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Development of two stage culture process by optimization of inorganic salts for improving catharanthine production in hairy root cultures of Catharanthus roseus
JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 77, No. I, 57-61. 1994
Development of Two Stage Culture Process by Optimization of Inorganic Salts for Improving Catharanthine Production in Hairy Root Cultures of C a t h a r a n t h u s r o s e u s KYUNG-HEE JUNG, 1,2 SANG-SOO KWAK, I CHA-YONG CHOI, 2 AND JANG RYOL LIU l* Plant Cell Biology Laboratory, Genetic Engineering Research Institute, Korea Institute of Science and Technology, P.O. Box 17, Taedok Science Town, Taejon, 305-606,1 and Biochemical Engineering Major, School o f Engineering, Seoul National University, Seoul, 151-742, 2 Korea Received 1 February 1993/Accepted 3 October 1993 The effects of the concentrations of inorganic salts in Schenk and Hildebrandt (SH) medium on catharanthine production in hairy root cultures of Catharanthus roseus were investigated. The inorganic salt components could be categorized into four groups. The first group (nitrate) supported both the growth of and catharanthine production by hairy roots with incremental increases in the concentration. The second (ammonium and phosphate) yielded contradictory effects with respect to growth and production. The third (borate and molibdate) inhibited both growth and production, while the fourth (potassium iodide, sulfate, and iron) did not exhibit any significant effects. Through optimization of the concentrations of inorganic salts in the medium, a two stage process of hairy root cultures with different media for growth and production was developed which enabled us to enhance the volumetric yield of catharanthine up to 60.5 mg/l. This productivity was 5.4 times higher than that of a one stage culture in the original SH medium.
Hairy root cultures have advantages over cell suspension cultures for the production of useful secondary metabolites due to their genetic and biochemical stability (1). Catharanthus roseus produces commercially important anticancer agents such as vinblastine and vincristine at an extremely low level, compounds which are dimeric indole alkaloids produced by the coupling of two precursors of catharanthine and vindoline. Hairy root cultures of C. roseus producing various indole alkaloids have been reported (2-4). The effects of nitrogen, phosphate, and other inorganic salts in culture medium on indole alkaloid production by C. roseus cell cultures have been extensively studied (5). For hairy root cultures, serially diluted original formula of well known media such as Murashige and Skoog (MS), Gamborg (BS), and Schenk and Hildebrandt (SH), have been widely used (6-8). However, only a few reports have described the effects of the inorganic salt components on the growth of and secondary metabolite production by hairy root cultures. Recently, Toivonen et al. (9) attempted to systematically determine nutrient concentrations necessary for supporting both the growth of and indole alkaloid production by hairy root cultures of C. roseus, however they did not succeed due to their contradictory effects of major nutrient components on the growth and production. The present study describes the effects of inorganic salts in SH medium on catharanthine production and the development of a two stage culture process to improve the catharanthine productivity.
for high yield of catharanthine was used in this study (4). Culture conditions of hairy roots Hairy roots were subcultured every two weeks in hormone-free 1/3 SH (Schenk and Hildebrandt (10) medium consisting of onethird strength of the original inorganic salts, 30g/l sucrose, 1 g/l myo-inositol, 5 mg/1 thiamine, 5 m g / l nicotinic acid, and 0.5 mg/1 pyridoxine) medium. About 0.5 g (fresh weight) of hairy roots were inoculated into 50 ml of medium in a 250 ml Erlenmeyer flask. All cultures were maintained in the dark at 25°C on a gyratory shaker (100 rpm). To investigate the effects of SH medium inorgallic salts, hairy roots were first cultured in 1/4 SH, 1/3 SH, 1/2 SH (SH medium consisting of one-fourth, onethird, and one-half strength of original inorganic salts, respectively), or the original strength SH medium. Next, to determine suitable levels for inorganic salts in the medium, various concentrations of each inorganic salt in 1/3 SH medium were examined with respect to growth of and catharanthine production by hairy roots. From these results, a new growth medium (GM) and production medium (PM) were formulated for a two stage culture. For the two stage culture, after 18 d of culture in GM, the spent medium was replaced with fresh PM. The hairy roots were harvested and washed with double-distilled water before measuring the fresh weight. The dry weight was measured after desiccation at 60°C for 24 h. Analysis of alkaloids The solvent extraction procedures and quantitative analysis of indole alkaloids were performed according to a previous report (4). For the quantitative analysis of catharanthine, the alkaloid extracts were loaded onto a/~-Bondapak Cts column (3.9 × 300 mm) connected to a Spectra-Physics H P L C system. Indole alkaloids were eluted using a solvent mixture of methanol, acetonitrile and 5 mM diammonium hydrogen phosphate (3/4/3, v / v / v , pH 7.3) at a flow rate of 1 m l / min, and detected at 298 nm.
