Upregulation of phytosterol and triterpene biosynthesis in Centella asiatica hairy roots overexpressed ginseng farnesyl diphosphate synthase.

Farnesyl diphosphate synthase (FPS) plays an essential role in organ development in plants. However, FPS has not previously been identified as a key regulatory enzyme in triterpene biosynthesis. To elucidate the functions of FPS in triterpene biosynthesis, C. asiatica was transformed with a construct harboring Panax ginseng FPS (PgFPS)-encoding cDNA coupled to the cauliflower mosaic virus 35S promoter. Higher levels of CaDDS (C. asiatica dammarenediol synthase) and CaCYS (C. asiatica cycloartenol synthase) mRNA were detected in all hairy root lines overexpressing when compared with the controls. However, no differences were detected in any expression of the CaSQS (C. asiatica squalene synthase) gene. In particular, the upregulation of CaDDS transcripts suggests that FPS may result in alterations in triterpene biosynthesis capacity. Squalene contents in the T17, T24, and T27 lines were increased to 1.1-, 1.3- and 1.5-fold those in the controls, respectively. The total sterol contents in the T24 line were approximately three times higher than those of the controls. Therefore, these results indicated that FPS performs a regulatory function in phytosterol biosynthesis. To evaluate the contribution of FPS to triterpene biosynthesis, we applied methyl jasmonate as an elicitor of hairy roots expressing PgFPS. The results of HPLC analysis revealed that the content of madecassoside and asiaticoside in the T24 line was transiently increased by 1.15-fold after 14 days of MJ treatment. This result may indicate that FPS performs a role not only in phytosterol regulation, but also in triterpene biosynthesis.

Characterization of a dammarenediol synthase in Centella asiatica (L.) Urban.

To elucidate the exact function of CabAS in Centella asiatica, which was previously reported as a putative beta-amyrin synthase [Plant Cell Rep, 24:304-311, 2005], this gene was functionally expressed in the lanosterol synthase-deficient yeast mutant (erg7). After inducing the CabAS gene with galactose, a peak consistent with the dammarenediol standard was detected in LC/APCIMS analyses and the accumulated product was confirmed as dammarenediol. CabAS should therefore be renamed to C. asiatica dammarenediol synthase (CaDDS). The confirmation of this gene function may allow us to better understand the generation of numerous triterpene carbon skeletons.

Enhanced production of asiaticoside from hairy root cultures of Centella asiatica (L.) Urban elicited by methyl jasmonate.

Transformed root ("hairy root") cultures have been shown to be a good model for the study of many secondary metabolites. However, economically important compounds such as asiaticoside and madecassoside are produced in insignificant amounts in the root of Centella asiatica (L.) Urban. To overcome this problem, C. asiatica was transformed using Agrobacterium rhizogenes strain R1000 that harbors pCAMBIA1302 encoding the hygromycin phosphotransferase (hpt) and green fluorescence protein (mgfp5) genes and the hairy culture was coupled with elicitation technique. Hairy roots were obtained at a frequency of up to 14.1% from a tissue junction between the leaf and petiole. Abundant hairy roots were observed when co-cultivation of the plant with A. rhizogenes was done for 7 days (36.1%). Transformation was confirmed by PCR and Southern blot analyses. Five weeks after inoculation, no asiaticoside was detected in the hairy root samples. However, when 0.1 mM methyl jasmonate (MJ) was applied as an elicitor to the culture medium for 3 weeks, a large quantity of asiaticoside was generated (7.12 mg/g, dry wt). In the case of gene expression, 12 h after MJ treatment the expression of the CabAS (C. asiatica putative beta-amyrin synthase) gene in the hairy roots is significantly different from that of the control and this level of transcripts was maintained for 14 days. Our results showed that production of C. asiatica hairy roots could be optimized and the resulting cultures could be elicited with MJ treatment for enhanced production of asiaticoside.

Production of antioxidant compounds by culture of Panax ginseng C.A. Meyer hairy roots: I. Enhanced production of secondary metabolite in hairy root cultures by elicitation.

Ginseng (Panax ginseng C.A. Meyer) hairy root cultures, established by infecting ginseng root discs with Agrobacterium rhizogenes, were used for secondary metabolite production. In this study, several elicitors [salicylic acid (SA), acetylsalicylic acid (ASA), yeast elicitor, and bacterial elicitor] were used to improve the productivity of useful metabolite in P. ginseng hairy root cultures. In SA elicitation, total ginseng saponin content increased slightly at lower elicitor dosages (0.1 to 0.5 mM). Also, the use of ASA as an elicitor resulted in the inhibition of biomass growth and an increase in total ginseng saponin content at every elicitor dosage (0.1 to 1.0 mM) by about 1.1 times. With yeast elicitor addition, hairy root growth was inhibited about 0.8-fold on a dry weight basis compared to the control, but total ginseng saponin content increased by about 1.17 times when compared to the control. The bacterial elicitor showed a slight inhibition of biomass growth, but total ginseng saponin content increased by about 1.23 times upon the addition of 1 mL.

Cloning and expression of a farnesyl diphosphate synthase in Centella asiatica (L.) Urban.

