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Effect of various stress factors on indole alkaloid formation by C. Roseus (periwinkle) cells
Effect of various stress factors on indole alkaloid formation by C. r o s e u s (periwinkle) cells F. Kargi and Parrish Potts Biotechnology Engineering Laboratory, D e p a r t m e n t o f Chemical Engineering, Washington University, St. Louis, M O
Catharanthus roseus cells producing indole alkaloids were grown for 21 days in suspension cultures. Three stress factors--fungal elicitors, vanadyl sulfate, and potassium chloride--were added to the production media at various concentrations. Alkaloid production and cell viability were followed and compared to a control experiment without stress factors. A Box-Wilson experimental design was used to determine the effects of these stress factors on cell performance. The stress factors used at low concentrations produced higher levels of indole alkaloids. This is thought to be due to the stimulatory effect of the stress imposed on the cells by the added components. The percent ofintracellular alkaloids produced in the presence of stress factors was lower than that of the control experiment. This is probably due to increased excretion of indole alkaloids in the presence of stress factors.
Keywords:Indolealkaloids;plant cell cultures
Introduction Plant cells may be used as biocatalysts to produce a wide variety of chemical products. These products include pharmaceuticals, flavors and fragrances, oils and sweeteners) Pharmaceuticals are the most valuable plant cell products, since approximately 25% of all prescription medicines are derived from plants. 2 At present, most plant products are derived from whole plants. The extraction of chemicals from intact plants has several inherent problems, including seasonal variations, pests, diseases, and inconsistent product quality and yield. Large-scale plant cell culture may become a viable economic alternative for production, if the product formation rate, yield, and final concentrations are increased significantly. Until recently, the production of chemical products from plant cells has primarily been of academic interest. The only industrial plant cell culture process, at the present time, belongs to the Mitsui Company of Japan for production of shikonin, an anti-inflammatory agent. 2 Suspension cultures are the most widely used method of plant cell cultivation. Suspension cultures provide a homogeneous growth environment with uniform aeration and agitation. 1 Nutrient limitations can Address reprint requests to Dr. Kargi at the BiotechnologyEngineeringLaboratory,Departmentof ChemicalEngineering,Washington University, St. Louis, MO 63130, USA. Received 21 June 1990; revised 7 February 1991
760
also be achieved in continuous suspension cultures. The problems of plant cell cultures include slow growth and product formation rates, low product concentrations in fermentation broth, limited excretion of products, and the shear sensitivity of cells. Secondary metabolites are high-value plant products formed under nutrient-limited and slow growth conditions. Though the exact functions of secondary metabolites are not known, it is likely that they are involved in establishing and maintaining interactions between a plant and its environment as a defense mechanism. 3 These products are primarily intracellular and are produced and deposited in vacuoles in small quantities. The addition of stress factors such as potassium chloride, vanadyl sulfate, and fungal elicitors has been reported to increase the accumulation of secondary metabolites in plant cells.l,4,5 Fungal elicitors are cell wall components of various fungi. A homogenate of Geotrichium candidum was used as a fungal elicitor throughout the experiments. When plant cells are exposed to elicitors, they produce secondary metabolites as a defense response. Vanadyl sulfate may act by increasing the cell wall permeability so that intracellular alkaloids are excreted into the medium, or it may act as an enzyme cofactor. Potassium chloride increases the osmotic pressure of the medium, thereby increasing cell wall permeability. In this study, Catharanthus roseus cells were grown for 21 days in suspension culture with varying levels of the stress factors to determine if these three factors
Enzyme Microb. Technol., 1991, vol. 13, September
© 1991 Butterworth-Heinemann
Effect of stress on indole alkaloid formation: F. Kargi and P. Potts Table1 Concentrations of stress factors used in Box-Wilson experimental design
Expt. number
Elicitor (X~) (mg g-1 dry cell)
VaS04 (X2) mg g-1 dry cell)
KCI (X3) mg g-1 dry cell)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Control
100 10 55 55 55 55 81 81 81 81 29 29 29 29 55 0
2.75 2.75 5 0.5 2.75 2.75 4.05 4.05 1.45 1.45 4.05 4.05 1.45 1.45 2.75 0
275 275 275 275 500 50 405 145 405 145 405 145 405 145 275 0
triplicate. Average values are reported and deviations from the average were less than 7%. A Box-Wilson experimental design8 was used to determine the response of final alkaloid titer (Y, mg 100 mg- 1cells), as a function of the medium concentration of the three variables: elicitor (x~, mg g-l dry cell), vanadyl sulfate (x2, mg g-~ dry cell), and potassium chloride (x3, mg g-~ dry cell). The response function (alkaloid titer) has the following form: y = a o + alx 1 + a2x 2 + a3x 3 + allX ~ + az2 x2
+ a33x ] + a l 2 x l X 2 + + a l 3 X l X 3 + a23x2x 3 The range of concentrations for the three variables tested were as follows:
Elicitor Vanadyl sulfate KC1
10-100 mg g-~ dry cell 0.5-5 mg g-1 dry cell 50-500 mg g-1 dry cell
The concentrations of stress factors used in each experiment are presented in Table 1.