MATERIALS AND M E T H O D S Plant material A hairy root clone LB1 of C. roseus (L.) G. Don cv. Little Bright Eye induced by infection with Agrobacterium rhizogenes strain 15834 and then screened
* Corresponding author. 57
58
JUNG ET AL.
J. FERMENT.BIOENG.,
RESULTS AND DISCUSSION D i l u t i o n effect o f S H i n o r g a n i c salts When the hairy roots were cultured in serially diluted SH media, the growth of hairy roots increased in proportion to the concentration of inorganic salts, up to half the strength of the original SH medium (Fig. 1). However, growth was slightly reduced in the original strength medium, suggesting that the ionic strength of the original SH medium is too high for the growth of hairy roots or that some inorganic salt compounds are inhibitory. Simultaneously, the catharanthine content (catharanthine yield, mg/g dry wt) increased inversely to the inorganic salt strength. In 1/3 SH medium, the catharanthine content and total catharanthine (volumetric yield, mg//) values were the highest (1.75 mg/g dry wt and 15.75 mg/1, respectively). Effect o f nitrate As shown in Fig. 2, the growth and catharanthine content of hairy roots were proportional to the initial concentration of nitrate. The catharanthine content increased with an increase in the concentration of nitrate up to 6.2 raM, but it leveled off at higher concentrations of nitrate. The initial concentration of nitrate (25 mM) was optimal for the growth of hairy roots, while total catharanthine production was maximum at 12.5 mM nitrate. Effect o f a m m o n i u m Likewise, higher concentrations of ammonium elevated the growth of hairy roots within the range of concentrations tested (Fig. 3). The limitation of ammonium reduced the final cell mass of hairy roots, whereas it increased the catharanthine content up to 2.5 mg/g dry wt. The optimal concentration of ammonium for the production of catharanthine was 1.3 mM. Effect o f p h o s p h a t e The increased concentration over the initial phosphate (up to 2.6mM) enhanced the growth of hairy roots, but its growth was inhibited at a higher concentration of phosphate (5 mM) (Fig. 4). It is of interest to point out that even though the hairy roots were cultured in phosphate free medium, they continued to grow to half the level of the maximum growth level in the medium containing 2.6 mM phosphate, and that the catharanthine content increased to its highest level (3.5 mg/g dry wt). The increment of the concentration of phosphate decreased the catharanthine content proportionally.
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FIG. 2. Effects of the initial concentration of nitrate on the growth of and catharanthine production by LBI hairy root cultures. Hairy roots were harvested after 3 weeks. Symbols:O, biomass; O, catharanthine content. Line: - - , total catharanthine. The catharanthine content was highest in the phosphate free medium, whereas total catharanthine was highest at a phosphate level of 0.65 mM. The increased biomass seemingly compensated for the loss of the catharanthine content in the medium containing phosphate. Effects o f o t h e r i n o r g a n i c salts The optimal concentration of CaC12 for catharanthine production was 1.36 mM (Fig. 5A). Although increased concentrations of calcium did not affect the catharanthine content, it sharply reduced the total catharanthine yield because of the decreased growth of hairy roots. The optimal concentration of cobalt (CoCl2) was 1.6 pM (Fig. 5B), which is 4 times higher than that in the original SH medium. Concentrations of borate (H3BO3) above 80pM inhibited both growth and catharanthine production (Fig. 5C). Molibdate (Na2MoOD showed effects similar to borate on growth and catharanthine production (data not shown). Potassium iodide (KI) did not clearly influence either growth or catharanthine production within the range of concentrations tested (Fig. 5D). In the same manner, the other inorganic salt components, including sulfate and iron, did not have a clear effect on catharanthine production (data not shown). All inorganic salt components tested in the present study could be categorized into one of four groups. The first group supported both the growth of and catharanthine
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Dilution ratio of SH inorganic salts FIG. 1. Effectsof the dilution ratio of SH inorganic salts on the growth of and catharanthine production by LBI hairy root cultures. The concentrations of inorganic salts in the media were serially diluted. Hairy roots were harvested after 3 weeks. Symbols: o, biomass; o, catharanthine content.
Ammonium (aM)
FIG. 3. Effectsof the initial concentration of ammonium on the growth of and catharanthine production by LB1 hairy root cultures. Hairy roots were harvested after 3 weeks. Symbols:o, biomass; o, catharanthine content. Line: - - , total catharanthine.
