Plant Cell Reports
P l a n t Cell R e p o r t s (1993) 1 3 : 9 5 - 9 8
© Springer-Verlag 1993
Comparison of terpenoid indole alkaloid production and degradation in two cell lines of Tabernaemontana divaricata Denise Dagnino, Jan Schripsema, and Robert Verpoorte B i o t e c h n o l o g y Delft Leiden, Project G r o u p P l a n t Cell Biotechnology, Center for B i o - P h a r m a c e u t i c a l Sciences, Division o f P h a r m a c o g n o s y , Leiden University; G o r l a e u s L a b o r a t o r i e s , P.O. B o x 9502, 2300 R A Leiden, T h e N e t h e r l a n d s Received 18 M a r c h 1993/Revised version received 13 A u g u s t 1993 - C o m m u n i c a t e d by W. Barz
A b s t r a c t . T w o cell lines o f Tabernaemontana divaricata derived f r o m the s a m e s u s p e n s i o n culture a c c u m u l a t e different a m o u n t s o f the terpenoid indole alkaloids O - a c e t y l v a l l e s a m i n e and voaphylline. [15N]O-acetylvallesamine a n d [l~N]voaphylline were a d d e d to the s u s p e n s i o n cultures to investigate w h e t h e r the lack o f a c c u m u l a t i n g capacity o f o n e o f the cell lines w a s due to a low biosynthetic ability or to high turnover rates. T h e difference w a s s h o w n to be d u e to the inability o f the cell culture to b i o s y n t h e s i z e both alkaloids. Both cell lines were able to metabolize O-acetylvallesamine. This metabolisation occurred m a i n l y d u r i n g the stationary phase. T h e alkaloids a d d e d were c h e m i c a l l y unstable u n d e r culture conditions. U n d e r n o r m a l batch cell culture conditions c h e m i c a l b r e a k d o w n is t h o u g h t to play a m i n o r role in the total a m o u n t o f c o m p o u n d transformed.
Materials and Methods
Abbreviations: GC-MS, gas chromatography-mass spectrometry; HPLC-UV, high performance liquid chromatography-ultraviolet; rpm, rotations per minute.
Addition of the alkaloids to the culture medium: To avoid the effect of the solvent on alkaloid accumulation observed in previous experiments (Dagnino et al. 1993, dos Santos et al. in press, 1993) a new procedure was developed to eliminate this extra variable. Each alkaloid was dissolved in a small volume of methanol. These solutions were transferred to 50 ml Erlenmeyer flasks and spread over the inner surface of the flasks. After this the methanol was evaporated leaving a thin film of the alkaloid on the inner walls of the Erlenmeyers. 50 ml of previously sterilized culture medium was added to each flask under aseptic conditions and then the flasks were left for 1-3 h in an ultrasonic bath at 40 *C. In a next step each solution was filtered through a cellulose acetate membrane filter (Schleieher & SchueU, FP 030/3, pore size 0.2 )un) in to larger amounts of culture medium (around 160 ml). After mixing, accurate amounts of medium (around 20 ml) were distributed between nine 100 ml Erlenmeyer flasks. For the control culture medium the same procedure as described above was followed but no alkaloid was added. To determine the initial alkaloid contents of the different media 200 ~ul were analyzed by HPLC-UV as described below.
Introduction
Product a c c u m u l a t i o n in s u s p e n s i o n cultures is d e p e n d e n t on the rate o f b i o s y n t h e s i s (rb), t u r n o v e r (r0 a n d c h e m i c a l b r e a k d o w n (r~) o f the c o m p o u n d s o f interest. A c c u m u l a t i o n o f a c o m p o u n d c a n o n l y o c c u r if r b - (r, + r~) > 0. In previous studies it w a s s h o w n that the a m o u n t s o f terpenoid indole alkaloids accumulated by suspension cultures of Tabernaemontana divaricata a n d Catharanthus roseus can be considerably i n f l u e n c e d by t r a n s f o r m a t i o n occurring during the culture period ( D a g n i n o et al. 1993, d o s Santos et al. in press, 1993). Efforts to m a n i p u l a t e s e c o n d a r y m e t a b o l i s m in order to obtain h i g h e r p r o d u c t yields s h o u l d include attempts to decrease transformation. Cell lines o f T. divaricata originally d e r i v e d f r o m the s a m e s u s p e n s i o n culture a n d m a i n t a i n e d u n d e r identical culture conditions c a n a c c u m u l a t e v e r y different a m o u n t s o f indole alkaloids. T h i s c a n be d u e to the different biosynthetic abilities or to different t u r n o v e r capacities o f the cell lines. In the f o l l o w i n g e x p e r i m e n t two cell lines o f T. divaricata were c o m p a r e d with r e s p e c t to their biosynthetical potential a n d turnover ability. One is the h i g h e s t indole alkaloid a c c u m u l a t i n g cell line m a i n t a i n e d at o u r laboratory. It a c c u m u l a t e s m a i n l y O - a c e t y l v a l l e s a m i n e (in about 15 d a y s up to 25 ;umol/1, a r o u n d 8 0 % o f the total alkaloids), but also voaphylline. In the o t h e r culture t h e s e c o m p o u n d s c a n n o t be detected. C h e m i c a l a n d biological b r e a k d o w n were separately investigated. T h e i r i n f l u e n c e o n the total a m o u n t o f alkaloid t r a n s f o r m e d is d i s c u s s e d . Correspondence to: D. D a g n i n o
Two cell lines of Tabernaemontana divaricata (L.) R.Br. ex Roem. et SchulL were chosen for the experiment; one with the highest accumulation of alkaloids maintained at our laboratory and another in which accumulation is below detection limit. The accumulation of alkaloids in these two cell lines has been followed during three years and, during the period investigated, the cultures have always shown this same accumulation pattern. Both cell lines were obtained 4 years ago from the same suspension culture and were subeultured in parallel every 3 to 4 weeks in MS medium (Murashige and Skoog 1962) without growth regulators and maintained under the same culture conditions (1500 lux, 25 *(2, on gyratory shakers at 100-120 rpm). Source and purity of the alkaloids added: [~SN]O-acetytvallesamine and [15N]voaphylline were obtained as described before (Dagnino et al. 1993). The alkaloids were analyzed by HPLC-UV before addition to the culture medium and were found to be 95 % pure.