could be used in combination to stimulate indole alkaloid production. The effects of these stress factors on cell viability and product excretion were determined. A wide range of concentrations was used to determine if high levels of any of these components would be toxic to the cells. A Box-Wilson experimental design was used to determine the optimal levels of stress factors resulting in maximum alkaloid titer.
Materials and methods Culture C. roseus cells were maintained in callus and suspen-
sion cultures. The growth medium used in suspension and callus culture was the Murashige and Skoog medium supplemented with 0.5 mg 1-~ 2,4-dichlorophenoxy-acetic acid. 6 The pH of the medium was adjusted to 5.8. The cells were maintained at 26°C under continuous lighting. The production medium used in the actual suspension culture experiments was the NB5 medium, which consisted of Gamborg's B5 basal medium supplemented with 1.0 mg 1- ~naphthaleneacetic acid and 0.1 mg 1-1 kinetin. 7 The pH of the medium was adjusted to 5.8. Since the production medium was used throughout the experiments, growth was less than 10% of the initial dry weight of the cells throughout the experiments. Experimental procedure
Fifteen suspension culture experiments were conducted with initial cell concentrations of I0 g dry cell 1-1. The stress factors were added into the culture in specified concentrations (Table 1). The elicitor used was a hydrolysate of a 10-day old Geotrichium culture which was cultivated in our laboratory. The plant cells were aseptically grown for 21 days in 100 ml of production medium, containing stress factors, in 500-ml shake flasks. Duplicate samples were taken every 3 days for analysis. The last experiment (center point) was run in
Analytical methods
The dry weight of the cells was estimated by filtering cells through 0.45/z glass fiber filter paper. After filtration, the filter paper was dried in a vacuum oven at 60°C until constant weight. The viability (respiration activity) ofceUs was determined throughout each experimental run. A 2,3,5-triphenyltetrazolium chloride (TTC) assay was used, which was based on reduction of tetrazolium salts to a water-insoluble red formazan. 9 Ethanol was used to extract the formazan, and the absorbance was determined at 485 nm. Both extracellular and intracellular indole alkaloid concentrations were determined. The cells were extracted for 24 h with 95% ethanol to remove intracellular alkaloids. Following centrifugation, the ethanol was separated from the cells and was evaporated. The residue was redissolved in 0.1 M HCI. The alkaloids were then separated from hydrophobic compounds at low pH followed by separation from hydrophilic compounds at high pH. The organic solvent used was ethyl acetate. Following the extraction procedure, the ethyl acetate was evaporated, and the residue was redissolved in methanol. Total alkaloid concentration was determined by absorbance measurement at 280 nm. ~0,H A calibration curve was prepared using ajmalicine as the standard alkaloid. Analysis of the product by HPLC indicated that ajmalicine was the only alkaloid produced by the cell line used.
Results and discussion Table 2 summarizes the intracellular, extracellular, and total alkaloid concentrations and the percent final viabilities obtained in each experiment. High concentrations of stress factors caused significant drops in cell viability as measured by respiration activity (experiments 1, 3, 5, 7, and 11). High cell viabilities were obtained with low levels of stress factors, as seen in experiments 2, 6, 12, and 14.
