Enhanced Biotransformation Capacity of Rhodiola rosea Callus Cultures for Glycosid Production (original) (raw)

Abstract

Rhodiola rosea is a promising medicinal plant that produces various glycosides. Recently we developed a successful method for cultivating it in liquid cultures of compact callus aggregates. In a previous study we reported the successful production of the glycosides of R. rosea by biotransformation of cinnamyl alcohol and tyrosol. In the present study we investigated the possibility of further increasing the yields of the biotransformation products by addition of glucose to the culture medium aside from sucrose, which was used earlier as carbon source. Surprisingly, glucose addition doubled the yield of cinnamyl alcohol glycosides. Rosavin was not produced at all when only sucrose was used. When glucose was added the accumulation dynamics of rosin and a recently described derivative glycoside (designed as compound 321) were similar. Both increased during the first days and then remained constant, while other glycoside compounds increased continuously throughout the cultivation. Rosavin reached its maximum concentration after nine days. In contrast to the beneficial effect on cinnamyl alcohol related glycosides the addition of glucose did not affect the accumulation of the tyrosol derivative salidroside.

Figures (5)

Figure 1. The most important secondary metabolites of R. rosea.

Figure 1. The most important secondary metabolites of R. rosea.

Figure 2. The four new compounds, found by the LCT analyses. The initiation of compact callus aggregate (CCA) cultures of R. rosea L. was made as previously described (GyGrgy et al., 2004). The cultures were maintained in Murashige and Skoog medium (1962), supplemented with 0.5 mg 1”! 6-benzylade- nine (BA), | mg I”! naphthalene acetic acid (NAA) and 30 g 1”! sucrose (named MS-Rh) ona gyratory shaker at 135 rpm, in 25 °C under 16 h light (warm white fluorescent lamps, ca. 18 mol m~ s~'). Sub- cultures were done every 10th day. In the present study, we investigated the possibility of further increasing the yield of the biotransformation products. Both rosin and sal- idroside are formed as a consequence of the glycosylation of cinnamyl alcohol and _ tyrosol respectively. Since the Murashige and Skoog (1962) medium, which we used in our earlier studies contains only sucrose as sugar source we

Figure 2. The four new compounds, found by the LCT analyses. The initiation of compact callus aggregate (CCA) cultures of R. rosea L. was made as previously described (GyGrgy et al., 2004). The cultures were maintained in Murashige and Skoog medium (1962), supplemented with 0.5 mg 1”! 6-benzylade- nine (BA), | mg I”! naphthalene acetic acid (NAA) and 30 g 1”! sucrose (named MS-Rh) ona gyratory shaker at 135 rpm, in 25 °C under 16 h light (warm white fluorescent lamps, ca. 18 mol m~ s~'). Sub- cultures were done every 10th day. In the present study, we investigated the possibility of further increasing the yield of the biotransformation products. Both rosin and sal- idroside are formed as a consequence of the glycosylation of cinnamyl alcohol and _ tyrosol respectively. Since the Murashige and Skoog (1962) medium, which we used in our earlier studies contains only sucrose as sugar source we

Figure 3. The wet and dry weight of the callus during 14 days, cultivated with 2 mM tyrosol in the original MS-Rh medium (con- taining only sucrose) or in the glucose containing medium.

Figure 3. The wet and dry weight of the callus during 14 days, cultivated with 2 mM tyrosol in the original MS-Rh medium (con- taining only sucrose) or in the glucose containing medium.

Figure 4. The content of rosin, compound 337, 481, 483, 321 and rosavin in the callus cultivated with 2 mM cinnamyl alcohol in the original MS-Rh medium (containing only sucrose) or in the glucose containing medium, during 14 days.

Figure 4. The content of rosin, compound 337, 481, 483, 321 and rosavin in the callus cultivated with 2 mM cinnamyl alcohol in the original MS-Rh medium (containing only sucrose) or in the glucose containing medium, during 14 days.

Figure 5. The content of salidroside in the callus cultivated with 2 mM tyrosol in the original MS-Rh medium (containing only su- crose) or in the glucose containing medium, during 14 days. shown, that higher levels of sugars, usually sucrose, results higher levels of secondary metab- olites like in the case of Catharanthus roseus for the production of arbutin (Yokoyama and Yanagi, 1991), in the case of Coleus blumei for the rosmarinic acid production, or in the case of Eschscholtzia californica for the production of benzophenanthridine alkaloids (Ramachandra Rao and Ravishankar, 2002). However there are also evidence that higher sugar concentrations reduce the production like in the case of Dioscorea doryophora where at lower sucrose level more diosgenin was produced (Vanisree et al., 2004) or in the case of Aralia cordata where also the lower sucrose level favoured the anthocyanin production (Ramachandra Rao and Ravishankar, 2002). In addition, the optimal type of the sugar source depends on the plant species. Usually sucrose is used, but in the case of Taxus brevifolia fructose was found to be the optimal for paclitaxel pro- duction or in the case of Angelica dahurica var. formosana glucose was found to be the best carbon source for imperatorin production (Vanisree et al., 2004). As one can see from these varying, incon- sistent results, the optimal sugar source should be optimised for each plant species even to each secondary compound to be produced separately. The effect of sugars on the production of second- ary metabolites is ambiguous. Several studies have

Figure 5. The content of salidroside in the callus cultivated with 2 mM tyrosol in the original MS-Rh medium (containing only su- crose) or in the glucose containing medium, during 14 days. shown, that higher levels of sugars, usually sucrose, results higher levels of secondary metab- olites like in the case of Catharanthus roseus for the production of arbutin (Yokoyama and Yanagi, 1991), in the case of Coleus blumei for the rosmarinic acid production, or in the case of Eschscholtzia californica for the production of benzophenanthridine alkaloids (Ramachandra Rao and Ravishankar, 2002). However there are also evidence that higher sugar concentrations reduce the production like in the case of Dioscorea doryophora where at lower sucrose level more diosgenin was produced (Vanisree et al., 2004) or in the case of Aralia cordata where also the lower sucrose level favoured the anthocyanin production (Ramachandra Rao and Ravishankar, 2002). In addition, the optimal type of the sugar source depends on the plant species. Usually sucrose is used, but in the case of Taxus brevifolia fructose was found to be the optimal for paclitaxel pro- duction or in the case of Angelica dahurica var. formosana glucose was found to be the best carbon source for imperatorin production (Vanisree et al., 2004). As one can see from these varying, incon- sistent results, the optimal sugar source should be optimised for each plant species even to each secondary compound to be produced separately. The effect of sugars on the production of second- ary metabolites is ambiguous. Several studies have

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