Plant Cell Reports
Plant Cell Reports (1985) 4:259-262
© S p r i n g e r - V e r l a g 1985
Alkaloid production in C a t h a r a n t h u s r o s e u s (L.) G. Don cell cultures. XIII. Effects of bioregulators on indole alkaloid biosynthesis James P. Kutney 1, Barbara Aweryn 1, Kenneth B. Chatson 2, Lewis S.L. Choi %and Woifgang G. W. Kurz 2 1 Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Y6 2 Plant Biotechnology Institute, National Research Council, 110 Gymnasium Road, University Campus, Saskatoon, Saskatchewan, Canada S7N OW9 Received December 5, 1984 / Revised version received August 10, 1985 - Communicated by F. Constabel
ABSTRACT A study on the effect of various bioregulators on the biosynthesis of ajmalicine (8) and catharanthine (9) in plant tissue cultures of Catharanthus roseus is described. It is shown that l,l-dimethylpiperidine bromide (3) and 2-diethylaminoethyl-3,4dimethylphenylether (7) are effective in increasing these alkaloid levels in the cell line PRL #200. Such studies may prove beneficial in larger scale experiments designed for the production of these alkaloids.
{I)
T
(2)
INTRODUCTION ;Lv¢ou[
It is well established that tryptamine (i) and secologanin (2) are the biosynthetic building units for a variety of indole alkaloids (Scheme i). Furthermore various laboratories including our own [i] have developed tissue cultures of Catharanthus roseus (L°) G. Don which are capable of producing alkaloids within the Corynanthe, Strychnos, Aspidosperma, and Iboga families. Our efforts in recent studies have been directed towards a detailed investigation of fermentation conditions with selected cell lines in order to optimize production of target alkaloids. In this regard, we have considered the utilization of certain bioregulators which have been shown to induce isoprenoid production in the hope that such substances may stimulate the biosynthesis of secologanin (2) and, in turn, the increased production of the desired alkaloid systems. Yokoyama et al. [2] have shown that rubber biosynthesis is induced by certain aminoethyl phenylethers, implying that this type of synthetic bioregulators may be general inducers of terpenoid biosynthesis. Recently, Scott et al. [3] also demonstrated that some of these compounds are effective in promoting indole alkaloid formation in one of their C. roseus cell lines. Here we wish to report our results on experiments with some of these bioregulators and an established C. roseus cell line (PRL #200) described previously [4]. MATERIAL AND METHODS Catharanthus Roseus Cell Culture Conditions for initiation and maintenance of cell line PRL #200 were as described previously [4].
Offprint requests to: J. P. Kutney
AI~DOS~RUA
(3)
(4)
~O~A
(5)
.oc~ "°~'_ c~
(61
n • a~,c~(a~,,),
(71
u
(8)
(9) Scheme i
Bioregulators Five bioregulators, l,l-dimethylpiperidine bromide (3), 2-diethylaminoethyl-3,4-dichlorophenylether (4), 2-diethylaminoethyl-2,4-dichlorophenylether (5), 2-diethylaminoethyl-~-naphthylether (6) and 2-diethylaminoethyl-3,4-dimethylphenylether (7) were obtained as gifts from Dr. H. Yokoyama (Fruit and Vegetable Chemistry Laboratory, Western Region, Agricultural Research Service, Pasadena, California, U.S.A.). The bioregulators were added as the free base (except (3) as the bromide), dissolved in methanol/
260 water and filter-sterilized brane.
through a 0.45 Dm mem-
Time Course Studies Shake flask cultures of PRL #200 cells (500 m£ in 1 £ flask) were grown in the alkaloid production medium [4] for 7 days when the cultures were near or at stationary phase of growth as monitored by the mitotic activity of the cells. At this stage the average cell dry weight was 25 mg/m% culture. For the first time course experiment all five bioregulators (3), (4), (5), (6) and (7) were examined. These were added separately to the above cultures at a concentration of 2 mg/~ and further incubated for four different time periods (12, 18, 24 and 31 days). Parallel control cultures (no bioregulator added) were also set up. Samples were withdrawn at indicated intervals, extracted and analysed for alkaloid content.