VoL. 77, 1994
TWO STAGE CULTURE OF C. ROSEUS HAIRY ROOTS
59
TABLE 1. Inorganic salt compositions of media for the growth and production of hairy roots Component
°
KNO3 MgSO4 MnSO4 ZnSO4 CuSO4 CaC12 KI
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0
1
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4
5
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Phosphate (mM)
FIG. 4. Effects of the initial concentration of phosphate on the growth of and catharanthine production by LBI hairy root cultures. Hairy roots were harvested after 3 weeks. Symbols: ©, biomass; O, catharanthine content. Line: - - , total catharanthine. p r o d u c t i o n b y hairy roots, the second showed contradictory effects on growth and catharanthine content, the third inhibited b o t h growth and catharanthine p r o d u c t i o n , while the f o u r t h did not exhibit any clear effects within the range o f concentrations tested. Nitrate belonged to the first group, a m m o n i u m and p h o s p h a t e to the second, b o r a t e and m o l i b d a t e to the third, a n d potassium iodide, sulfate, and iron to the fourth. Since the callus culture o f C. r o s e u s by Babcock and Carew (11), improvements in indole alkaloid p r o d u c t i o n b y cell cultures have been made. The l a n d m a r k r e p o r t by Z e n k e t al. (12) showed that an a p p r o p r i a t e selection o f m e d i u m constituents is required to induce indole alkaloid p r o d u c t i o n . This revised m e d i u m , referred to as UZenk's m e d i u m " , contains inorganic salts at roughly half the level o f the original MS m e d i u m . K n o b l o c h a n d Berlin (13) f o u n d that the only prerequisite to induce indole
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NH4H2PO4 KH2PO4 (NH4)2SO4 H3BO3 Na2MoO4 FeSO4 Na2-EDTA
alkaloid p r o d u c t i o n is a high concentration o f sucrose, while the a d d i t i o n o f certain levels o f p h o s p h a t e or nitrogen to the m e d i u m inhibit p r o d u c t i o n . Serially diluted original f o r m u l a such as 1/2 B5 and 1/4 B5 have been widely used for hairy r o o t cultures (6-8). A few reports have described the effects o f inorganic salt c o m p o n e n t s on the growth o f and secondary metabolite p r o d u c t i o n by hairy r o o t cultures. Toivonen et al. (9) investigated the optimization o f concentrations o f sucrose, phosphate, nitrate, and a m m o n i a with respect to the growth o f C. r o s e u s hairy roots. They f o u n d c o n t r a d i c t o r y effects for these nutrients on the growth to the indole alkaloid production, in which the specific p r o d u c t i o n o f alkaloid is the highest at the lowest levels o f all nutrients tested. T w o stage culture o f hairy r o o t s The concentrations
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Concentration ~M) of GM PM 25000 12500 540 540 20 20 1.2 1.2 0.26 0.26 1360 1360 12 12 0.4 1.6 --1300 330 1300 330 27 -0.13 -18 18 18 18
SH, Original Schenk and Hildebrandt medium. GM, PM, Media revised for growth and production, respectively.
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O0 38 125 25O H3BOa (DM) KU (~vt) FIG. 5. Effects of the initial concentration of CaC12 (A), CoCIz (B), H3BO3 (C), and KI (D) on the growth of and catharanthin¢ production by LB1 hairy root cultures. Hairy roots were harvested after 3 weeks. Symbols: ©, biomass; O, catharanthine content. Line: - - , total catharanthine. •
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JUNGET AL.
