Identification of the protopanaxatriol synthase gene CYP6H for ginsenoside biosynthesis in Panax quinquefolius.

Panax quinquefolius is one of perennial herbs and well known for its outstanding pharmacological activity. Ginsenosides are thought to be the main active ingredients in P. quinquefolius and exist in many kinds of plant genus Panax (ginseng). Protopanaxatriol synthase, which is considered cytochrome P450 (CYP450) in ginsenoside biosynthesis pathway can convert protopanaxadiol into protopanaxatriol. However, the protopanaxatriol synthase gene in P. quinquefolius has not been identified. Here, we cloned and identified a protopanaxatriol synthase gene from P. quinquefolius (CYP6H, GenBank accession no. KC190491) at the first time, reverse transcription-PCR (RT-PCR) analysis showed no obvious transcription change of CYP6H in methyl jasmonate (MeJA)-induced hairy roots. Ectopic expression of CYP6H in Saccharomyces cerevisiae resulted in the production of protopanaxatriol with added exogenous protopanaxadiol and confirmed by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Moreover, high-performance liquid chromatography (HPLC) analysis shows that RNA interferences of CYP6H in transgenic hairy roots could increase the accumulation of protopanaxadiol-type ginsenosides and decrease the accumulation of protopanaxatriol-type ginsenosides, whereas the effect of overexpression CYP6H in transgenic hairy roots was contrary. Our study indicated that CYP6H is a gene encoding protopanaxadiol 6-hydroxylase which could convert protopanaxadiol into protopanaxatriol in P. quinquefolius ginsenoside biosynthesis, we also have confirmed the function of CYP6H on effect accumulation of ginsenosides.

The isolation and characterization of dammarenediol synthase gene from Panax quinquefolius and its heterologous co-expression with cytochrome P450 gene PqD12H in yeast.

Panax quinquefolius is one of perennial herbs and well known for its outstanding pharmacological activity. Ginsenosides are thought to be the main active ingredients in Panax quinquefolius and exist in many kinds of plant genus Panax (ginseng). Dammarenediol synthase, which is considered as a key enzyme in ginsenoside biosynthesis pathway can convert 2, 3-oxidosqualene into dammarenediol-II. However, the dammarenediol synthase gene in Panax quinquefolius has not been identified. Here, we cloned and identified a dammarenediol synthase gene from Panax quinquefolius (PqDS, GenBank accession No. KC316048) at the first time, and reverse transcription-PCR (RT-PCR) analysis also showed an obvious transcription increase of PqDS in the methyl jasmonate (MeJA)-induced hairy roots. Ectopic expression of PqDS in yeast resulted in the production of dammarenediol-II was confirmed by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Moreover, overexpression of PqDS in transgenic hairy roots could increase the transcription of gene PqDS and another P450 gene PqD12H (encoding protopanaxadiol synthase in Panax quinquefolius), the accumulation of ginsenosides also increased at the same time. In addition, both PqDS and PqD12H gene co-expressed in recombinant yeast result in the production of protopanaxadiol was detected by LC/APCIMS; this result also provides a new strategy for the abundant production of protopanaxadiol in vitro.

[Induction of hairy roots of Panax ginseng and studies on suitable culture condition of ginseng hairy roots].

Ginseng is a valuable medicinal plant with ginsenosides as its mian effective components. Because ginseng is a perennial plant and has a very strict demand for soil conditions, the way of cultivating ginseng by cutting woods is still used in China at present and thus forest resources has been extremely destroyed. Increasing attention has been paid to the hairy roots induced by the infection of Agrobacterium rhizogenes in the production of plant secondary metabolic products for the hairy roots are characterized by rapid growth and stable hereditary and biochemical traits. That has opened a new way for the industrial production of ginseosides. However, there is little report for such studies from China. In this paper, hairy roots of ginseng were induced from the root explants of two-year-old ginseng by Agrobacterium rhizogenes A4 with directly inoculating. The transformed hairy roots could grow rapidly on MS medium and 1/2 MS medium without hormones. The cultured clones of the hairy roots were established on a solid 1/2 MS medium. After 4 - 5 subcultures the hairy roots still maintained a vigorous growth. A pair of primers were designed and synthesized according to the analytical results of RiA4TL-DNA sequence by Slightom et al . 0.8kb rolC was obtained by PCR using the genome DNA of hairy root of ginseng. Transformation was confirmed by PCR amplification of rolC genes from the hairy roots of P. ginseng. Growth rate of hairy roots on liquid medium increased by 2 times then that of the solid medium. The growth of the hairy roots can be divided into three stages: high speed in the first two weeks, middle speed in the 3 - 4 weeks and low speed hereafter. Changing the culture solution at 2 weeks regular intervals is conductive to maintaining the rapid growth of the hairy roots. By means of determination for specific growth rate and ginsenosides content, the high-yield hairy root clone R9923 was selected. The content of monomer gisenoside of Rg1, Re, Rf, Rbl, Rc, Rb2 and Rd in hairy root clone R9923 was determined by the HPLC. The total ginsenosides content in the hairy toot clone R9923 came up to 15.2 mg/g. The suitable culture conditions for ginseng hairy roots growing were 1/2 MS liquid medium (30 g/L glucose), in a shaker at 110 r/min, changing the culture solution at 2 weeks and subculture time 4 weeks. In the liquid fermented culture of 2L medium, the yield of the hairy roots could amount to 270.10 g in 4 weeks. The industrial production of ginsenosides has been preliminarily realized. Effect factors on biomass and ginsenosides content such as culture volume, inoculation, in steps cultural technology at the scale-up process of hairy roots culture were also explorated. Our results have laid a foundation for defining optimum culture manner for large-scale cultivation and large-scale production of ginsenosides.