II
A
~9 U
5
, (4)
(3)
(4) , ~
(5)
(6)
, fS) o 161
(7)
Days of Incubation with Biorecjulators
A second time course experiment was carried out to investigate the effects of the two most promising bioregulators, (3) and (7), after shorter incubation time (4, 6, 8 and i0 days) with the PRL #200 cultures. Samples were harvested at indicated intervals. Extraction and analyses of the alkaloid content were carried out according to the standard procedure described below.
Figure I. Effect of bioregulators (3) to (7) on cell dry weight over an incubation period of 12 to 31 days. second time course experiment) and then levelled off from day 12 on (Figure i). Total alkaloid obtained from the cell extracts, expressed as percent of cell dry weight, showed substantial differences (Figure 2) especially after 12 and 18 days of incubation. Of
Alkaloid Extraction and Analysis A detailed account of the extraction procedure was given in an earlier publication [5]. The alkaloid extract from each sample was then analysed by HPLC (Waters Associates system) equipped with a Radial Compression Module, I0~ C18 cartridge, eluted with 2 different solvent systems at room temperature. (A)
(B)
45% H20 in MeOH with 0.1% Et3N at a flow rate of 4 m£/min. Alkaloids were monitored at 280 and 254 nm (Waters Associates 440 UV detector). Retention times for catharanthine (9) and ajmalicine (8) are 13.2 min and 17.7 min respectively. 32% H20 in MeOH with 0.1% Et3N at a flow rate of 1 m~/min. Retention times for (9) and (8) are 19.6 min and 21.7 min respectively.
12
I0 =
(3)
(4)
(5)
a~ )
• Control • (S) .(4) • (5} o 161
O.IB
"6 o-
O.E
o ~
o.4
o ~-
0.2
='4
Quantitative determinations of (8) and (9) were made on the basis of peak areas on HPLC output, compared to standard curves. RESULTS AND DISCUSSION We have shown earlier that cell line PRL #200 was capable of producing (8) and (9) among other alkaloids [4]. This cell line was selected for further investigation to improve and optimize the yield of these alkaloids with special emphasis on (9) because of its important role in both the biosynthesis and chemical formation of vinblastine-type dimeric alkaloids [i]. It is now appropriate to present results of the first time course experiment. Mitotic index of all samples remained at or near zero, while the pH of the culture medium maintained within a fairly constant value (pH = 5.0-5.6). There was no significant fluctuation of biomass in terms of cell dry weight from day 12 to day 31 of incubation in the presence of bioregulator (Figure i). This indicated that the five bioregulators (3), (4), (5), (6) and (7) when added separately at a concentration of 2 mg/£ to cell cultures which had already reached the stationary phase, did not affect further mitotic activity nor inhibit the normal growth of the cultures. Cell biomass continued to increase for 6 to 8 days (Figure 5, from
(6)
~1
Days of Incubation with BiorecJulators
Figure 2. Effect of bioregulators (3) to (7) on total alkaloid yield over an incubation period of 12 to 31 days. the five bioregulators used, (3), (6) and (7) were effective in increasing the total alkaloid production from the cell culture as compared with the control sample, while the other bioregulators showed the op~ posite effect. Yields of the two specific alkaloids, ajmalicine (8) and catharanthine (9), as monitored by HPLC analyses (Figures 3 and 4) indicated the different effects of the bioregulators on the biosynthesis of these two alkaloids by cultures of cell line PRL #200. The results suggested that bioregulators (3) and (7) affected the best improvement especially for compound (9). Results of the second time course experiment carried out to investigate the effects of the two most promising bioregulators, (3) and (7), after shorter incubation time (4, 6, 8 and i0 days) with the PRL #200 cultures are now described. The mitotic index and pH of the cultures again remained quite constant. Increases in biomass were observed after 4 days of incubation and maximum cell dry weight was reached a few days later (Figure 5). The mitotic index indi-
261 ~
~
~ =
mC~rol °(3; O~
,(4)
(3)
(4)
"(51 o(61
(5)
azc
"(71 "~
(6) I~• CH~4=N(O~pl=l=
=~ ^(71
,,~-..=
~
cated that although cell division had ceased at the time of bioregulator addition and did not reinitiate again, dry weight increase continued for 6 to 8 days at the stationary phase (Figure 5). Total alkaloid production expressed as percent of cell dry weight (Figure 6) reached a maximum level in all three cases after 6 days of incubation with or without bioregulator. The actual yield was highest for bioregulator
~
~
• (31
O.SI" 0.1
12
18
24
° (71
31
Days of Incubation with Bioregulators
. . . . . .