Enzyme Microb. Technol., 1991, vol. 13, September
761
Papers Table 2 Indole alkaloid concentrations [intracellular (ICA), extracellular (ECA), and total (TAC)] and final percent viabilities obtained at each experiment of the Box-Wilson design
Expt. number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Control
Max. % Via
ICA (mg 100 mg -1 dry cell)
ECA (mg 100 mg -~ dry cell)
TAC (mg 100 mg -1 dry cell)
20 86 24 75 20 90 20 67 28 84 29 92 78 88 72 100
0.063 0.017 0.001 0.005 0 0.011 0.058 0.044 0.046 0.056 0.038 0.022 0.049 0.117 0.046 0.058
0.021 0.049 0.027 0.08 0.038 N.A. 0.007 0.018 0.021 0.031 0.064 0.013 0.012 0.031 0.008 0.011
0.084 0.066 0.028 0.085 0.038 0.011 0.065 0.062 0.067 0.087 0.102 0.035 0.061 0.148 0.058 0.069
N.A. = not available.
The intracellular alkaloid content of the cells in 12 experiments was lower than that of the control, which did not contain stress factors; on the other hand, the extracellular alkaloid content was significantly higher in most of the experiments as compared to the control. The results clearly indicated that stress factors stimulated excretion of alkaloids. The total indole alkaloid content of the cells was higher in five of the experiments as compared to the control experiment. Experiment 14, which included the lowest level of all three stress factors (elicitor, 29 mg g-~ dry cell; vanadyl sulfate, 1.45 mg g-1 dry cell; KCI, 145 mg g-1 dry cell) resulted in a total alkaloid content of 0.148 mg g- ~dry cell which was nearly 2.2 times higher than that of the control run. Nearly 80% of the total alkaloids produced in this run were intracellular, due to low levels of stress factors. Experiments 3 and 5, which contained the highest amount of vanadyl sulfate (5 mg g- 1 cell) and KCI (500 mg g-~cell), resulted in significantly lower levels of alkaloid content as compared to the control run, indicating toxic effects of high levels of stress factors. A typical alkaloid formation profile from experiment 10 (X l = 81, X 2 = 1.45, X 3 = 145 m g g - I dry cell) is depicted in Figure 1. Total alkaloid concentration reached a maximum within the first 12-15 days and then declined. The drop in total alkaloid concentration later on during the experiments may be due to degradation of alkaloids outside the cells, since the pH of the media was nearly 6, as compared to the vacuolar pH of 4.0, where alkaloids are stored intracellularly, The least-square error fit of the experimental data to the aforementioned objective function resulted in the following coefficients for the alkaloid titer function: Y = 0.368 - 3.7 × 10 - 3 S l - 0.0719X2 - 7 ×
10 - 4 X 3 + 1.8 • 1 0 - 5 X ~ +
3 •5
,~ ~ u
0.1
o O
0.0~
t~ vE
0.06
~ t-, -o ~ ~o .~ ,--4 < -,--I
• 10-3X 2
+ 3 × 10 - 7 g ~ + 3 . 5 × 10 -4 X 1 X 2 + 2 . 9 × 10 - 6 X I X 3 q- 7 • 4 ×
762
The projections of the response function on different planes of XI, X2, and X3 are depicted in Figures 2--4. Figure 2 is a plot of alkaloid titer versus elicitor concentration for different values of V a S O 4 concentration at a KCI concentration of 50 mg g- ~dry cell. Lower elicitor (10 mg g-~ dry cell) and V a S O 4 concentrations (0.5 mg g- ~dry cell) resulted in higher alkaloid titers. Final alkaloid titers decreased with increasing elicitor and VaSO4 concentrations. Similar trends can be observed in Figures 3 and 4. Alkaloid titers increased with dec r e a s i n g V a S O 4, KCI, and elicitor concentrations. The optimal set of values of stress factors resulting in maximum final alkaloid titer was determined by using a computer program. A standard curve fitting/error minimizing program was used to determine the coefficients of the objective function and the optimal set of variables. The theoretical optimum was found to be at
10 - 6 X 2 X 3
0.1 ¢
0.04 0.02
o
=
0
3
i
=
6
9 Time
=
=
i
12
15
18
21
(days)
Figure 1 Typical indole alkaloid formation profile (experiment 10)
Enzyme Microb. Technol., 1991, vol. 13, September
Effect of stress on indole alkaloid formation: F. Kargi and P. Potts 0.30
0.30
0.2 5 ~
.