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TABLE 2. Production of catharanthine in hairy root cultures of C. roseus by optimization of inorganic salt levels Culture condition One stage culture SH 1/3 SH GM PM Two stage culture GM-+PM 1/3 SH~I/3 SH
Biomass (g//)
Catharanthine content (mg/g dry wt)
Culture (d) t i m e
Volumetric (rag//) yield
Relativeproductivitya
8.5 9.5 12.5 7.5
0.75 1.8 1.5 3.4
20 20 20 20
6.38 16.63 18.75 25.5
1.0 2.7 2.9 4.0
19.5 19.7
3.1 1.9
35 35
60.5 37.4
5.4 3.4
1/3 SH, Medium containing one-third diluted inorganic salts of SH. GM, PM, Media revised for growth and production, respectively. Relative productivity was calculated by comparing the volumetric productivity (mg/l/d) for each process to that of SH. and composition of inorganic salts favorable for the growth of hairy roots were not the same as that for catharanthine production. Thus, the SH medium favorable for the growth of (GM) and catharanthine production (PM) by hairy roots, respectively, were revised (Table 1). Hairy root culture growth and catharanthine production in the four different media, SH, 1/3 SH, GM, and PM were compared. The catharanthine content of hairy roots cultured in PM increased up to 3.4 mg/g dry wt, which is about two times higher than that in 1/3 SH medium (Table 2). However, with subsequent culturing of the hairy roots in PM, growth was considerably limited and catharanthine content was not further enhanced. As mentioned above, Toivonen et al. (9) failed to improve indole alkaloid pro3o
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duction by transferring hairy roots cultured in their GM into PM. They found that the growth of hairy roots in production medium continued despite the lack of nutrients, although production did not improve over the control. Since they did not describe in detail the production medium and culture conditions of the two stage process, we could not ascertain the reason for their failure. Thus, we attempted the two stage culture of hairy roots to ensure both maximum growth in GM and maximum production in PM. In Fig. 6, two stage culture from GM to PM was compared to that from 1/3 SH to 1/3 SH. After 18 d of culture in GM, which was during the late exponential phase of hairy root growth, the spent medium was replaced with fresh PM (Fig. 6A). In the GM, the growth rate of hairy roots was faster than that in 1/3 SH, however, catharanthine content decreased slightly. After the hairy roots were transferred from GM to PM, the growth rate slowed, but the catharanthine content increased up to twice that in GM (Fig. 6B). After 35 d of culture, the catharanthine content of the hairy roots had increased up to 3.1 mg/g dry wt. The concentration of biomass reached 19.5 g dry wt/1, and total catharanthine was enhanced to 60.5 m g / l (Fig. 6C). Catharanthine productivity of the two stage culture from GM to PM was about 60% higher than that of 1/3 SH-~-I/3 SH. This finding indicates that the optimization of inorganic salt levels in the medium results in a large increase in indole alkaloid production. Through the optimization of the composition and concentration of inorganic salts in the culture medium, we developed a two stage process for culturing hairy roots which resulted in an enhancement of catharanthine productivity, catharanthine content (4.1 fold) and total productivity (5.4 fold) higher than those for one stage culture in the original SH medium. Research on a scaie-up process is currently under progress. ACKNOWLEDGMENTS This work was supported by Grants-in-Aid (G70320) from the Korean Ministry of Science and Technology. We thank Dr. Hyouk Joung for his critical reading of this manuscript.
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50 REFERENCES
Culture time (d) FIG. 6. Profileof the growth of and catharanthine production by LBI hairy roots in two stage cultures from GM to PM (e) and from 1/3 SH to 1/3 SH (©). Arrows indicate the time of medium change from GM (or 1/3 SH) to PM (or 1/3 SH) media. (A) Profile of hairy root growth; (B) profile of catharanthine content; (C) profile of total catharanthine.
I. Hamfll, J. D., Parr, A. J., Rhodes, M. J. C., Robins, R. J., and
Walton, N.J.: New routes to plant secondary products. Bio/ Technology, 5, 800-804 (1987). 2. Parr, A . J . , Peerles, A . C . J . , Hamii, J . D . , Walton, N.J., Robins, R.J., and Rhodes, M. J. C.: Alkaloid production by transformed root cultures of C. roseus. Plant Cell Reports, 7,
309-312 (1988).
Vog. 77, 1994 3. Brillancean, M. H., David, C., and Tempe, L : Genetic transformation of C. roseus G. Don by Agrobacterium rhizogenes. Plant Cell Reports, 8, 63-66 (1989). 4. Jung, K. H., Kwak, S. S., Klm, S. W., Choi, C. Y., Lee, H., and Llu, J . R . : Improvement of the catharanthine productivity in hairy root cultures of C. roseus by using monosaccharides as a carbon source. Biotechnol. Lett., 14, 695-700 (1992). 5. Ganapathi, B. and Kargi, F.: Recent advances in indole alkaloid production by C. roseus. J. Exp. Bot., 41, 259-267 (1990). 6. Hilton, M. G. and Rhodes, M. J. C.: Growth and hyoscyamine production of hairy root cultures of Datura stramonium in a modified stirred tank reactor. Appl. Microbiol. Biotechnol., 33, 132-138 (1990). 7. Toivonen, L., Ojala, M., and Kanppinen, V.: Indole alkaloid production by hairy root cultures of C. roseus. Biotechnol. Lett., 12, 519-524 0990). 8. Christen, P., Aoki, T., and Shimomura, K.: Characteristics of growth and tropane alkaloid production in H y o s c y a m u s albus hairy roots transformed with A . rhizogenus A4. Plant Cell Reports, 11, 597-600 0992).