Inoculation procedure: Cells used as inoculum were obtained from the stock cultures described above. Culture medium was separated from the biomass by filtration through a glass filter. An accurately weighed amount, around 1.6 g (fresh weight), of biomass was inoculated in each Erlenmeyer flask containing culture medium with or without the added alkaloid. No cells were added to the chemical degradation controls. During the experiment all the flasks were maintained like the stock cultures except that the Erlenmeyers were closed with silicon stoppers (T-22, Shin-Etsu Polymer Co., Ltd.) instead of cotton plugs to allow the determination of dissimilation curves (Schripsema et al. 1990). Sampling procedure: The time curve of dissimilation of sugars from a culture flask is correlated to the concentration of sugars in the medium and the dry weight of the biomass accumulated during the culture period. The developmental stage of the cultures was monitored daily by the dissimilation curve (Schripsema et al. 1990). A flask of each of the geaUnents was harvested at the beginning and end of the growth phase and in the stationary phase. Culture medium and cells were separated through a glass filter with the aid of vacuum and
96 the cells were washed three times with 10 ml of distilled water. Biomass was stored at -15 "12 before freeze drying and freeze dried biomass was stored at the same temperature until analysis. Culture medium was stored at -15 *C until analysis (0 to 4 days after harvesting). Investigation of the stability of O-acetylvallesamine in fresh culture medium: O-acetylvallesamine was added to fresh culture medium (MS without growth regulators). The culture medium containing the alkaloids was kept under the same culture conditions as described above. Samples were taken periodically under aseptic conditions and 200 jal of the sampled medium was injected directly into the HPLC system to determine the Oacetylvallesamine content and to detect breakdown products. The same procedure was followed for fresh culture medium without alkaloid. Investigation of the stability of O-acetylvallesamine in used culture medium: Culture medium was separated from the biomass under aseptic conditions at two different times of the culture period. One part of the collected medium was used to determine the alkaloid content and the other part was kept under the normal culture conditions described above until the time of harvest. Analyzing procedure: For quantification purposes alkaloids were extracted from the biomass (Schripsema and Verlx)orte 1992, Dagnino et al. 1993). The culture medium was injected directly into the HPLC system. The cell extracts and the medium were analyzed by HPLC-UV as described before (Dagnino et al. 1991). The analysis was monitored at 220, 280, 310 and 340 nm. GCMS analysis was carried out as described before (Dagnino et al. 1991).
The time course of dissimilation, dry weight and fresh weight accumulation of the control cultures is shown in Fig. 1. Both cell lines show a similar pattern of biomass accumulation. The maximum dry weight (350 mg) occurred 13 days after inoculation as indicated by the bending point of the dissimilation curve which coincides with the exhaustion of sugars from the culture medium (Schripsema et al. 1990). Dry weight accumulation of the non accumulating cell line was slightly retarded in relation to the producing one. Although the fresh weight inoculated was about the same for both cell lines, the inoculum of the non producing cell line was found to be less exhausted from culture medium when the dry weight/fresh weight of the inocula were compared.
10
400
!o
300
200
~oo
r'" 0
i 5
400
~" 300 E -~ 200 "~ 100 0
Results and Discussion
•
compounds in the culture. The accumulation starts during the growth phase and the final level of O-acetylvallesamine reaches 260 nmol/flask. Most of the terpenoid indole alkaloids is found in the culture medium (nearly 100 % in the first days of the culture cycle decreasing to around 70 % after 19 days in culture). In the culture of the other cell line these compounds cannot be detected. On the other hand tryptamine, a precursor of the terpenoid indole alkaloids, is accumulated to a higher extent by this cell line but the amount of this precursor is lower than the total amount of indole alkaloids accumulated by the other culture. Since both cell lines are kept under identical culture conditions, factors other than environmental are influencing the amount of compound accumulated.
10
15
20
time (days)
Fig 1. Time courses of dry (O) and fresh weight (A) and dissimilation ( I ) of the control cultures from the terpenoid indole alkaloid accumulating ( ~ ) and non accumulating (---) cell lines.
The only observed difference between the two cell lines investigated is their indole alkaloid accumulating capacity (Fig. 2). One cell line is able to accumulate the terpenoid indole alkaloids O-acetylvallesamine and voaphylline, which account for around 90 % of the total amount of these
~.
