Biosynthesis of ginsenoside compound K in <i>Panax japonicus</i> by metabolic regulation

Ginsenoside compound K (CK) exhibits valuable pharmacological activity and has potential applications in the development of antitumor and immunity-enhancing drugs. As a metabolite of ginsenosides in the gut, ginsenoside CK is generally considered a kind of non-natural ginsenoside that cannot be synthesized in Panax species. In this study, necessary genetic modules for ginsenoside CK biosynthesis were found in Panax species, more specifically, in Panax japonicas (P. japonicus) for the first time. Based on the new findings, RNA sequencing was conducted on P. japonicus cells, and two UDP-glycosyltransferases (UGTs), UGTPj3 and UGTPj20, involved in the biosynthesis of protopanaxadiol (PPD)-type ginsenosides were identified. UGTPj3 and UGTPj20 can convert propanaxanediol to ginsenosides Rh2 and CK, respectively. Further analyses showed that UGTPj20 exhibited a lower affinity for propanaxanediol, compared with UGTPj3. Therefore, propanaxanediol tended to be converted to ginsenoside Rh2 by UGTPj3, which led to the absence of ginsenoside CK in P. japonicus. Moreover, ginsenoside CK was successfully synthesized in P. japonicus cells with CYP716A53V2 and UGTPj3 RNA interference and UGTPj20 overexpression in this study. The titer of ginsenoside CK in the P. japonicus cell suspension culture reached 85 mg L^-1. This study has achieved ginsenoside CK biosynthesis in Panax species for the first time by regulating the metabolic pathway without introducing any foreign genes. The findings of this study also show that the variety of saponins synthesized by Panax species may be far richer than expected.