Figure 3. Effect of bioregulators (3) to (7) on yield of ajmalicine over an incubation period of 12
i/
i
~ o.4~ ~~o 03 /|
to 31 days. 0.014 '~
\
,,,
Days oflncubat~nwith B ~ g u l a t o r s
(s,
(.,
Figure 6. Effect of bioregulators (3) and (7) on total alkaloid yield over an incubation period of 4 to i0 days. I ~
~
0 o.o~ - - - - - ~ ,
\.
~\
\\
\
ooos \
~
\
\ \
\
\
• Control
\ \
.(31 , (41
"
"(5)
(7), while (3) also produced more than the control sample. Yields of alkaloids (8) and (9) as shown in Figures 7 and 8 respectively indicated both bioregulators provided quite similar effects on the biosynthetic capacity of cell line PRL #200 suspension cultures. Comparing with control samples, significant
o(., ° (71
OD03
o.oo
,,,.
o ool
,~
o 12
=p
~4
Days oflncubation with Bioregulators
(3)
Figure 4. Effect of bioregulators (3) to (7) on yield of catharanthine over an incubation period of 12 to 31 days.
n • o.~(oV:~),
(rl
o.i
Control • (3) a (71
•
0.09 0.08
~', 0 0 7 a =c~ OJO6 o ~6 O.C~
(3) 14
~C.~
cn
0.04
) 13
• Control ° (3) = (7)
o~ 12
0.03
"8 0.02 E
Plant Cell Reports (1985) 4:259-262
© S p r i n g e r - V e r l a g 1985
Alkaloid production in C a t h a r a n t h u s r o s e u s (L.) G. Don cell cultures. XIII. Effects of bioregulators on indole alkaloid biosynthesis James P. Kutney 1, Barbara Aweryn 1, Kenneth B. Chatson 2, Lewis S.L. Choi %and Woifgang G. W. Kurz 2 1 Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Y6 2 Plant Biotechnology Institute, National Research Council, 110 Gymnasium Road, University Campus, Saskatoon, Saskatchewan, Canada S7N OW9 Received December 5, 1984 / Revised version received August 10, 1985 - Communicated by F. Constabel
ABSTRACT A study on the effect of various bioregulators on the biosynthesis of ajmalicine (8) and catharanthine (9) in plant tissue cultures of Catharanthus roseus is described. It is shown that l,l-dimethylpiperidine bromide (3) and 2-diethylaminoethyl-3,4dimethylphenylether (7) are effective in increasing these alkaloid levels in the cell line PRL #200. Such studies may prove beneficial in larger scale experiments designed for the production of these alkaloids.
{I)
T
(2)
INTRODUCTION ;Lv¢ou[
It is well established that tryptamine (i) and secologanin (2) are the biosynthetic building units for a variety of indole alkaloids (Scheme i). Furthermore various laboratories including our own [i] have developed tissue cultures of Catharanthus roseus (L°) G. Don which are capable of producing alkaloids within the Corynanthe, Strychnos, Aspidosperma, and Iboga families. Our efforts in recent studies have been directed towards a detailed investigation of fermentation conditions with selected cell lines in order to optimize production of target alkaloids. In this regard, we have considered the utilization of certain bioregulators which have been shown to induce isoprenoid production in the hope that such substances may stimulate the biosynthesis of secologanin (2) and, in turn, the increased production of the desired alkaloid systems. Yokoyama et al. [2] have shown that rubber biosynthesis is induced by certain aminoethyl phenylethers, implying that this type of synthetic bioregulators may be general inducers of terpenoid biosynthesis. Recently, Scott et al. [3] also demonstrated that some of these compounds are effective in promoting indole alkaloid formation in one of their C. roseus cell lines. Here we wish to report our results on experiments with some of these bioregulators and an established C. roseus cell line (PRL #200) described previously [4]. MATERIAL AND METHODS Catharanthus Roseus Cell Culture Conditions for initiation and maintenance of cell line PRL #200 were as described previously [4].