-,
-= 5
10
15
-20
time (days)
Fig. 2. Time courses of the amount of O-acetylvallesamine (11), voaphylline ( O ) and tryptamine ( ~ ) in the flasks of the control cultures from the terpenoid indole alkaloid accumulating ( ~ ) and non accumulating (----) cell lines. No O-acetylvallesamine and voaphylline could be found in the non producing cell line as indicated by the symbols going through the abscissa.
From the comparison of the loss of weight by dissimilation and the dry weight and fresh weight accumulation of the control cultures with those to which labelled alkaloid had been added it was concluded that the addition of the alkaloid to the culture medium did not affect the growth of any of the two cell lines. In Fig. 3 the time courses of alkaloid content in the flasks to which alkaloid was added is shown. O-acetylvaUesamine was not stable in fresh culture medium and abiotic breakdown products were found to accumulate. These compounds could not be detected in the presence of the cultures. No further increase in the amount of O-acetylvallesamine per flask was observed after the addition of this compound to the cultures. In the culture of the producing cell line there was a slight decrease in the amount originally added. The OacetylvaUesamine content of the non producing cell line decreased about the same way as in the flasks without any cell culture (chemical degradation). Voaphylline accumulation in these cultures occurred to the same extent as in the controls while tryptamine contents were reduced. Voaphylline was also not stable in fresh culture medium (Fig. 3b). After 19 days no voaphylline was detected in the chemical degradation control; instead another compound was found to accumulate which was not detected in the presence of any of the cell cultures. No further accumulation of voaphylline was observed after its addition to the cultures. The addition did not influence O-acetylvallesamine accumulation but resulted, as after the addition of O-
97 acetylvallesamine, control cultures.
in lower tryptamine levels than in the
Table 1. Dilution of the 15N labelled alkaloids added and the amount of non labelled compound accumulated during 19 days of culture. The dilution was calculated by dividing the labelling percentage at time 0 by the final labelling percentage measured.
400 -'A ~" 300 E
in the biosynthesis will be found before the splitting of the pathway to give the different products.
-::: m
labelled alkaloid added
200
dilution of labelled compound
amount of non labelled compound (nmol/flask)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
O-acetylvallesamine voaphylline
o..'" -..
100
a.line' 2.3 1.7
non a.lineb 1.1 1.1
a.line" 242 39
non a.lineb 13 3
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
10
15
20
• terpenoid indole alkaloid accumulating cell line b terpenoid indole alkaloid non accumulating cell line
400
B ~" 300 E
~ 200 _ 100 0 -
= ,
5
""'m~'l--l[............. ~I ....................
t0 time (days)
15
=
20
Fig. 3. Time courses of the amount of O-acetylvallesamine (11), voaphylline ( 0 ) and tryptamine (A) in the flasks of the cultures from the terpenoid indole alkaloid accumulating ( ~ ) and non accumulating (o--) cell lines to which [15N]O-acetylvallesamine ( A ) and ['SN]voaphylline ( B ) had been added. The stability of these compounds in fresh medium under normal culture conditions but in the absence of cells is also indicated (.,...). Note that no Oacetylvallesamine or voaphylline can be detected in the cultures of the non producing cell line unless these compounds have been added to the culture.
Indole alkaloids are known to suffer autoxidation under several conditons (Sundberg 1970). They are also transformed by a variety of enzyme systems (Goodbody 1988). Thus the observed degradation of the alkaloids in the cultures of both cell lines can be a result of the chemical instability of these compounds under the culture conditions and/or their biotransformation to other compounds by the cells. The stability of O-acetylvallesamine in fresh culture medium at normal culture conditions was investigated in more detail (Fig. 4). O-acetylvaltesamine was found to be unstable under these circumstances. While the amount of O-acetylvallesamine decreased~ at least 8 compounds presenting several chromophores could be detected by HPLC-UV. Degradation did not follow first order rate kinetics; the decrease in concentration showed a pattern similar to the previously found in the presence of the cell cultures (Dagnino et al. 1993), a slow rate of breakdown in the first days and afterwards increasing. Unfortunately, for obvious reasons, the time course of chemical transformation found in fresh culture medium cannot be expected to be the same as in used medium. 70 60
40 30
Although biosynthesis of voaphylline and O-acetylvallesamine could not be confirmed by the analysis of product accumulation in the cultures to which these compounds had been added, it was possible to confirm it by measuring the labelling percentages of the exlyacted alkaloids (Table 1). From the dilution of the labelled compounds it is clear that the producing cell line was biosynthesizing Oacetylvallesamine and voaphylline also after these compounds had been added to the culture medium. The amount of non labelled compound accumulated during this period was comparable to the amount accumulated by the control cultures of this cell line; thus the presence of high amounts of this alkaloid at the beginning of the batch cell culture cycle did not alter the balance between biosynthesis and breakdown. Degradation of the alkaloids occurred since the total amount of alkaloids remained about constant. As for the non producing cell line, hardly any dilution of the labelling was observed. Thus it can be concluded that under these conditions nearly no biosynthesis took place. From the results above it can be concluded that the inability of the non producing cell line to accumulate O-acetylvallesamine and voaphylline is due to its low biosynthetic capacity. The fact that this was demonstrated for both alkaloids suggests that the limiting step
1(1 o time (days)
Fig. 4. Time course of the stability of O-acetylvallesamine in fresh culture medium.