~cz - 50 =g/g
"~
=••
0.25
O
0.20
0.20~ o o ,-4
0.15
ws°4(=~/s)
O.i 0 ~
2.o ____
~
E
0.15
o 4-J
O.IC
-,-I 0
O.
VaS0& = 0 , 5
l:tl~:t t o r
mg/g
(mg/g)
3.5
0.05
.
o
<
i
i
i
i
i
i
i
i
I0
20
30
40
50
60
70
80
Elicitor
05
,--t
i
90 100
<
i
i
r
,
i
i
i
i
50 i00 150 200 250 300 350 400 450 500
(mg/ g DW cells)
KCl (mg/ g DW cells)
Figure 2 Total final alkaloid titer (mg alkaloid 100 mg -1 cells) as a function of elicitor concentration (mg g-~ dry cell wt) at
Figure 4 Total final alkaloid titer (mg alkaloid 100 mg -~ cells) as a function of KCI concentration (mg g-1 dry cell wt) at different
different VaS04 concentrations as predicted by the response function. [KCI] = 50 mg g-~ dry cell w t
elicitor concentrations as predicted by the response function.
elicitor concentrations of 10 mg g-~ dry cell, VaSO 4 concentrations of 0.5 mg g-~ dry cell, and KCI concentrations of 50 mg g- l dry cell, which were the minimum concentrations tested. The final alkaloid titer at this optimal point is expected to be 0.30 mg 100 mg -l dry cell, which is approximately two times higher than that obtained with the control experiment containing no stress factors.
KCI within the experiments listed in Table 1 were 29, 1.45, and 145 mg g- ] dry cell, respectively, resulting in a total alkaloid titer of approximately 0.148 mg alkaloid 100 mg-] dry cell. The response function obtained on the basis of experimental data predicts an even higher alkaloid titer (0.3 mg 100 mg-l dry cell) at the lowest tested levels of stress factors. High concentrations of stress factors were toxic to the cells, resulting in loss of viability. Therefore, low concentrations of the stress factors, i.e. I0 mg g-i < [E] -< 30 mg g-l, 0.5 mg g-l -%<[VaSO4] ~< 1.45 mg g-l, 50 mg g-i < [KC1] -< 145 mg g-l, should be used in suspension culture of C. r o s e u s in order to obtain high cell viabilities, high alkaloid titers, and excretion from cells into the medium.
Conclusion The use of stress factors at low levels stimulated indole alkaloid formation and excretion by C. r o s e u s cells. The optimal concentrations of elicitor, VaSO4, and 0.30
O
0 •2
E
0
O O
E
[VaS04] = 0.5 mg g-~ dry cell wt
Acknowledgement
5
.
-
~
2
This study was supported by National Science Foundation Grant No. ECE-8612936.
Elicitor = i0 =g/g
0
~
References
~
1 2
0.15_
3 KCI (mg/g)
0
4 5
73 O
6
2~
<
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Vanadyl Sulfate (mg/g DW cells)
Figure 3 Total final alkaloid titer (mg alkaloid 100 mg -1 cells) as a function of VaS04 concentration (rng g-1 dry cell wt) at different KCI concentrations as predicted by the response function. [Elicitor] = 10 mg g-1 dry cell w t
7 8 9 10 11
Kargi, F., and Rosenberg, M. Biotechnol Prog. 1987, 3, 1-8 Stafford, A., Morris, P. and Fowler, M. Enzyme Microb. Technol. 1986, 8, 578-587 Smith, J. 1., Smart, N. J., Kurz, W. G. W. and Misawa, M. J. Exp. Bot. 1987, 38, 1501-1506 Eilert, U., Constabel, F. and Kurz, W. G. W. J Plant Physiol. 1986, 127, 11-12 Smith, J. I., Smart, N. J., Misawa, M., Kurz, W. G. W., Tullevi, S. G., and DiCosmo, F. Plant Cell Rep. 1987, 6, 142-145 Dixon, R. in Plant Cell Culture: A Practical Approach (Dixon, R., ed.) IRL Press, Oxford, 1985, pp. 1-20 Morris, P. Planta Medica 1986, 121-126 Perry's Chemical Engineering Handbook, 6th ed. (Perry, R. H. and Green, D. W., eds.) 1984, 2.74, 75 Towill, L. and Mazur, P. Can. J. Bot. 1975, 53, 1097-1102 Payne, G. Personal communication, University of Maryland, 1986 Shuler, M. L. Personal Communication, Cornell University, 1986
Enzyme Microb. Technol., 1991, vol. 13, September
763
Catharanthus roseus cells producing indole alkaloids were grown for 21 days in suspension cultures. Three stress factors--fungal elicitors, vanadyl sulfate, and potassium chloride--were added to the production media at various concentrations. Alkaloid production and cell viability were followed and compared to a control experiment without stress factors. A Box-Wilson experimental design was used to determine the effects of these stress factors on cell performance. The stress factors used at low concentrations produced higher levels of indole alkaloids. This is thought to be due to the stimulatory effect of the stress imposed on the cells by the added components. The percent ofintracellular alkaloids produced in the presence of stress factors was lower than that of the control experiment. This is probably due to increased excretion of indole alkaloids in the presence of stress factors.
