct the transgenic system of many medicinal plants, such as _D. zingibernensis_ and _P. polyphylla_, which leads to difficulty in the verification of many biosynthetic pathway genes. Therefore, people use microorganisms to explore the biosynthetic mechanism of steroidal saponins [28; 30], and the construction of engineering strains seems to be the most effective and economical method to produce natural plant-based drugs. [PERSON] et al. first constructed a yeast chassis strain (DG-Cho) capable of producing cholesterol and then co-expressed the _DzinCYP90G6_ and _DzinCYP94D144_ (identified from _D. zingibernsis_) and _VvCPR_ (cytochrome P450 reductases) genes from _Vitis vinifera_ in the constructed strain. Finally, engineering strain DG002, which can convert cholesterol into diosgenin, was obtained [30]. The DG002 strain constructed by [PERSON] et al. significantly improved the conversion rate of cholesterol into diosgenin [30]. In addition, the constructed engineering strains can also be used to verify the function of the key genes involved in steroidal saponin biosynthesis isolated and identified from different plants.