Study On The Synthesis Of Ginsenoside CK In Panax Japonicus Cells

Ginsenosides are the main active ingredients of Panax medicinal materials.However,most ginsenosides with high content in Panax plants have low bioavailability and must be decomposed and converted into rare ginsenosides by specific enzymes before they can be easily absorbed and utilized by the human body.Ginsenoside CK(Compound K),referred to as CK,is a degradation product of PPD-type ginsenosides in the human intestinal tract.CK has high biological activity and shows good value in the development of proprietary drugs in anti-tumor and improving the body’s immune activity.But unfortunately,CK is not contained in the medicinal materials.Through metabolic network analysis,we found that P.japonicus has all the gene elements needed to synthesize CK.However,why P.japonicus cannot effectively synthesize CK is worthy of further exploration.This study explained the possible mechanism of Panax not synthesizing CK through experiments,and on this basis,through synthetic biology technology,the synthesis of ginsenoside CK was realized for the first time in the cells of P.japonicus.The main results obtained in this study are as follows:(1)Two glycosyltransferase genes,UGTPj3 and UGTPj20,were first cloned from P.japonicus.The recombinant plasmids of p Cold TF-UGTPj3 and p Cold TF-UGTPj20 were constructed,transformed into Escherichia coli to induce expression,and soluble recombinant proteins were successfully obtained.The results of in vitro enzymatic reaction showed that UGTPj20 can glycosylate PPD into CK,and UGTPj3 can glycosylate PPD into ginsenoside Rh2.In addition,the kinetic parameters of the enzyme reaction showed that UGTPj3 had a higher affinity for PPD than UGTPj20.When both UGTPj20 and UGTPj3 existed in the enzymatic reaction system,only ginsenoside Rh2 could be detected in the reaction product.The above results preliminarily revealed the possible mechanism that gene elements needed for the synthesis of CK exist in P.japonicus,but it is difficult to effectively synthesize CK.UGTPj20 has a poor affinity with the substrate PPD.When the plant contains UGTPj3,which has a higher affinity to PPD,PPD is extensively utilized for synthesizing ginsenoside Rh2,thereby resulting in an ineffective enzymatic reaction from PPD to CK.(2)The RNA interference vectors p Hellsgate-UGTPj3 and p HellsgateCYP716A53v2 of UGTPj3 and CYP716A53v2 genes,as well as the overexpression vector p CAMBIA2300s-UGTPj20 of UGTPj20 genes were successfully constructed.The three expression vectors were respectively transformed into Agrobacterium LBA4404 competent cells,and then introduced into P.japonicus cells via genetic transformation.After positive strain screening,four transgenic ginseng cell lines(T3-1～T3-4)were obtained,which realized dual gene expression interference of UGTPj3 and CYP716A53v2,while overexpressing UGTPj20.Compared with non-transgenic cells,the expression level of UGTPj20 gene was significantly increased in T3-1～T3-4,while the expression level of UGTPj3 and CYP716A53v2 gene was decreased in different degrees.The coordinated regulation of gene overexpression and interference was successfully achieved in the four P.japonicus cell lines mentioned above to synthesize ginsenosides.Further detection and analysis of the biosynthesis of saponins in the transgenic lines showed that ginsenoside CK was detected in Panax japonicus cells,achieving the synthesis and accumulation of ginsenoside CK in P.japonicus cells.Ginsenoside CK is an important active metabolite of natural ginsenosides in the human body,represents the core pharmacological effects of Panax species herbs and has significant research value.Academia generally believes that ginsenoside CK,as an unnatural ginsenoside,cannot be synthesized in Panax plants.Our research has broken with traditional fixed ideas and explored a new approach,achieving for the first time the synthesis of non-natural ginsenoside CK in Panax species plant cells without introducing exogenous genes.This study provides technical methods and new strategies for a deeper understanding of the biosynthetic network of ginsenosides.