Keywords:Indolealkaloids;plant cell cultures
Introduction Plant cells may be used as biocatalysts to produce a wide variety of chemical products. These products include pharmaceuticals, flavors and fragrances, oils and sweeteners) Pharmaceuticals are the most valuable plant cell products, since approximately 25% of all prescription medicines are derived from plants. 2 At present, most plant products are derived from whole plants. The extraction of chemicals from intact plants has several inherent problems, including seasonal variations, pests, diseases, and inconsistent product quality and yield. Large-scale plant cell culture may become a viable economic alternative for production, if the product formation rate, yield, and final concentrations are increased significantly. Until recently, the production of chemical products from plant cells has primarily been of academic interest. The only industrial plant cell culture process, at the present time, belongs to the Mitsui Company of Japan for production of shikonin, an anti-inflammatory agent. 2 Suspension cultures are the most widely used method of plant cell cultivation. Suspension cultures provide a homogeneous growth environment with uniform aeration and agitation. 1 Nutrient limitations can Address reprint requests to Dr. Kargi at the BiotechnologyEngineeringLaboratory,Departmentof ChemicalEngineering,Washington University, St. Louis, MO 63130, USA. Received 21 June 1990; revised 7 February 1991
760
also be achieved in continuous suspension cultures. The problems of plant cell cultures include slow growth and product formation rates, low product concentrations in fermentation broth, limited excretion of products, and the shear sensitivity of cells. Secondary metabolites are high-value plant products formed under nutrient-limited and slow growth conditions. Though the exact functions of secondary metabolites are not known, it is likely that they are involved in establishing and maintaining interactions between a plant and its environment as a defense mechanism. 3 These products are primarily intracellular and are produced and deposited in vacuoles in small quantities. The addition of stress factors such as potassium chloride, vanadyl sulfate, and fungal elicitors has been reported to increase the accumulation of secondary metabolites in plant cells.l,4,5 Fungal elicitors are cell wall components of various fungi. A homogenate of Geotrichium candidum was used as a fungal elicitor throughout the experiments. When plant cells are exposed to elicitors, they produce secondary metabolites as a defense response. Vanadyl sulfate may act by increasing the cell wall permeability so that intracellular alkaloids are excreted into the medium, or it may act as an enzyme cofactor. Potassium chloride increases the osmotic pressure of the medium, thereby increasing cell wall permeability. In this study, Catharanthus roseus cells were grown for 21 days in suspension culture with varying levels of the stress factors to determine if these three factors
Enzyme Microb. Technol., 1991, vol. 13, September
© 1991 Butterworth-Heinemann
Effect of stress on indole alkaloid formation: F. Kargi and P. Potts Table1 Concentrations of stress factors used in Box-Wilson experimental design
Expt. number
Elicitor (X~) (mg g-1 dry cell)
VaS04 (X2) mg g-1 dry cell)
KCI (X3) mg g-1 dry cell)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Control
100 10 55 55 55 55 81 81 81 81 29 29 29 29 55 0
2.75 2.75 5 0.5 2.75 2.75 4.05 4.05 1.45 1.45 4.05 4.05 1.45 1.45 2.75 0
275 275 275 275 500 50 405 145 405 145 405 145 405 145 275 0
triplicate. Average values are reported and deviations from the average were less than 7%. A Box-Wilson experimental design8 was used to determine the response of final alkaloid titer (Y, mg 100 mg- 1cells), as a function of the medium concentration of the three variables: elicitor (x~, mg g-l dry cell), vanadyl sulfate (x2, mg g-~ dry cell), and potassium chloride (x3, mg g-~ dry cell). The response function (alkaloid titer) has the following form: y = a o + alx 1 + a2x 2 + a3x 3 + allX ~ + az2 x2
+ a33x ] + a l 2 x l X 2 + + a l 3 X l X 3 + a23x2x 3 The range of concentrations for the three variables tested were as follows:
Elicitor Vanadyl sulfate KC1
10-100 mg g-~ dry cell 0.5-5 mg g-1 dry cell 50-500 mg g-1 dry cell
The concentrations of stress factors used in each experiment are presented in Table 1.
