Taxol production in suspension cultures of Taxus baccata

Plant Cell, llssue and Organ Culture 44: 95-102, 1996. 95 © 1996 Kluwer Academic Publishers. Printed in the Netherlands.

Taxol production in suspension cultures of Taxus baccata

Thomas J. Hirasuna 1,3, Luis J. Pestchanker 2, Venkatesh Srinivasan 1 & Michael L. Shuler l'* 1 School of Chemical Engineering, Cornell University, Ithaca, NY 14853-5201; 2 Catedra de Fisiologia Chacabuco y Pedernera, Universidad, Nacional de San Luis, San Luis, Argentina (3present address: The International Food Network, 95 Brown Rd., Ithaca, NY 14850-1257) (* requests for offprints)

Received 8 November 1994; accepted in revised form 25 October 1995

Key words: Excretion, natural products, plant tissue culture, secondary metabolites, taxanes

Abstract

The response of Taxus baccata (PC2) to basic manipulations of culture conditions is described. Suspension cultures of Taxus baccata (PC2) were maintained at 25 °C on a modified B5 medium with two-week transfers. Under these conditions, no taxol® is formed. However, if the cells are left in the same medium for 7 or more additional days, taxol is produced and released (ca. 90%) into the extracellular medium. Levels as high as 13 mg 1-1 extracellular taxol were achieved in shake flask cultures and taxol was the primary taxane formed representing between 50 and 80% of total taxane in the medium. The cells are sensitive to changes in culture conditions and cultures cycle through periods of high (13 mg 1-1) and low (<0.1 mg 1-1) levels of taxol production during extended culture. Picloram was the most effective of the auxins tested with respect to cell growth but it suppressed taxol production. Addition of fructose to moderately-productive cultures (ca. 4 mg 1-1) improved taxol production, but cultures in a high producing state did not respond. Glucose suppressed taxane production. Two isoprenoids (geraniol and pinene) had a modest effect on taxol production when added to cultures at 10 mg 1-1 .

Introduction

Taxol is a complex diterpenoid from the Pacific yew tree (Taxus brevifolia) that was recently approved for treatment against ovarian and breast cancers and shows promise against some other cancers (Holmes et al., 1995) and malaria (Pouvelle et al., 1994). Kingston (1994) summarizes the history of taxol, describes the chemistry and structure-activity relationships of the taxol molecule, and summarizes the alternatives for taxol supply.

The supply problems with taxol exemplify the need for large scale processes to make natural products so that clinical studies can proceed unimpeded (Cragg et al., 1993). Although the crisis in the supply of taxol has eased and it is commercially available, control- lable alternative sources are still needed. One alterna-

®Taxol is a registered trademark of Bristol Meyer Squibb for paclitaxel

tive source is taxol from plant cell culture (Christen et al., 1991; Fett-Neto et al., 1992; Fett-Neto et al., 1993; Fett-Neto et al., 1994a; Fett-Neto et al., 1994b; Srinivasan et al., 1995; Shuler, 1994; Mirjalili & Lin- den, 1995; Wickremesinke & Arteca, 1993 and 1994). Although the ability of plant cell culture to produce taxol is well-established, the current levels of productivity reported in the literature are too low to be useful as a practical alternative source of taxol. New cell lines and culture conditions may yield higher productivity.

Our objective has been to gain a broad-based under- standing of how the manipulation of nutritional and environmental conditions affect the ability of a Taxus baccata cell line to grow and to produce taxol. In this paper, we report on a cell line and culture conditions that can result in synthesis at moderate rates with substantial release of taxol into the extracellular medium.

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Materials and methods

Cultures

Suspension cultures of T. baccata were originated and supplied to us by Dr. V. Bringi (Phyton Catalytic, Itha- ca, NY) in December 1991. Cells were designated as "PC2." Cells were maintained as callus and suspension cultures using conventional plant tissue culture techniques (i.e., Evans et al., 1983).

Medium

Cell cultures were maintained on a modified B5 medium (Gamborg et al., 1968). A sucrose level of 20 g 1-l was used in most experiments. Plant growth regulators used were 5.0 #M 1-naphthaleneacetic acid (NAA) and 0.01 #M 6-benzylaminopurine (BA). Essential to the growth of the cells was the post-autoclave addition of filter-sterilized glutamine at a level of 2.0 mM. As suggested by Fett-Neto et al. (1992), the use of antioxidants is important to prevent the accumulation of toxic phenolic oxidation products. Filter-sterilized ascorbic acid was added at a level of 50 mg 1- l also after autoclaving. The initial pH of the medium was adjusted to 5.5.

Culture conditions

The cultures were maintained in 250 ml erlenmeyer flasks with silicone/foam closures (Bellco, Vineland, NJ). Cells were kept in the dark. Temperature was maintained at 25 °C; shaker speed was set at 120 rpm. Routine subculture was performed every 14 days; 20 ml of suspension was added to 50 ml of fresh medium using a wide-bore sterile pipette. This procedure yields an initial fresh weight of 18 -4- 3 g 1-1 and by day 14 fresh weight is 60 to 75 g 1-1.