The main cause of transformation was determined by comparing the transformation which occurred under normal culture conditions with the transformation which occurred in used culture medium separated from the biomass and the transformation in fresh culture medium (Table 2). In the first period investigated transformation was comparable under all conditions; in the presence or absence of cells and in fresh culture medium O-acetylvallesamine was transformed to the same extent. In fresh and used culture medium the same type of degradation products were found and so the chemical
98 Table 2. An attempt to distinguish between chemical and biological transformation by comparing the rate of O-acetylvallesamine transformation in fresh culture medium, in used culture medium in the absence of the cultures with the transformation occurring in the presence of the cultures. Abbreviations as in Table 1. time interval 4 - 13 days 13 - 19 days
% of O-acetylvallesamine transformed during the time interval with the culture without the culture fresh medium a. line' non a. lineb a. line' non a. lineb 38 36 45 34 40 39 31 10 9 41
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Legends as in Table 1. instability of O-acetylvallesamine can be regarded to be the main cause of transformation during this period. In the next period a different situation was observed; transformation in used culture medium separated from the cells occurred at a slower rate than in the presence of the cells..From these results it becomes clear that the instability of the alkaloid in used culture medium cannot explain the total amount transformed in the presence of the cultures and that turnover of these compounds must have occurred.
400
A
~'300
i 200 100 "0
It is well known that alkaloids are often not end products of metabolism but can be turned over to compounds of the primary metabolism (Luckner 1990, Ghini et al. 1991). Indole alkaloids are also known to suffer unspecific oxidation by peroxidases (Sariaslani 1985, Goodbody 1988). The ability of horseradish peroxidase to transform O-acetylvallesamine has been tested before (Sierra 1991) and it was found to be very stable when compared to other terpenoid indole alkaloids. The presence of enzymes capable of transforming Oacetylvallesamine was confirmed by incubating this compound with crude enzyme extracts. The nature of the reaction is still under investigation. By combining the data of the O-acetylvallesamine content of a flask and the labeling percentages found at each sampling time, the total amount of O-acetylvallesamine transformed during the period investigated could be calculated. An estimation of the role of the chemical instability of Oacetylvallesamine and its transformation by the suspension cultures on the total amount of O-acetylvallesamine transformed is given in Fig. 5. The main cause of transformation in the first 13 days in the cultures to which Oacetylvallesamine was added was the chemical instability of O-acetylvallesamine. Biotransformation occurred mainly during the stationary phase. While biotransformation occurred no alkaloid was observed to accumulate and so rapid metabolisation of this compound is assumed to occur. Both cell lines were able to biotransform O-acetylvallesamine although the amounts biotransformed differed. In spite of the inability of the non accumulating cell line to store Oacetylvallesamine, it was able to biotransform this compound. Whether this ability was triggered by the addition of the compound to the culture is not clear. During a normal culture cycle the amount of alkaloids increases during the culture period; therefore, under the usual culture conditions, the chemical instability of the alkaloids in culture medium will play a minor role in the total amount transformed. From the results presented above it becomes clear that there are several approaches to increase the amount of alkaloid accumulated by the producing culture. The cause of alkaloid instability in the culture medium can be investigated and perhaps eliminated. Furthermore the cells can be kept in the growth phase for longer periods to try to avoid the turnover verified during the stationary phase. Last but not least attempts can be made to block the turnover.
0 400
0
5
10
15
20
13
E 300 v
200 100
0
m
i
,
5
10
i
i
15
20
time (days)
Fig. 5. Estimation of the role of the chemical instability of OacetylvaUesamine and its turnover by the suspension cultures of the producing (5A) and non producing cell line (5B) on the total amount of O-acetytvallesamine transformed (11). The main cause of transformation in the first 13 days of culture was the chemical instability ( 0 ) of O-acetylvaUesamine. In the cultures of both cell lines biotransformation (A) occurred mainly during the stationary phase. Acknowledgement. We thank CNPq, Brazil for the grant given to Denise Dagnino. References Dagnino D, Schripsema J, Peltenburg-Looman A, Verpoorte R, Teunis K (1991) J Nat Prod 54:1558-1563 Dagnino D, Schripsema J, Verpoorte R (1993) Phytochemistry 32:325-329 dos Santos RI, Schripsema J, Verpoorte R Phytochemistry, in press (1993) Ghini AA, Burton G, Gros EG (1991) Phytochemistry 30:779-784 Goodbody A, Endo T, Vukovic J, Misawa M (1988) Planta Medica 210-214 Luckner M (1990) Secondary Metabolism in Microorganisms Plants and Animals. 3'~ edn, Springer, Berlin Heidelberg New York Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Sariaslani FS, Duffel MW Rosazza JP (1985) J Meal Chem 28:629633 Schripsema J, Meijer AH, lren Fvan, Hoopen HJGten, Verlx)orte R (1990) Plant Cell Tissue Org Cult 8:153-161 Schripsema J, Verlx~rte R (1992) Planta Med 58:245-249 Sierra MI (1991) Ph D Thesis, University of Leiden, The Netherlands, pp 67-75 Sundberg RJ (1970) The Chemistry of Indoles. Academic Press, London
P l a n t Cell R e p o r t s (1993) 1 3 : 9 5 - 9 8
© Springer-Verlag 1993