Offprint requests to: J. P. Kutney
AI~DOS~RUA
(3)
(4)
~O~A
(5)
.oc~ "°~'_ c~
(61
n • a~,c~(a~,,),
(71
u
(8)
(9) Scheme i
Bioregulators Five bioregulators, l,l-dimethylpiperidine bromide (3), 2-diethylaminoethyl-3,4-dichlorophenylether (4), 2-diethylaminoethyl-2,4-dichlorophenylether (5), 2-diethylaminoethyl-~-naphthylether (6) and 2-diethylaminoethyl-3,4-dimethylphenylether (7) were obtained as gifts from Dr. H. Yokoyama (Fruit and Vegetable Chemistry Laboratory, Western Region, Agricultural Research Service, Pasadena, California, U.S.A.). The bioregulators were added as the free base (except (3) as the bromide), dissolved in methanol/
260 water and filter-sterilized brane.
through a 0.45 Dm mem-
Time Course Studies Shake flask cultures of PRL #200 cells (500 m£ in 1 £ flask) were grown in the alkaloid production medium [4] for 7 days when the cultures were near or at stationary phase of growth as monitored by the mitotic activity of the cells. At this stage the average cell dry weight was 25 mg/m% culture. For the first time course experiment all five bioregulators (3), (4), (5), (6) and (7) were examined. These were added separately to the above cultures at a concentration of 2 mg/~ and further incubated for four different time periods (12, 18, 24 and 31 days). Parallel control cultures (no bioregulator added) were also set up. Samples were withdrawn at indicated intervals, extracted and analysed for alkaloid content.
II
A
~9 U
5
, (4)
(3)
(4) , ~
(5)
(6)
, fS) o 161
(7)
Days of Incubation with Biorecjulators
A second time course experiment was carried out to investigate the effects of the two most promising bioregulators, (3) and (7), after shorter incubation time (4, 6, 8 and i0 days) with the PRL #200 cultures. Samples were harvested at indicated intervals. Extraction and analyses of the alkaloid content were carried out according to the standard procedure described below.
Figure I. Effect of bioregulators (3) to (7) on cell dry weight over an incubation period of 12 to 31 days. second time course experiment) and then levelled off from day 12 on (Figure i). Total alkaloid obtained from the cell extracts, expressed as percent of cell dry weight, showed substantial differences (Figure 2) especially after 12 and 18 days of incubation. Of
Alkaloid Extraction and Analysis A detailed account of the extraction procedure was given in an earlier publication [5]. The alkaloid extract from each sample was then analysed by HPLC (Waters Associates system) equipped with a Radial Compression Module, I0~ C18 cartridge, eluted with 2 different solvent systems at room temperature. (A)
(B)
45% H20 in MeOH with 0.1% Et3N at a flow rate of 4 m£/min. Alkaloids were monitored at 280 and 254 nm (Waters Associates 440 UV detector). Retention times for catharanthine (9) and ajmalicine (8) are 13.2 min and 17.7 min respectively. 32% H20 in MeOH with 0.1% Et3N at a flow rate of 1 m~/min. Retention times for (9) and (8) are 19.6 min and 21.7 min respectively.