A cDNA encoding farnesyl diphosphate synthase (FPS; EC2.5.1.1/EC2.5.1.10) was isolated from Centella asiacita (L.) Urban, using degenerate primers based on two highly conserved domains. A full-length cDNA clone was subsequently isolated by rapid amplification of cDNA ends (RACE) PCR. The sequence of the CaFPS (C. asiatica farnesyl diphosphate synthase) cDNA contains an open reading frame of 1029 nucleotides encoding 343 amino acids with a molecular mass of 39.6 kDa. The deduced CaFPS amino acid sequence exhibits 84, 79, and 72%, identity to the FPSs of Artemisia annua, Arabidopsis thaliana, and Oryza sativa, respectively. Southern blot analysis suggested that the C. asiatica genome contains only one FPS gene. An artificially expressed soluble form of the CaFPS was identified by SDS-PAGE. It had high specific activity and produced farnesyl diphosphate as the major isoprenoid.

Cloning of a cDNA probably encoding oxidosqualene cyclase associated with asiaticoside biosynthesis from Centella asiatica (L.) Urban.

A homology-based PCR method was used to clone a cDNA encoding oxidosqualene cyclase from Centella asiatica, which produces a large quantity of triterpene saponins such as asiaticoside and madecassoside. Sequence analysis of one clone found sequences related to beta-amyrin synthase. An open reading frame in the full-length clone was named CabAS (Centella asiatica putative beta-amyrin synthase). On the basis of amino acid sequence, CabAS appears to be an enzyme (beta-amyrin synthase) that synthesizes beta-amyrin. Southern analysis showed that the C. asiatica genome contains one copy of the CabAS gene. Northern blot analysis demonstrated that the CabAS gene is expressed in leaves with no detectable transcript in other plant tissues, consistent with the organ-specific accumulation of the asiaticoside. Up-regulation of expression of CabAS by methyl jasmonate in leaves was also demonstrated.

Effects of inoculum conditions on growth of hairy roots of Panax ginseng C.A. Meyer.

Plants have a potential to produce a large number of important metabolites such as pharmaceuticals, food additives, pigments, flavors, fragrances, and fine chemicals. Large-scale plant cell and tissue cultures for producing useful products has been considered an attractive alternative to whole plant extraction for obtaining valuable chemicals. In plant cell and tissue cultures, cell growth and metabolite production are influenced by nutritional and environmental conditions as well as physical properties of the culture system. To obtain a high growth rate of plant cell and tissue cultures, the culture conditions should be maintained at an optimum level. We studied the relationship between inoculum conditions and the growth of Panax ginseng hairy root culture, and found that the growth rate varied with the inoculum conditions such as the number of root tips, the length of root tips, the part of root tips, and the inoculum size and age of hairy roots.

Comparison of growth characteristics of Panax ginseng hairy roots in various bioreactors.

This study investigated the effects of flask-to-liquid volume ratio on the growth of Panax ginseng hairy root, transformed by Agrobacterium rhizogenes, in flask cultures and compared the characteristics of various bioreactors for scale-up. The flask-to-liquid volume ratio was optimum at 1.5 mL of air/mL of medium in flask cultures, and hairy root growth was not affected above the optimum ratio. In 500-mL flask culture, hairy root showed two growth phases. After the first exponential growth, specific growth rate decreased. The growth characteristics of P. ginseng hairy root in various bioreactors were investigated. Hairy root growth was about 55- fold of inoculum after 39 d in a 5-L bioreactor and about 38-fold of inoculum after 40 d in a 19-L bioreactor. Carbon yield was higher in a 19-L bioreactor than in others, but it did not show any linear relationship to the growth rate of hairy roots in bioreactors.

Studies on mass production of transformed Panax ginseng hairy roots in bioreactor.

The growth properties of Panax ginseng hairy roots transformed by Agrobacterium rhizogenes were compared between flask and aerated column or stirred bioreactor. In flask cultures, sucrose, initially 30 g/L, was nearly exhausted after 45 d of culture. The pH of the medium dropped from 5.5 to 4.96 after 10 d, but afterward it gradually increased to 6.4. After 45 d, hairy roots grew about 16-folds. The growth rate of hairy roots in air-bubble column or stirred bioreactor cultures was 1.13 (1.11) to 1.23 (1.20) g fresh wt (dry wt)/(g of cells x d), respectively. For both bioreactors, growth was about three times as high as in the flask cultivation.

Optimum conditions for transformed Panax ginseng hairy roots in flask culture.

Panax ginseng hairy roots were transformed by Agrobacterium rhizogenes KTCT 2744. They showed an active branching pattern and fast growth in hormone-free medium, and good growth at 23 degrees C, pH 5.8, 1/2 MS medium, and 3% sucrose. Sucrose provided the highest growth among seven carbon sources tested. Six complex media were also tested. In the combined sugar study, hairy roots grew better on sucrose without glucose or fructose than with glucose or fructose. In the 1/2 MS basal medium, 30 mM in nitrogen and 0.62 mM phosphate salt concentration was the optimum. The growth ratio was maximal at an inoculum size of 0.4% (w/v). Crude saponin and polysaccharide levels were also measured.