TWO STAGE CULTURE OF C. R O S E U S HAIRY ROOTS
61
9. Toivonen, L., Ojala, M., and Kanppinen, V.: Studies on the optimization of growth and indole alkaloid production by hairy root cultures of C. roseus. Biotechnol. Bioeng., 37, 6?3-680
0990. 10. Sehenk, R. V. and Hildebrandt, A. C.: Medium and techniques for induction and growth of monocotyledones cell cultures. Can. J. Bot., 50, 197-204 0972). 11. Babcock, P . A . and Carew, D.P.: Tissue culture of the Apocynaceae. I. Culture requirements and alkaloid analysis. Lloydia, 25, 209-213 (1962). 12. Zenk, M.H., EI-Shagi, H., Arens, H., Stoeckigt, J., Weiler, E.W., and Deus, B.: Formation of the indole alkaloids serpentine and ajmalicine in cell suspension cultures of C. roseus, p. 27--43. In Barz, W., Reinhard, E., and Zenk, M . H . (ed.), Plant tissue culture and its biological application. SpringerVerlag, New York (1977). 13. Knobloeh, K. H. and Berlin, J.: Influence of medium composition on the formation of secondary compounds in cell suspension cultures of C. roseus (L.) G. Don. Z. Naturforch., 35, 551-556 (1980).
Development of Two Stage Culture Process by Optimization of Inorganic Salts for Improving Catharanthine Production in Hairy Root Cultures of C a t h a r a n t h u s r o s e u s KYUNG-HEE JUNG, 1,2 SANG-SOO KWAK, I CHA-YONG CHOI, 2 AND JANG RYOL LIU l* Plant Cell Biology Laboratory, Genetic Engineering Research Institute, Korea Institute of Science and Technology, P.O. Box 17, Taedok Science Town, Taejon, 305-606,1 and Biochemical Engineering Major, School o f Engineering, Seoul National University, Seoul, 151-742, 2 Korea Received 1 February 1993/Accepted 3 October 1993 The effects of the concentrations of inorganic salts in Schenk and Hildebrandt (SH) medium on catharanthine production in hairy root cultures of Catharanthus roseus were investigated. The inorganic salt components could be categorized into four groups. The first group (nitrate) supported both the growth of and catharanthine production by hairy roots with incremental increases in the concentration. The second (ammonium and phosphate) yielded contradictory effects with respect to growth and production. The third (borate and molibdate) inhibited both growth and production, while the fourth (potassium iodide, sulfate, and iron) did not exhibit any significant effects. Through optimization of the concentrations of inorganic salts in the medium, a two stage process of hairy root cultures with different media for growth and production was developed which enabled us to enhance the volumetric yield of catharanthine up to 60.5 mg/l. This productivity was 5.4 times higher than that of a one stage culture in the original SH medium.
Hairy root cultures have advantages over cell suspension cultures for the production of useful secondary metabolites due to their genetic and biochemical stability (1). Catharanthus roseus produces commercially important anticancer agents such as vinblastine and vincristine at an extremely low level, compounds which are dimeric indole alkaloids produced by the coupling of two precursors of catharanthine and vindoline. Hairy root cultures of C. roseus producing various indole alkaloids have been reported (2-4). The effects of nitrogen, phosphate, and other inorganic salts in culture medium on indole alkaloid production by C. roseus cell cultures have been extensively studied (5). For hairy root cultures, serially diluted original formula of well known media such as Murashige and Skoog (MS), Gamborg (BS), and Schenk and Hildebrandt (SH), have been widely used (6-8). However, only a few reports have described the effects of the inorganic salt components on the growth of and secondary metabolite production by hairy root cultures. Recently, Toivonen et al. (9) attempted to systematically determine nutrient concentrations necessary for supporting both the growth of and indole alkaloid production by hairy root cultures of C. roseus, however they did not succeed due to their contradictory effects of major nutrient components on the growth and production. The present study describes the effects of inorganic salts in SH medium on catharanthine production and the development of a two stage culture process to improve the catharanthine productivity.