Comparison of terpenoid indole alkaloid production and degradation in two cell lines of Tabernaemontana divaricata Denise Dagnino, Jan Schripsema, and Robert Verpoorte B i o t e c h n o l o g y Delft Leiden, Project G r o u p P l a n t Cell Biotechnology, Center for B i o - P h a r m a c e u t i c a l Sciences, Division o f P h a r m a c o g n o s y , Leiden University; G o r l a e u s L a b o r a t o r i e s , P.O. B o x 9502, 2300 R A Leiden, T h e N e t h e r l a n d s Received 18 M a r c h 1993/Revised version received 13 A u g u s t 1993 - C o m m u n i c a t e d by W. Barz
A b s t r a c t . T w o cell lines o f Tabernaemontana divaricata derived f r o m the s a m e s u s p e n s i o n culture a c c u m u l a t e different a m o u n t s o f the terpenoid indole alkaloids O - a c e t y l v a l l e s a m i n e and voaphylline. [15N]O-acetylvallesamine a n d [l~N]voaphylline were a d d e d to the s u s p e n s i o n cultures to investigate w h e t h e r the lack o f a c c u m u l a t i n g capacity o f o n e o f the cell lines w a s due to a low biosynthetic ability or to high turnover rates. T h e difference w a s s h o w n to be d u e to the inability o f the cell culture to b i o s y n t h e s i z e both alkaloids. Both cell lines were able to metabolize O-acetylvallesamine. This metabolisation occurred m a i n l y d u r i n g the stationary phase. T h e alkaloids a d d e d were c h e m i c a l l y unstable u n d e r culture conditions. U n d e r n o r m a l batch cell culture conditions c h e m i c a l b r e a k d o w n is t h o u g h t to play a m i n o r role in the total a m o u n t o f c o m p o u n d transformed.
Materials and Methods
Abbreviations: GC-MS, gas chromatography-mass spectrometry; HPLC-UV, high performance liquid chromatography-ultraviolet; rpm, rotations per minute.
Addition of the alkaloids to the culture medium: To avoid the effect of the solvent on alkaloid accumulation observed in previous experiments (Dagnino et al. 1993, dos Santos et al. in press, 1993) a new procedure was developed to eliminate this extra variable. Each alkaloid was dissolved in a small volume of methanol. These solutions were transferred to 50 ml Erlenmeyer flasks and spread over the inner surface of the flasks. After this the methanol was evaporated leaving a thin film of the alkaloid on the inner walls of the Erlenmeyers. 50 ml of previously sterilized culture medium was added to each flask under aseptic conditions and then the flasks were left for 1-3 h in an ultrasonic bath at 40 *C. In a next step each solution was filtered through a cellulose acetate membrane filter (Schleieher & SchueU, FP 030/3, pore size 0.2 )un) in to larger amounts of culture medium (around 160 ml). After mixing, accurate amounts of medium (around 20 ml) were distributed between nine 100 ml Erlenmeyer flasks. For the control culture medium the same procedure as described above was followed but no alkaloid was added. To determine the initial alkaloid contents of the different media 200 ~ul were analyzed by HPLC-UV as described below.
Introduction
Product a c c u m u l a t i o n in s u s p e n s i o n cultures is d e p e n d e n t on the rate o f b i o s y n t h e s i s (rb), t u r n o v e r (r0 a n d c h e m i c a l b r e a k d o w n (r~) o f the c o m p o u n d s o f interest. A c c u m u l a t i o n o f a c o m p o u n d c a n o n l y o c c u r if r b - (r, + r~) > 0. In previous studies it w a s s h o w n that the a m o u n t s o f terpenoid indole alkaloids accumulated by suspension cultures of Tabernaemontana divaricata a n d Catharanthus roseus can be considerably i n f l u e n c e d by t r a n s f o r m a t i o n occurring during the culture period ( D a g n i n o et al. 1993, d o s Santos et al. in press, 1993). Efforts to m a n i p u l a t e s e c o n d a r y m e t a b o l i s m in order to obtain h i g h e r p r o d u c t yields s h o u l d include attempts to decrease transformation. Cell lines o f T. divaricata originally d e r i v e d f r o m the s a m e s u s p e n s i o n culture a n d m a i n t a i n e d u n d e r identical culture conditions c a n a c c u m u l a t e v e r y different a m o u n t s o f indole alkaloids. T h i s c a n be d u e to the different biosynthetic abilities or to different t u r n o v e r capacities o f the cell lines. In the f o l l o w i n g e x p e r i m e n t two cell lines o f T. divaricata were c o m p a r e d with r e s p e c t to their biosynthetical potential a n d turnover ability. One is the h i g h e s t indole alkaloid a c c u m u l a t i n g cell line m a i n t a i n e d at o u r laboratory. It a c c u m u l a t e s m a i n l y O - a c e t y l v a l l e s a m i n e (in about 15 d a y s up to 25 ;umol/1, a r o u n d 8 0 % o f the total alkaloids), but also voaphylline. In the o t h e r culture t h e s e c o m p o u n d s c a n n o t be detected. C h e m i c a l a n d biological b r e a k d o w n were separately investigated. T h e i r i n f l u e n c e o n the total a m o u n t o f alkaloid t r a n s f o r m e d is d i s c u s s e d . Correspondence to: D. D a g n i n o
Two cell lines of Tabernaemontana divaricata (L.) R.Br. ex Roem. et SchulL were chosen for the experiment; one with the highest accumulation of alkaloids maintained at our laboratory and another in which accumulation is below detection limit. The accumulation of alkaloids in these two cell lines has been followed during three years and, during the period investigated, the cultures have always shown this same accumulation pattern. Both cell lines were obtained 4 years ago from the same suspension culture and were subeultured in parallel every 3 to 4 weeks in MS medium (Murashige and Skoog 1962) without growth regulators and maintained under the same culture conditions (1500 lux, 25 *(2, on gyratory shakers at 100-120 rpm). Source and purity of the alkaloids added: [~SN]O-acetytvallesamine and [15N]voaphylline were obtained as described before (Dagnino et al. 1993). The alkaloids were analyzed by HPLC-UV before addition to the culture medium and were found to be 95 % pure.