Sample preparation for taxane analysis: Culture broth was filtered using Miracloth (Calbiochem, La Jolla, CA) to remove cells. Cells were washed with deionized water and samples were disintegrated using a glass homogenizer and extracted with methanol. Sam- pies were then centrifuged and the supernatant ill- tered with 0.2 #m polymeric filters (Gelman Acrodisc Tuffryn HT). Medium samples for determination of taxanes were filtered and extracted with methylene chloride (25 ml : 25 ml) in a separatory funnel. The water phase was removed, the methylene chloride phase was evaporated using a rotovap, and the residue

was resuspended in methanol and filtered with 0.2/~m polymeric filters prior to analysis.

Taxane analysis

Determination of taxol levels and other selected taxanes was done by HPLC. Samples were first screened for taxol production using reversed-phase HPLC (Spectra Physics). A Curosil G column (Phenomenex, Torrance, CA), which is a proprietary modified PFP- type column, was used. Isocratic operation with a mobile phase of 45% acteonitrile: 55% 12.5 mM sodi- um acetate buffer (adjusted to pH 4.0 with acetic acid), was used. Flow was 1.0 ml min -1, and the column was maintained at 30 °C with a column heater. A UV/Visible light detector was set at a wavelength of 227 nm. Standards, supplied by Hauser Chemical and NCI, gave the following retention times: 10-deacetyl baccatin III (4.6 min), baccatin III (6.2 min), 7-xylosyl- 10-deacetyl taxol (7.4 min), 10-deacetyl taxol (9.7 min), cephalomannine (12.9 min), 7-epi-10-deacetyl taxol (14.0 min), taxol (14.8 min) and 7-epi-taxol (22.3 min). Retention time is not adequate for identification as false positive values for taxol can be observed due to co-eluting compounds. Samples showing significant taxol levels were then sent to other laboratories for confirmation of presumption values. These laboratories had HPLC systems equipped with diode array so the UV absorption spectrum could be scanned; deviations from the known adsorption scan indicated contamina- tion with co-elutants. Procedures varied slightly from laboratory to laboratory. The procedures used by the USDA laboratory (Ketchum & Gibson, 1993) and by Hauser Chemical (Riehheimer et al., 1992) have been published, while the procedure at Phyton, Inc. is proprietary. Hauser Chemical, Inc. has been a primary sup- plier of taxol for clinical use. The authenticity of taxol production from cell cultures has been confirmed by Phyton, Inc. using tandem mass spectroscopy (Bringi, personal communication). All data reported here has been analyzed with the screening method and inde- pendently by at least one of the above organizations. Samples analyzed by all groups differed by less than 20% and usually differences were much smaller.

Assays

Routine assays of samples included measurement of fresh weight (filtered on Miracloth) and taxol and taxane level in medium (as determined above). Other test performed (although not on a routine basis) included

cell viability by fluorescien diacetate (FDA) staining, cell dry weight (using lyophilization for 24 h), taxol/taxane in cell fraction (intracellular levels), DNA (assay kit from Pierce), lactate dehydrogenease (LDH, assay kit from Sigma), and carbohydrate analysis of medium (assay kit for glucose/fructose/sucrose from B oehringer-Mannheim).

Experimental

All of the reported data are averages from at least two independent experiments conducted in parallel with each other. Because the culture would cycle through periods of low and high productivity, it was difficult to reproduce quantitative results from time to time, but the treatment effect could be reproduced. Thus, response to some experiments are given with respect to the intrinsic productivity displayed in the control flasks. Cultures yielding less than 1 mg 1- l were "low" producers, while "moderate" producers yield about 1 to 6 mg I-1 taxol and "high" producing cultures had the intrinsic capacity to produce about 10 mg 1-1 taxol in control cultures. When the culture was in a low producing phase, all control cultures would yield low values, while in a high producing phase all control cultures produced high levels. Results at any time were reproducible (4- 20%) in independent control flasks, but the levels at times two or three months apart could be different.

Results and discussion

Characteristics of callus and suspension culture

PC2 was received as a suspension. 'Healthy' suspension cultures are an off-white color with moderate cell clusters. Any browning of the cells or reddening of the cells or the medium indicates a problem with the culture. We found that the suspensions transferred well at 14 day intervals--for a 250 ml flask size: 20 ml of inoculum into 50 ml fresh medium. If the inoculum were too low, the cells would turn a light red color. The red color usually an indicated a problem with the culture, and was also noted by Wickremesinhe & Arteca (1994). Figure 1 is an example of batch flask growth. The mean doubling time of these cultures for the 26 day period was approximately 8 days based on fresh weight. In the second growth phase the minimum doubling time (maximum specific growth rate) was in the

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200 "] 5.00 180 1 0 Fr.kvl(lllrl I ~4.50

,4o

i ,,o °o 1:::Oo 100 ~-

60 -~ ! .50 40 -:] 1.00 20 "10.50

Oo . . . . . . . . - . . . . . . . . . . , ,-oo.oo

Daye Fig. 1. Typical kinetics curve for growth and taxol production of Taxus baccata PC2 suspension culture.

range of 4-5 days. The final dry weight of the culture is approximately 10 to 11 g 1-1.