for high yield of catharanthine was used in this study (4). Culture conditions of hairy roots Hairy roots were subcultured every two weeks in hormone-free 1/3 SH (Schenk and Hildebrandt (10) medium consisting of onethird strength of the original inorganic salts, 30g/l sucrose, 1 g/l myo-inositol, 5 mg/1 thiamine, 5 m g / l nicotinic acid, and 0.5 mg/1 pyridoxine) medium. About 0.5 g (fresh weight) of hairy roots were inoculated into 50 ml of medium in a 250 ml Erlenmeyer flask. All cultures were maintained in the dark at 25°C on a gyratory shaker (100 rpm). To investigate the effects of SH medium inorgallic salts, hairy roots were first cultured in 1/4 SH, 1/3 SH, 1/2 SH (SH medium consisting of one-fourth, onethird, and one-half strength of original inorganic salts, respectively), or the original strength SH medium. Next, to determine suitable levels for inorganic salts in the medium, various concentrations of each inorganic salt in 1/3 SH medium were examined with respect to growth of and catharanthine production by hairy roots. From these results, a new growth medium (GM) and production medium (PM) were formulated for a two stage culture. For the two stage culture, after 18 d of culture in GM, the spent medium was replaced with fresh PM. The hairy roots were harvested and washed with double-distilled water before measuring the fresh weight. The dry weight was measured after desiccation at 60°C for 24 h. Analysis of alkaloids The solvent extraction procedures and quantitative analysis of indole alkaloids were performed according to a previous report (4). For the quantitative analysis of catharanthine, the alkaloid extracts were loaded onto a/~-Bondapak Cts column (3.9 × 300 mm) connected to a Spectra-Physics H P L C system. Indole alkaloids were eluted using a solvent mixture of methanol, acetonitrile and 5 mM diammonium hydrogen phosphate (3/4/3, v / v / v , pH 7.3) at a flow rate of 1 m l / min, and detected at 298 nm.
MATERIALS AND M E T H O D S Plant material A hairy root clone LB1 of C. roseus (L.) G. Don cv. Little Bright Eye induced by infection with Agrobacterium rhizogenes strain 15834 and then screened
* Corresponding author. 57
58
JUNG ET AL.
J. FERMENT.BIOENG.,
RESULTS AND DISCUSSION D i l u t i o n effect o f S H i n o r g a n i c salts When the hairy roots were cultured in serially diluted SH media, the growth of hairy roots increased in proportion to the concentration of inorganic salts, up to half the strength of the original SH medium (Fig. 1). However, growth was slightly reduced in the original strength medium, suggesting that the ionic strength of the original SH medium is too high for the growth of hairy roots or that some inorganic salt compounds are inhibitory. Simultaneously, the catharanthine content (catharanthine yield, mg/g dry wt) increased inversely to the inorganic salt strength. In 1/3 SH medium, the catharanthine content and total catharanthine (volumetric yield, mg//) values were the highest (1.75 mg/g dry wt and 15.75 mg/1, respectively). Effect o f nitrate As shown in Fig. 2, the growth and catharanthine content of hairy roots were proportional to the initial concentration of nitrate. The catharanthine content increased with an increase in the concentration of nitrate up to 6.2 raM, but it leveled off at higher concentrations of nitrate. The initial concentration of nitrate (25 mM) was optimal for the growth of hairy roots, while total catharanthine production was maximum at 12.5 mM nitrate. Effect o f a m m o n i u m Likewise, higher concentrations of ammonium elevated the growth of hairy roots within the range of concentrations tested (Fig. 3). The limitation of ammonium reduced the final cell mass of hairy roots, whereas it increased the catharanthine content up to 2.5 mg/g dry wt. The optimal concentration of ammonium for the production of catharanthine was 1.3 mM. Effect o f p h o s p h a t e The increased concentration over the initial phosphate (up to 2.6mM) enhanced the growth of hairy roots, but its growth was inhibited at a higher concentration of phosphate (5 mM) (Fig. 4). It is of interest to point out that even though the hairy roots were cultured in phosphate free medium, they continued to grow to half the level of the maximum growth level in the medium containing 2.6 mM phosphate, and that the catharanthine content increased to its highest level (3.5 mg/g dry wt). The increment of the concentration of phosphate decreased the catharanthine content proportionally.
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FIG. 2. Effects of the initial concentration of nitrate on the growth of and catharanthine production by LBI hairy root cultures. Hairy roots were harvested after 3 weeks. Symbols:O, biomass; O, catharanthine content. Line: - - , total catharanthine. The catharanthine content was highest in the phosphate free medium, whereas total catharanthine was highest at a phosphate level of 0.65 mM. The increased biomass seemingly compensated for the loss of the catharanthine content in the medium containing phosphate. Effects o f o t h e r i n o r g a n i c salts The optimal concentration of CaC12 for catharanthine production was 1.36 mM (Fig. 5A). Although increased concentrations of calcium did not affect the catharanthine content, it sharply reduced the total catharanthine yield because of the decreased growth of hairy roots. The optimal concentration of cobalt (CoCl2) was 1.6 pM (Fig. 5B), which is 4 times higher than that in the original SH medium. Concentrations of borate (H3BO3) above 80pM inhibited both growth and catharanthine production (Fig. 5C). Molibdate (Na2MoOD showed effects similar to borate on growth and catharanthine production (data not shown). Potassium iodide (KI) did not clearly influence either growth or catharanthine production within the range of concentrations tested (Fig. 5D). In the same manner, the other inorganic salt components, including sulfate and iron, did not have a clear effect on catharanthine production (data not shown). All inorganic salt components tested in the present study could be categorized into one of four groups. The first group supported both the growth of and catharanthine
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1
2
3
4
5
6
Dilution ratio of SH inorganic salts FIG. 1. Effectsof the dilution ratio of SH inorganic salts on the growth of and catharanthine production by LBI hairy root cultures. The concentrations of inorganic salts in the media were serially diluted. Hairy roots were harvested after 3 weeks. Symbols: o, biomass; o, catharanthine content.