could be used in combination to stimulate indole alkaloid production. The effects of these stress factors on cell viability and product excretion were determined. A wide range of concentrations was used to determine if high levels of any of these components would be toxic to the cells. A Box-Wilson experimental design was used to determine the optimal levels of stress factors resulting in maximum alkaloid titer.
Materials and methods Culture C. roseus cells were maintained in callus and suspen-
sion cultures. The growth medium used in suspension and callus culture was the Murashige and Skoog medium supplemented with 0.5 mg 1-~ 2,4-dichlorophenoxy-acetic acid. 6 The pH of the medium was adjusted to 5.8. The cells were maintained at 26°C under continuous lighting. The production medium used in the actual suspension culture experiments was the NB5 medium, which consisted of Gamborg's B5 basal medium supplemented with 1.0 mg 1- ~naphthaleneacetic acid and 0.1 mg 1-1 kinetin. 7 The pH of the medium was adjusted to 5.8. Since the production medium was used throughout the experiments, growth was less than 10% of the initial dry weight of the cells throughout the experiments. Experimental procedure
Fifteen suspension culture experiments were conducted with initial cell concentrations of I0 g dry cell 1-1. The stress factors were added into the culture in specified concentrations (Table 1). The elicitor used was a hydrolysate of a 10-day old Geotrichium culture which was cultivated in our laboratory. The plant cells were aseptically grown for 21 days in 100 ml of production medium, containing stress factors, in 500-ml shake flasks. Duplicate samples were taken every 3 days for analysis. The last experiment (center point) was run in
Analytical methods
The dry weight of the cells was estimated by filtering cells through 0.45/z glass fiber filter paper. After filtration, the filter paper was dried in a vacuum oven at 60°C until constant weight. The viability (respiration activity) ofceUs was determined throughout each experimental run. A 2,3,5-triphenyltetrazolium chloride (TTC) assay was used, which was based on reduction of tetrazolium salts to a water-insoluble red formazan. 9 Ethanol was used to extract the formazan, and the absorbance was determined at 485 nm. Both extracellular and intracellular indole alkaloid concentrations were determined. The cells were extracted for 24 h with 95% ethanol to remove intracellular alkaloids. Following centrifugation, the ethanol was separated from the cells and was evaporated. The residue was redissolved in 0.1 M HCI. The alkaloids were then separated from hydrophobic compounds at low pH followed by separation from hydrophilic compounds at high pH. The organic solvent used was ethyl acetate. Following the extraction procedure, the ethyl acetate was evaporated, and the residue was redissolved in methanol. Total alkaloid concentration was determined by absorbance measurement at 280 nm. ~0,H A calibration curve was prepared using ajmalicine as the standard alkaloid. Analysis of the product by HPLC indicated that ajmalicine was the only alkaloid produced by the cell line used.
Results and discussion Table 2 summarizes the intracellular, extracellular, and total alkaloid concentrations and the percent final viabilities obtained in each experiment. High concentrations of stress factors caused significant drops in cell viability as measured by respiration activity (experiments 1, 3, 5, 7, and 11). High cell viabilities were obtained with low levels of stress factors, as seen in experiments 2, 6, 12, and 14.