12
I0 =
(3)
(4)
(5)
a~ )
• Control • (S) .(4) • (5} o 161
O.IB
"6 o-
O.E
o ~
o.4
o ~-
0.2
='4
Quantitative determinations of (8) and (9) were made on the basis of peak areas on HPLC output, compared to standard curves. RESULTS AND DISCUSSION We have shown earlier that cell line PRL #200 was capable of producing (8) and (9) among other alkaloids [4]. This cell line was selected for further investigation to improve and optimize the yield of these alkaloids with special emphasis on (9) because of its important role in both the biosynthesis and chemical formation of vinblastine-type dimeric alkaloids [i]. It is now appropriate to present results of the first time course experiment. Mitotic index of all samples remained at or near zero, while the pH of the culture medium maintained within a fairly constant value (pH = 5.0-5.6). There was no significant fluctuation of biomass in terms of cell dry weight from day 12 to day 31 of incubation in the presence of bioregulator (Figure i). This indicated that the five bioregulators (3), (4), (5), (6) and (7) when added separately at a concentration of 2 mg/£ to cell cultures which had already reached the stationary phase, did not affect further mitotic activity nor inhibit the normal growth of the cultures. Cell biomass continued to increase for 6 to 8 days (Figure 5, from
(6)
~1
Days of Incubation with BiorecJulators
Figure 2. Effect of bioregulators (3) to (7) on total alkaloid yield over an incubation period of 12 to 31 days. the five bioregulators used, (3), (6) and (7) were effective in increasing the total alkaloid production from the cell culture as compared with the control sample, while the other bioregulators showed the op~ posite effect. Yields of the two specific alkaloids, ajmalicine (8) and catharanthine (9), as monitored by HPLC analyses (Figures 3 and 4) indicated the different effects of the bioregulators on the biosynthesis of these two alkaloids by cultures of cell line PRL #200. The results suggested that bioregulators (3) and (7) affected the best improvement especially for compound (9). Results of the second time course experiment carried out to investigate the effects of the two most promising bioregulators, (3) and (7), after shorter incubation time (4, 6, 8 and i0 days) with the PRL #200 cultures are now described. The mitotic index and pH of the cultures again remained quite constant. Increases in biomass were observed after 4 days of incubation and maximum cell dry weight was reached a few days later (Figure 5). The mitotic index indi-
261 ~
~
~ =
mC~rol °(3; O~
,(4)
(3)
(4)
"(51 o(61
(5)
azc
"(71 "~
(6) I~• CH~4=N(O~pl=l=
=~ ^(71
,,~-..=
~
cated that although cell division had ceased at the time of bioregulator addition and did not reinitiate again, dry weight increase continued for 6 to 8 days at the stationary phase (Figure 5). Total alkaloid production expressed as percent of cell dry weight (Figure 6) reached a maximum level in all three cases after 6 days of incubation with or without bioregulator. The actual yield was highest for bioregulator
~
~
• (31
O.SI" 0.1
12
18
24
° (71
31
Days of Incubation with Bioregulators
. . . . . .
Figure 3. Effect of bioregulators (3) to (7) on yield of ajmalicine over an incubation period of 12
i/
i
~ o.4~ ~~o 03 /|
to 31 days. 0.014 '~
\
,,,
Days oflncubat~nwith B ~ g u l a t o r s
(s,
(.,
Figure 6. Effect of bioregulators (3) and (7) on total alkaloid yield over an incubation period of 4 to i0 days. I ~
~
0 o.o~ - - - - - ~ ,
\.
~\
\\
\
ooos \
~
\
\ \
\
\
• Control
\ \
.(31 , (41
"
"(5)
(7), while (3) also produced more than the control sample. Yields of alkaloids (8) and (9) as shown in Figures 7 and 8 respectively indicated both bioregulators provided quite similar effects on the biosynthetic capacity of cell line PRL #200 suspension cultures. Comparing with control samples, significant
o(., ° (71
OD03
o.oo
,,,.
o ool
,~
o 12
=p
~4
Days oflncubation with Bioregulators
(3)
Figure 4. Effect of bioregulators (3) to (7) on yield of catharanthine over an incubation period of 12 to 31 days.
n • o.~(oV:~),
(rl
o.i
Control • (3) a (71
•
0.09 0.08
~', 0 0 7 a =c~ OJO6 o ~6 O.C~
(3) 14
~C.~
cn
0.04
) 13
• Control ° (3) = (7)
o~ 12
0.03
"8 0.02 E