Ammonium (aM)
FIG. 3. Effectsof the initial concentration of ammonium on the growth of and catharanthine production by LB1 hairy root cultures. Hairy roots were harvested after 3 weeks. Symbols:o, biomass; o, catharanthine content. Line: - - , total catharanthine.
VoL. 77, 1994
TWO STAGE CULTURE OF C. ROSEUS HAIRY ROOTS
59
TABLE 1. Inorganic salt compositions of media for the growth and production of hairy roots Component
°
KNO3 MgSO4 MnSO4 ZnSO4 CuSO4 CaC12 KI
'"
0
1
2
3
4
5
CoCl 2
6
Phosphate (mM)
FIG. 4. Effects of the initial concentration of phosphate on the growth of and catharanthine production by LBI hairy root cultures. Hairy roots were harvested after 3 weeks. Symbols: ©, biomass; O, catharanthine content. Line: - - , total catharanthine. p r o d u c t i o n b y hairy roots, the second showed contradictory effects on growth and catharanthine content, the third inhibited b o t h growth and catharanthine p r o d u c t i o n , while the f o u r t h did not exhibit any clear effects within the range o f concentrations tested. Nitrate belonged to the first group, a m m o n i u m and p h o s p h a t e to the second, b o r a t e and m o l i b d a t e to the third, a n d potassium iodide, sulfate, and iron to the fourth. Since the callus culture o f C. r o s e u s by Babcock and Carew (11), improvements in indole alkaloid p r o d u c t i o n b y cell cultures have been made. The l a n d m a r k r e p o r t by Z e n k e t al. (12) showed that an a p p r o p r i a t e selection o f m e d i u m constituents is required to induce indole alkaloid p r o d u c t i o n . This revised m e d i u m , referred to as UZenk's m e d i u m " , contains inorganic salts at roughly half the level o f the original MS m e d i u m . K n o b l o c h a n d Berlin (13) f o u n d that the only prerequisite to induce indole
~
12 ~"
9
U
o
0
,
-
NH4H2PO4 KH2PO4 (NH4)2SO4 H3BO3 Na2MoO4 FeSO4 Na2-EDTA
alkaloid p r o d u c t i o n is a high concentration o f sucrose, while the a d d i t i o n o f certain levels o f p h o s p h a t e or nitrogen to the m e d i u m inhibit p r o d u c t i o n . Serially diluted original f o r m u l a such as 1/2 B5 and 1/4 B5 have been widely used for hairy r o o t cultures (6-8). A few reports have described the effects o f inorganic salt c o m p o n e n t s on the growth o f and secondary metabolite p r o d u c t i o n by hairy r o o t cultures. Toivonen et al. (9) investigated the optimization o f concentrations o f sucrose, phosphate, nitrate, and a m m o n i a with respect to the growth o f C. r o s e u s hairy roots. They f o u n d c o n t r a d i c t o r y effects for these nutrients on the growth to the indole alkaloid production, in which the specific p r o d u c t i o n o f alkaloid is the highest at the lowest levels o f all nutrients tested. T w o stage culture o f hairy r o o t s The concentrations
9 :-
,o
-
,
-
3
2
q
-
,
4
-
$
,
6
-
7
_L,,
o
°
o
i
CaCI2 (mM)
i'
0
i
~
4
cocl2 ( ~ ) 20
12
6-
Concentration ~M) of GM PM 25000 12500 540 540 20 20 1.2 1.2 0.26 0.26 1360 1360 12 12 0.4 1.6 --1300 330 1300 330 27 -0.13 -18 18 18 18
SH, Original Schenk and Hildebrandt medium. GM, PM, Media revised for growth and production, respectively.
!
A
,
1
SH 25000 1620 59 3.5 0.8 1360 6 0.4 2600 --80 0.4 54 54
12 ~(~
C
16
~
12
_~
D
9 2.0
6 A
w
4
~
°
~
_L 1.0
W
v
s o
-~
O0 38 125 25O H3BOa (DM) KU (~vt) FIG. 5. Effects of the initial concentration of CaC12 (A), CoCIz (B), H3BO3 (C), and KI (D) on the growth of and catharanthin¢ production by LB1 hairy root cultures. Hairy roots were harvested after 3 weeks. Symbols: ©, biomass; O, catharanthine content. Line: - - , total catharanthine. •
m
-
m
-
u
-
,
-
60
JUNGET AL.