Inoculation procedure: Cells used as inoculum were obtained from the stock cultures described above. Culture medium was separated from the biomass by filtration through a glass filter. An accurately weighed amount, around 1.6 g (fresh weight), of biomass was inoculated in each Erlenmeyer flask containing culture medium with or without the added alkaloid. No cells were added to the chemical degradation controls. During the experiment all the flasks were maintained like the stock cultures except that the Erlenmeyers were closed with silicon stoppers (T-22, Shin-Etsu Polymer Co., Ltd.) instead of cotton plugs to allow the determination of dissimilation curves (Schripsema et al. 1990). Sampling procedure: The time curve of dissimilation of sugars from a culture flask is correlated to the concentration of sugars in the medium and the dry weight of the biomass accumulated during the culture period. The developmental stage of the cultures was monitored daily by the dissimilation curve (Schripsema et al. 1990). A flask of each of the geaUnents was harvested at the beginning and end of the growth phase and in the stationary phase. Culture medium and cells were separated through a glass filter with the aid of vacuum and
96 the cells were washed three times with 10 ml of distilled water. Biomass was stored at -15 "12 before freeze drying and freeze dried biomass was stored at the same temperature until analysis. Culture medium was stored at -15 *C until analysis (0 to 4 days after harvesting). Investigation of the stability of O-acetylvallesamine in fresh culture medium: O-acetylvallesamine was added to fresh culture medium (MS without growth regulators). The culture medium containing the alkaloids was kept under the same culture conditions as described above. Samples were taken periodically under aseptic conditions and 200 jal of the sampled medium was injected directly into the HPLC system to determine the Oacetylvallesamine content and to detect breakdown products. The same procedure was followed for fresh culture medium without alkaloid. Investigation of the stability of O-acetylvallesamine in used culture medium: Culture medium was separated from the biomass under aseptic conditions at two different times of the culture period. One part of the collected medium was used to determine the alkaloid content and the other part was kept under the normal culture conditions described above until the time of harvest. Analyzing procedure: For quantification purposes alkaloids were extracted from the biomass (Schripsema and Verlx)orte 1992, Dagnino et al. 1993). The culture medium was injected directly into the HPLC system. The cell extracts and the medium were analyzed by HPLC-UV as described before (Dagnino et al. 1991). The analysis was monitored at 220, 280, 310 and 340 nm. GCMS analysis was carried out as described before (Dagnino et al. 1991).
The time course of dissimilation, dry weight and fresh weight accumulation of the control cultures is shown in Fig. 1. Both cell lines show a similar pattern of biomass accumulation. The maximum dry weight (350 mg) occurred 13 days after inoculation as indicated by the bending point of the dissimilation curve which coincides with the exhaustion of sugars from the culture medium (Schripsema et al. 1990). Dry weight accumulation of the non accumulating cell line was slightly retarded in relation to the producing one. Although the fresh weight inoculated was about the same for both cell lines, the inoculum of the non producing cell line was found to be less exhausted from culture medium when the dry weight/fresh weight of the inocula were compared.
10
400
!o
300
200
~oo
r'" 0
i 5
400
~" 300 E -~ 200 "~ 100 0
Results and Discussion
•
compounds in the culture. The accumulation starts during the growth phase and the final level of O-acetylvallesamine reaches 260 nmol/flask. Most of the terpenoid indole alkaloids is found in the culture medium (nearly 100 % in the first days of the culture cycle decreasing to around 70 % after 19 days in culture). In the culture of the other cell line these compounds cannot be detected. On the other hand tryptamine, a precursor of the terpenoid indole alkaloids, is accumulated to a higher extent by this cell line but the amount of this precursor is lower than the total amount of indole alkaloids accumulated by the other culture. Since both cell lines are kept under identical culture conditions, factors other than environmental are influencing the amount of compound accumulated.
10
15
20
time (days)
Fig 1. Time courses of dry (O) and fresh weight (A) and dissimilation ( I ) of the control cultures from the terpenoid indole alkaloid accumulating ( ~ ) and non accumulating (---) cell lines.
The only observed difference between the two cell lines investigated is their indole alkaloid accumulating capacity (Fig. 2). One cell line is able to accumulate the terpenoid indole alkaloids O-acetylvallesamine and voaphylline, which account for around 90 % of the total amount of these
~.
