Repeated evolution of cytochrome P450-mediated spiroketal steroid biosynthesis in plants

Abstract

Diosgenin is a spiroketal steroidal natural product extracted from plants and used as the single most important precursor for the world steroid hormone industry. The sporadic occurrences of diosgenin in distantly related plants imply possible independent biosynthetic origins. The characteristic 5,6-spiroketal moiety in diosgenin is reminiscent of the spiroketal moiety present in anthelmintic avermectins isolated from actinomycete bacteria. How plants gained the ability to biosynthesize spiroketal natural products is unknown. Here, we report the diosgenin-biosynthetic pathways in himalayan paris ( Paris polyphylla ), a monocot medicinal plant with hemostatic and antibacterial properties, and fenugreek ( Trigonella foenum–graecum ), an eudicot culinary herb plant commonly used as a galactagogue. Both plants have independently recruited pairs of cytochromes P450 that catalyze oxidative 5,6-spiroketalization of cholesterol to produce diosgenin, with evolutionary progenitors traced to conserved phytohormone metabolism. This study paves the way for engineering the production of diosgenin and derived analogs in heterologous hosts.

Characterization of steroid 5α-reductase involved in α-tomatine biosynthesis in tomatoes

“…Then, the duplication gives rise to SlS5αR2, which is specialized in α-tomatine biosynthesis. The duplication and neofunctionalization have been reported for several enzymes involved in phytosterols, BRs and cholesterol biosynthesis (Christ et al 2019;Sawai et al 2014;Sonawane et al 2017). For instance, potato and tomato have two DWF1 homologs named SSR1 and SSR2 (Sawai et al 2014).…”

An enormous Paris polyphylla genome sheds light on genome size evolution and polyphyllin biogenesis

“…Five saponins (i.e., polyphyllins I, II, V, VII and H) were detected, among which the diosgenin-derived polyphyllins accounted for ~90% of total saponins on average, and the rest minor saponins were pennogenin-derived polyphyllins in the storage tissue rhizome (Supplementary Table 10). Despite the recent elucidation of diosgenin biogenesis 19 , the genes responsible for biosynthesis of pennogenin and various glycosylations to form multiple polyphyllins remain unknown. Diosgenin was previously confirmed to be produced by a number of cytochrome P450 enzymes (CYPs) including CYP90G4 and CYP94D108/CYP94D109/CYP72A616 via C16, C22 and C26 hydroxylation and the subsequent 5,6-spiroketalization of cholesterol 19 (Fig.…”

“…Despite the recent elucidation of diosgenin biogenesis 19 , the genes responsible for biosynthesis of pennogenin and various glycosylations to form multiple polyphyllins remain unknown. Diosgenin was previously confirmed to be produced by a number of cytochrome P450 enzymes (CYPs) including CYP90G4 and CYP94D108/CYP94D109/CYP72A616 via C16, C22 and C26 hydroxylation and the subsequent 5,6-spiroketalization of cholesterol 19 (Fig. 4a).…”

“…Of the 259 identified PPY P450 genes ( Supplementary Fig. 17), eight CYPs including CYP90G4 and CYP94D108 19 , which were proved to be involved in diosgenin biosynthesis, and other 110 genes were located in a common co-expression module (module 35) (Fig. 4b).…”

“…Many plant P450s exhibit functional versatility. For example, the CYP90B subfamily has been well known to catalyze the rate-limiting sterol C22hydroxylation in brassinosteroid (BR) biosynthetic pathway 23,24 ; and it also displays low activity of sterol 16-hydroxylation 25 . Since the contents of pennogenin-derived saponins are low in the storage tissue rhizome throughout different living stages ( Supplementary Table 10) with diosgenin-based saponins being the main components 26 , we could not exclude the possibility that pennogenin might be a "side-product" that is generated by a moonlight P450 activity during the production of diosgenin.…”