J. FEINT. Blom~o.,
TABLE 2. Production of catharanthine in hairy root cultures of C. roseus by optimization of inorganic salt levels Culture condition One stage culture SH 1/3 SH GM PM Two stage culture GM-+PM 1/3 SH~I/3 SH
Biomass (g//)
Catharanthine content (mg/g dry wt)
Culture (d) t i m e
Volumetric (rag//) yield
Relativeproductivitya
8.5 9.5 12.5 7.5
0.75 1.8 1.5 3.4
20 20 20 20
6.38 16.63 18.75 25.5
1.0 2.7 2.9 4.0
19.5 19.7
3.1 1.9
35 35
60.5 37.4
5.4 3.4
1/3 SH, Medium containing one-third diluted inorganic salts of SH. GM, PM, Media revised for growth and production, respectively. Relative productivity was calculated by comparing the volumetric productivity (mg/l/d) for each process to that of SH. and composition of inorganic salts favorable for the growth of hairy roots were not the same as that for catharanthine production. Thus, the SH medium favorable for the growth of (GM) and catharanthine production (PM) by hairy roots, respectively, were revised (Table 1). Hairy root culture growth and catharanthine production in the four different media, SH, 1/3 SH, GM, and PM were compared. The catharanthine content of hairy roots cultured in PM increased up to 3.4 mg/g dry wt, which is about two times higher than that in 1/3 SH medium (Table 2). However, with subsequent culturing of the hairy roots in PM, growth was considerably limited and catharanthine content was not further enhanced. As mentioned above, Toivonen et al. (9) failed to improve indole alkaloid pro3o
J
A
y
1o o
i 0
c
duction by transferring hairy roots cultured in their GM into PM. They found that the growth of hairy roots in production medium continued despite the lack of nutrients, although production did not improve over the control. Since they did not describe in detail the production medium and culture conditions of the two stage process, we could not ascertain the reason for their failure. Thus, we attempted the two stage culture of hairy roots to ensure both maximum growth in GM and maximum production in PM. In Fig. 6, two stage culture from GM to PM was compared to that from 1/3 SH to 1/3 SH. After 18 d of culture in GM, which was during the late exponential phase of hairy root growth, the spent medium was replaced with fresh PM (Fig. 6A). In the GM, the growth rate of hairy roots was faster than that in 1/3 SH, however, catharanthine content decreased slightly. After the hairy roots were transferred from GM to PM, the growth rate slowed, but the catharanthine content increased up to twice that in GM (Fig. 6B). After 35 d of culture, the catharanthine content of the hairy roots had increased up to 3.1 mg/g dry wt. The concentration of biomass reached 19.5 g dry wt/1, and total catharanthine was enhanced to 60.5 m g / l (Fig. 6C). Catharanthine productivity of the two stage culture from GM to PM was about 60% higher than that of 1/3 SH-~-I/3 SH. This finding indicates that the optimization of inorganic salt levels in the medium results in a large increase in indole alkaloid production. Through the optimization of the composition and concentration of inorganic salts in the culture medium, we developed a two stage process for culturing hairy roots which resulted in an enhancement of catharanthine productivity, catharanthine content (4.1 fold) and total productivity (5.4 fold) higher than those for one stage culture in the original SH medium. Research on a scaie-up process is currently under progress. ACKNOWLEDGMENTS This work was supported by Grants-in-Aid (G70320) from the Korean Ministry of Science and Technology. We thank Dr. Hyouk Joung for his critical reading of this manuscript.
2o
o
lb
A
50 REFERENCES
Culture time (d) FIG. 6. Profileof the growth of and catharanthine production by LBI hairy roots in two stage cultures from GM to PM (e) and from 1/3 SH to 1/3 SH (©). Arrows indicate the time of medium change from GM (or 1/3 SH) to PM (or 1/3 SH) media. (A) Profile of hairy root growth; (B) profile of catharanthine content; (C) profile of total catharanthine.
I. Hamfll, J. D., Parr, A. J., Rhodes, M. J. C., Robins, R. J., and
Walton, N.J.: New routes to plant secondary products. Bio/ Technology, 5, 800-804 (1987). 2. Parr, A . J . , Peerles, A . C . J . , Hamii, J . D . , Walton, N.J., Robins, R.J., and Rhodes, M. J. C.: Alkaloid production by transformed root cultures of C. roseus. Plant Cell Reports, 7,
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TWO STAGE CULTURE OF C. R O S E U S HAIRY ROOTS
61
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