-,
-= 5
10
15
-20
time (days)
Fig. 2. Time courses of the amount of O-acetylvallesamine (11), voaphylline ( O ) and tryptamine ( ~ ) in the flasks of the control cultures from the terpenoid indole alkaloid accumulating ( ~ ) and non accumulating (----) cell lines. No O-acetylvallesamine and voaphylline could be found in the non producing cell line as indicated by the symbols going through the abscissa.
From the comparison of the loss of weight by dissimilation and the dry weight and fresh weight accumulation of the control cultures with those to which labelled alkaloid had been added it was concluded that the addition of the alkaloid to the culture medium did not affect the growth of any of the two cell lines. In Fig. 3 the time courses of alkaloid content in the flasks to which alkaloid was added is shown. O-acetylvaUesamine was not stable in fresh culture medium and abiotic breakdown products were found to accumulate. These compounds could not be detected in the presence of the cultures. No further increase in the amount of O-acetylvallesamine per flask was observed after the addition of this compound to the cultures. In the culture of the producing cell line there was a slight decrease in the amount originally added. The OacetylvaUesamine content of the non producing cell line decreased about the same way as in the flasks without any cell culture (chemical degradation). Voaphylline accumulation in these cultures occurred to the same extent as in the controls while tryptamine contents were reduced. Voaphylline was also not stable in fresh culture medium (Fig. 3b). After 19 days no voaphylline was detected in the chemical degradation control; instead another compound was found to accumulate which was not detected in the presence of any of the cell cultures. No further accumulation of voaphylline was observed after its addition to the cultures. The addition did not influence O-acetylvallesamine accumulation but resulted, as after the addition of O-
97 acetylvallesamine, control cultures.
in lower tryptamine levels than in the
Table 1. Dilution of the 15N labelled alkaloids added and the amount of non labelled compound accumulated during 19 days of culture. The dilution was calculated by dividing the labelling percentage at time 0 by the final labelling percentage measured.
400 -'A ~" 300 E
in the biosynthesis will be found before the splitting of the pathway to give the different products.
-::: m
labelled alkaloid added
200
dilution of labelled compound
amount of non labelled compound (nmol/flask)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
O-acetylvallesamine voaphylline
o..'" -..
100
a.line' 2.3 1.7
non a.lineb 1.1 1.1
a.line" 242 39
non a.lineb 13 3
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
10
15
20
• terpenoid indole alkaloid accumulating cell line b terpenoid indole alkaloid non accumulating cell line
400
B ~" 300 E
~ 200 _ 100 0 -
= ,
5
""'m~'l--l[............. ~I ....................
t0 time (days)
15
=
20
Fig. 3. Time courses of the amount of O-acetylvallesamine (11), voaphylline ( 0 ) and tryptamine (A) in the flasks of the cultures from the terpenoid indole alkaloid accumulating ( ~ ) and non accumulating (o--) cell lines to which [15N]O-acetylvallesamine ( A ) and ['SN]voaphylline ( B ) had been added. The stability of these compounds in fresh medium under normal culture conditions but in the absence of cells is also indicated (.,...). Note that no Oacetylvallesamine or voaphylline can be detected in the cultures of the non producing cell line unless these compounds have been added to the culture.
Indole alkaloids are known to suffer autoxidation under several conditons (Sundberg 1970). They are also transformed by a variety of enzyme systems (Goodbody 1988). Thus the observed degradation of the alkaloids in the cultures of both cell lines can be a result of the chemical instability of these compounds under the culture conditions and/or their biotransformation to other compounds by the cells. The stability of O-acetylvallesamine in fresh culture medium at normal culture conditions was investigated in more detail (Fig. 4). O-acetylvaltesamine was found to be unstable under these circumstances. While the amount of O-acetylvallesamine decreased~ at least 8 compounds presenting several chromophores could be detected by HPLC-UV. Degradation did not follow first order rate kinetics; the decrease in concentration showed a pattern similar to the previously found in the presence of the cell cultures (Dagnino et al. 1993), a slow rate of breakdown in the first days and afterwards increasing. Unfortunately, for obvious reasons, the time course of chemical transformation found in fresh culture medium cannot be expected to be the same as in used medium. 70 60
40 30
Although biosynthesis of voaphylline and O-acetylvallesamine could not be confirmed by the analysis of product accumulation in the cultures to which these compounds had been added, it was possible to confirm it by measuring the labelling percentages of the exlyacted alkaloids (Table 1). From the dilution of the labelled compounds it is clear that the producing cell line was biosynthesizing Oacetylvallesamine and voaphylline also after these compounds had been added to the culture medium. The amount of non labelled compound accumulated during this period was comparable to the amount accumulated by the control cultures of this cell line; thus the presence of high amounts of this alkaloid at the beginning of the batch cell culture cycle did not alter the balance between biosynthesis and breakdown. Degradation of the alkaloids occurred since the total amount of alkaloids remained about constant. As for the non producing cell line, hardly any dilution of the labelling was observed. Thus it can be concluded that under these conditions nearly no biosynthesis took place. From the results above it can be concluded that the inability of the non producing cell line to accumulate O-acetylvallesamine and voaphylline is due to its low biosynthetic capacity. The fact that this was demonstrated for both alkaloids suggests that the limiting step
1(1 o time (days)
Fig. 4. Time course of the stability of O-acetylvallesamine in fresh culture medium.
