Research On MVA And MEP Pathways Of Ginsenoside Biosynthesis By Utilizing Inhibitors

Ginsenoside is one of the main active ingredients of ginseng, it is of great medicinal valueand widely used in many aspects such as anti-aging, enhancing immunity, etc. It also has someeffects on prevention and treatment of cancer. Via increasing the contents of ginsenoside byregulation of ginsenoside biosynthesis, metabolic engineering has become one kind of effectivemethod to improve the quality of ginseng. Until now, the research of ginsenoside biosynthesismechanism is still in the initial stage. It was generally believed that ginsenosides aresynthesized via the mevalonate (MVA) pathway, but few relevant reports on MEP pathway(also called pyruvate pathway) of ginseng.In our study, ginseng hairy root was used as experimental material. we added mevalonatepathway（MVA） inhibitor lovastatin and the non-mevalonate pathway (MEP) inhibitorfosmidomycin into the MS medium. These two inhibitors were added respectively or together.Based on the contents of total saponin, monomer saponin and ginsenoside precursordammarenediol-II in ginseng, and changes of MVA and MEP pathway key enzyme activity, wewanted to find whether the MVA and MEP pathway were two of the ginsenoside biosynthesispathway, as well as the relationship between these two pathways to lay the foundation for theresearch on metabolic engineering of ginsenosides and the further study of ginsenosidebiosynthesis mechanism.Results of the study are as follows:1. Issued for the problem of high polysaccharide levels in ginseng hairy root, optimized theextraction and determination of total saponin and monomer saponin (Rg1, Re and Rb1),established an appropriate method for xtraction and content determination.2. In different concentration and feeding time of inhibitor for ginseng hairy root growth study,we found that lovastatin and fosmidomycin did not inhibit the growth of hairy root whenconcentrations were lower than50μmol/L and30μmol/L, and the most suitable feeding time was7days.3. Under the experimental conditions, the maximum inhibition rate of lovastatin on thebiosynthesis of total saponin was57.93%, the maximum inhibition rate of fosmidomycinon the biosynthesis of total saponin was42.17%, it was suggested that the both MVApathway which played a dominant role and MEP pathway exsited in the biosynthesis ofginsenosides.4. Experimental investigation on content of monomer saponin Rg1、Re and Rb1in Ginsenghairy root indicated that the inhibition on these monomer saponins roughly subject to thesame laws with the inhibition on total saponin, the maximum inhibition rate of lovastatinon Rg1、Re and Rb1was respectively50.40%、66.65%、40.12%, and the maximuminhibition rate of fosmidomycin on Rg1、Re and Rb1was respectively38.54%、38.84%、19.51%. This result indicated that the accumulatation rates of these monomer saponins aredifferent.5Experimental investigation indicated that both lovastatin and fosmidomycin cansignificantly inhibit the content of dammarenediol-II. The maximum inhibition rates oflovastatin and fosmidomycin on dammarenediol-II was respectively64.58%and46.83%,which coincided with the results of variation of total saponin.6. Enzyme activity determination of HMGR under lovastatin treatment indicated thatlovastatin has inhibition on HMGR with the maximum inhibition rate of96.82%comparedto the highest enzyme activity of10.393U/mg of control group; Enzyme activitydetermination of DXR under fosmidomycin treatment indicated that fosmidomycin hasinhibition on DXR with the maximum inhibition rate of96.25%compared to the highestenzyme activity of0.266U/mg of control group.