Enzyme Microb. Technol., 1991, vol. 13, September
761
Papers Table 2 Indole alkaloid concentrations [intracellular (ICA), extracellular (ECA), and total (TAC)] and final percent viabilities obtained at each experiment of the Box-Wilson design
Expt. number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Control
Max. % Via
ICA (mg 100 mg -1 dry cell)
ECA (mg 100 mg -~ dry cell)
TAC (mg 100 mg -1 dry cell)
20 86 24 75 20 90 20 67 28 84 29 92 78 88 72 100
0.063 0.017 0.001 0.005 0 0.011 0.058 0.044 0.046 0.056 0.038 0.022 0.049 0.117 0.046 0.058
0.021 0.049 0.027 0.08 0.038 N.A. 0.007 0.018 0.021 0.031 0.064 0.013 0.012 0.031 0.008 0.011
0.084 0.066 0.028 0.085 0.038 0.011 0.065 0.062 0.067 0.087 0.102 0.035 0.061 0.148 0.058 0.069
N.A. = not available.
The intracellular alkaloid content of the cells in 12 experiments was lower than that of the control, which did not contain stress factors; on the other hand, the extracellular alkaloid content was significantly higher in most of the experiments as compared to the control. The results clearly indicated that stress factors stimulated excretion of alkaloids. The total indole alkaloid content of the cells was higher in five of the experiments as compared to the control experiment. Experiment 14, which included the lowest level of all three stress factors (elicitor, 29 mg g-~ dry cell; vanadyl sulfate, 1.45 mg g-1 dry cell; KCI, 145 mg g-1 dry cell) resulted in a total alkaloid content of 0.148 mg g- ~dry cell which was nearly 2.2 times higher than that of the control run. Nearly 80% of the total alkaloids produced in this run were intracellular, due to low levels of stress factors. Experiments 3 and 5, which contained the highest amount of vanadyl sulfate (5 mg g- 1 cell) and KCI (500 mg g-~cell), resulted in significantly lower levels of alkaloid content as compared to the control run, indicating toxic effects of high levels of stress factors. A typical alkaloid formation profile from experiment 10 (X l = 81, X 2 = 1.45, X 3 = 145 m g g - I dry cell) is depicted in Figure 1. Total alkaloid concentration reached a maximum within the first 12-15 days and then declined. The drop in total alkaloid concentration later on during the experiments may be due to degradation of alkaloids outside the cells, since the pH of the media was nearly 6, as compared to the vacuolar pH of 4.0, where alkaloids are stored intracellularly, The least-square error fit of the experimental data to the aforementioned objective function resulted in the following coefficients for the alkaloid titer function: Y = 0.368 - 3.7 × 10 - 3 S l - 0.0719X2 - 7 ×
10 - 4 X 3 + 1.8 • 1 0 - 5 X ~ +
3 •5
,~ ~ u
0.1
o O
0.0~
t~ vE
0.06
~ t-, -o ~ ~o .~ ,--4 < -,--I
• 10-3X 2
+ 3 × 10 - 7 g ~ + 3 . 5 × 10 -4 X 1 X 2 + 2 . 9 × 10 - 6 X I X 3 q- 7 • 4 ×
762
The projections of the response function on different planes of XI, X2, and X3 are depicted in Figures 2--4. Figure 2 is a plot of alkaloid titer versus elicitor concentration for different values of V a S O 4 concentration at a KCI concentration of 50 mg g- ~dry cell. Lower elicitor (10 mg g-~ dry cell) and V a S O 4 concentrations (0.5 mg g- ~dry cell) resulted in higher alkaloid titers. Final alkaloid titers decreased with increasing elicitor and VaSO4 concentrations. Similar trends can be observed in Figures 3 and 4. Alkaloid titers increased with dec r e a s i n g V a S O 4, KCI, and elicitor concentrations. The optimal set of values of stress factors resulting in maximum final alkaloid titer was determined by using a computer program. A standard curve fitting/error minimizing program was used to determine the coefficients of the objective function and the optimal set of variables. The theoretical optimum was found to be at
10 - 6 X 2 X 3
0.1 ¢
0.04 0.02
o
=
0
3
i
=
6
9 Time
=
=
i
12
15
18
21
(days)
Figure 1 Typical indole alkaloid formation profile (experiment 10)
Enzyme Microb. Technol., 1991, vol. 13, September
Effect of stress on indole alkaloid formation: F. Kargi and P. Potts 0.30
0.30
0.2 5 ~
.