The main cause of transformation was determined by comparing the transformation which occurred under normal culture conditions with the transformation which occurred in used culture medium separated from the biomass and the transformation in fresh culture medium (Table 2). In the first period investigated transformation was comparable under all conditions; in the presence or absence of cells and in fresh culture medium O-acetylvallesamine was transformed to the same extent. In fresh and used culture medium the same type of degradation products were found and so the chemical
98 Table 2. An attempt to distinguish between chemical and biological transformation by comparing the rate of O-acetylvallesamine transformation in fresh culture medium, in used culture medium in the absence of the cultures with the transformation occurring in the presence of the cultures. Abbreviations as in Table 1. time interval 4 - 13 days 13 - 19 days
% of O-acetylvallesamine transformed during the time interval with the culture without the culture fresh medium a. line' non a. lineb a. line' non a. lineb 38 36 45 34 40 39 31 10 9 41
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Legends as in Table 1. instability of O-acetylvallesamine can be regarded to be the main cause of transformation during this period. In the next period a different situation was observed; transformation in used culture medium separated from the cells occurred at a slower rate than in the presence of the cells..From these results it becomes clear that the instability of the alkaloid in used culture medium cannot explain the total amount transformed in the presence of the cultures and that turnover of these compounds must have occurred.
400
A
~'300
i 200 100 "0
It is well known that alkaloids are often not end products of metabolism but can be turned over to compounds of the primary metabolism (Luckner 1990, Ghini et al. 1991). Indole alkaloids are also known to suffer unspecific oxidation by peroxidases (Sariaslani 1985, Goodbody 1988). The ability of horseradish peroxidase to transform O-acetylvallesamine has been tested before (Sierra 1991) and it was found to be very stable when compared to other terpenoid indole alkaloids. The presence of enzymes capable of transforming Oacetylvallesamine was confirmed by incubating this compound with crude enzyme extracts. The nature of the reaction is still under investigation. By combining the data of the O-acetylvallesamine content of a flask and the labeling percentages found at each sampling time, the total amount of O-acetylvallesamine transformed during the period investigated could be calculated. An estimation of the role of the chemical instability of Oacetylvallesamine and its transformation by the suspension cultures on the total amount of O-acetylvallesamine transformed is given in Fig. 5. The main cause of transformation in the first 13 days in the cultures to which Oacetylvallesamine was added was the chemical instability of O-acetylvallesamine. Biotransformation occurred mainly during the stationary phase. While biotransformation occurred no alkaloid was observed to accumulate and so rapid metabolisation of this compound is assumed to occur. Both cell lines were able to biotransform O-acetylvallesamine although the amounts biotransformed differed. In spite of the inability of the non accumulating cell line to store Oacetylvallesamine, it was able to biotransform this compound. Whether this ability was triggered by the addition of the compound to the culture is not clear. During a normal culture cycle the amount of alkaloids increases during the culture period; therefore, under the usual culture conditions, the chemical instability of the alkaloids in culture medium will play a minor role in the total amount transformed. From the results presented above it becomes clear that there are several approaches to increase the amount of alkaloid accumulated by the producing culture. The cause of alkaloid instability in the culture medium can be investigated and perhaps eliminated. Furthermore the cells can be kept in the growth phase for longer periods to try to avoid the turnover verified during the stationary phase. Last but not least attempts can be made to block the turnover.
0 400
0
5
10
15
20
13
E 300 v
200 100
0
m
i
,
5
10
i
i
15
20
time (days)
Fig. 5. Estimation of the role of the chemical instability of OacetylvaUesamine and its turnover by the suspension cultures of the producing (5A) and non producing cell line (5B) on the total amount of O-acetytvallesamine transformed (11). The main cause of transformation in the first 13 days of culture was the chemical instability ( 0 ) of O-acetylvaUesamine. In the cultures of both cell lines biotransformation (A) occurred mainly during the stationary phase. Acknowledgement. We thank CNPq, Brazil for the grant given to Denise Dagnino. References Dagnino D, Schripsema J, Peltenburg-Looman A, Verpoorte R, Teunis K (1991) J Nat Prod 54:1558-1563 Dagnino D, Schripsema J, Verpoorte R (1993) Phytochemistry 32:325-329 dos Santos RI, Schripsema J, Verpoorte R Phytochemistry, in press (1993) Ghini AA, Burton G, Gros EG (1991) Phytochemistry 30:779-784 Goodbody A, Endo T, Vukovic J, Misawa M (1988) Planta Medica 210-214 Luckner M (1990) Secondary Metabolism in Microorganisms Plants and Animals. 3'~ edn, Springer, Berlin Heidelberg New York Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Sariaslani FS, Duffel MW Rosazza JP (1985) J Meal Chem 28:629633 Schripsema J, Meijer AH, lren Fvan, Hoopen HJGten, Verlx)orte R (1990) Plant Cell Tissue Org Cult 8:153-161 Schripsema J, Verlx~rte R (1992) Planta Med 58:245-249 Sierra MI (1991) Ph D Thesis, University of Leiden, The Netherlands, pp 67-75 Sundberg RJ (1970) The Chemistry of Indoles. Academic Press, London