~cz - 50 =g/g
"~
=••
0.25
O
0.20
0.20~ o o ,-4
0.15
ws°4(=~/s)
O.i 0 ~
2.o ____
~
E
0.15
o 4-J
O.IC
-,-I 0
O.
VaS0& = 0 , 5
l:tl~:t t o r
mg/g
(mg/g)
3.5
0.05
.
o
<
i
i
i
i
i
i
i
i
I0
20
30
40
50
60
70
80
Elicitor
05
,--t
i
90 100
<
i
i
r
,
i
i
i
i
50 i00 150 200 250 300 350 400 450 500
(mg/ g DW cells)
KCl (mg/ g DW cells)
Figure 2 Total final alkaloid titer (mg alkaloid 100 mg -1 cells) as a function of elicitor concentration (mg g-~ dry cell wt) at
Figure 4 Total final alkaloid titer (mg alkaloid 100 mg -~ cells) as a function of KCI concentration (mg g-1 dry cell wt) at different
different VaS04 concentrations as predicted by the response function. [KCI] = 50 mg g-~ dry cell w t
elicitor concentrations as predicted by the response function.
elicitor concentrations of 10 mg g-~ dry cell, VaSO 4 concentrations of 0.5 mg g-~ dry cell, and KCI concentrations of 50 mg g- l dry cell, which were the minimum concentrations tested. The final alkaloid titer at this optimal point is expected to be 0.30 mg 100 mg -l dry cell, which is approximately two times higher than that obtained with the control experiment containing no stress factors.
KCI within the experiments listed in Table 1 were 29, 1.45, and 145 mg g- ] dry cell, respectively, resulting in a total alkaloid titer of approximately 0.148 mg alkaloid 100 mg-] dry cell. The response function obtained on the basis of experimental data predicts an even higher alkaloid titer (0.3 mg 100 mg-l dry cell) at the lowest tested levels of stress factors. High concentrations of stress factors were toxic to the cells, resulting in loss of viability. Therefore, low concentrations of the stress factors, i.e. I0 mg g-i < [E] -< 30 mg g-l, 0.5 mg g-l -%<[VaSO4] ~< 1.45 mg g-l, 50 mg g-i < [KC1] -< 145 mg g-l, should be used in suspension culture of C. r o s e u s in order to obtain high cell viabilities, high alkaloid titers, and excretion from cells into the medium.
Conclusion The use of stress factors at low levels stimulated indole alkaloid formation and excretion by C. r o s e u s cells. The optimal concentrations of elicitor, VaSO4, and 0.30
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[VaS04] = 0.5 mg g-~ dry cell wt
Acknowledgement
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.
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This study was supported by National Science Foundation Grant No. ECE-8612936.
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References
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0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Vanadyl Sulfate (mg/g DW cells)
Figure 3 Total final alkaloid titer (mg alkaloid 100 mg -1 cells) as a function of VaS04 concentration (rng g-1 dry cell wt) at different KCI concentrations as predicted by the response function. [Elicitor] = 10 mg g-1 dry cell w t
7 8 9 10 11
Kargi, F., and Rosenberg, M. Biotechnol Prog. 1987, 3, 1-8 Stafford, A., Morris, P. and Fowler, M. Enzyme Microb. Technol. 1986, 8, 578-587 Smith, J. 1., Smart, N. J., Kurz, W. G. W. and Misawa, M. J. Exp. Bot. 1987, 38, 1501-1506 Eilert, U., Constabel, F. and Kurz, W. G. W. J Plant Physiol. 1986, 127, 11-12 Smith, J. I., Smart, N. J., Misawa, M., Kurz, W. G. W., Tullevi, S. G., and DiCosmo, F. Plant Cell Rep. 1987, 6, 142-145 Dixon, R. in Plant Cell Culture: A Practical Approach (Dixon, R., ed.) IRL Press, Oxford, 1985, pp. 1-20 Morris, P. Planta Medica 1986, 121-126 Perry's Chemical Engineering Handbook, 6th ed. (Perry, R. H. and Green, D. W., eds.) 1984, 2.74, 75 Towill, L. and Mazur, P. Can. J. Bot. 1975, 53, 1097-1102 Payne, G. Personal communication, University of Maryland, 1986 Shuler, M. L. Personal Communication, Cornell University, 1986
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