The callus, on the other hand, stays a light brown color, often releasing a red compound into the solid medium. Transfers were performed at 21 day intervals. Measured average doubling time for dark grown callus was in the range of 13-23 days (based on initial and final weights). Usually if the callus culture grew at a slower rate, it did not survive. Early in the study, it was determined that the callus cultures and suspension cultures of PC2 could not be interconvert- ed easily from one type of culture to the other, unlike some other Taxus lines which are maintained in our lab. Thus, sufficient amounts of suspension cultures were always maintained since we could not depend on the callus cultures as a backup for the suspensions. Both callus and suspensions grow much better in the dark. No advantages were obtained either in cell growth or taxol production by applying light to the cultures. Dou- bling time of callus grown under continuous direct white light exceeded 50 days. Under prolonged expo- sure to continuous room light, the PC2 suspension cultures turned a lime-green color. Similar greening results were obtained from a taxol producing T. cusp- /data cell line. No taxol was produced under the light conditions.

Growth kinetics and taxol production

Figure 1 illustrates the profile of a typical shake flask experiment. In this experiment, cells displayed a char- acteristic, slightly biphasic pattern for the increase of fresh weight over time. taxol data are averages of measurements from two independent flasks. There is little fresh weight accumulation between day 11 and day 15. Note that these cells were regimented to a regu-

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lar 14-day transfer cycle. If the cells are held within culture past the transfer date, taxol release and a second phase of fresh weight accumulation begin nearly simultaneously. Sucrose is hydrolyzed to glucose and fructose during the first 10 days followed by preferential consumption of glucose (Srinivasan et al., 1995). For Taxus × media cv. Hicksii, Wickremesinhe & Arte- ca (1994) also noticed the consumption of glucose first and the preferential consumption of fructose in the lat- ter part of the cell culture period.

In this experiment with PC2, the maximum instan- taneous rate of taxol accumulation in the extracellular medium was 0.96 mg 1-1 d -1 (between day 21 and 26). The average value for taxol production over the complete batch cycle (3.9 mg 1-1 in 26 days) is 0.15 mg 1-1d - l . This rate is substantially higher than the value of about 0.15 mg 1-1 after 38 days (0.0039 mg 1-1d -1) observed by Fett-Neto et al. (1994a) for a suspension culture of T. cuspidata. In this example, we ended the culture at 26 days; however, based on later sampling of other batches, the taxol level was not expected to increase upon further incubation. For example, another set of cultures was sampled over a series of days bracketing the 26 day endpoint. The taxol in medium is presented below. Each sample is from a different flask; all the flasks were inoculated from the same culture and maintained together on one shaker.

Number of days 20 22 26 28 32 36

taxol(mg1-1 ) 1.2 1.6 3.2 3.2 3.2 2.8

The ratio of taxol made to dry weight accumulated is 0.04% for PC2 (at 4 mg 1-1 production) which com- pares to 0.0073% in the dried bark of T. brevifolia and 0.012% for the intracellular component of T. cuspidata cultures (Fett-Neto et al., 1992). In PC2, intracellular taxol levels are always a small fraction of the total taxol (< 10%). In comparison, T. cuspidata (Fett-Neto et al., 1994a) report 34% in the medium. Wickremesinhe & Arteca (1993) report less than 10% of the taxol in the medium. The hypothesis that taxol is released by viable cells rather than through cell lysis in PC2 is supported by several lines of evidence. The first is that staining by fluorescin diacetate (FDA) indicates that a high fraction of the cells are viable even at later stages of the culture. The second is that dry weight and fresh weight both accumulate at substantial rates even during taxol release. The third indication is that the level of LDH in the medium at day 15 and increases by a factor similar to the rate of dry weight accumulation.

A fourth indication is that extracellular levels of DNA in producing and non-producing cells are essentially identical indicating low levels of lysis.

Stability of taxol production

Over a three year period the PC2 culture has cycled from low to moderate to low to moderate to low to high to low producer of taxol (based on reproducible measurements of taxol levels in multiple control flasks). During the same period, the cultures have remained vir- tually unchanged with respect to growth rate, growth pattern, color, or degree of aggregation. As Kleinig (1989) notes, "Geranylgeranyl diphosphate serves as a key branching point in plastid isoprenoid metabolism. Delicate mechanisms of regulation and channeling to subsequent pathways must be postulated. These mechanisms are easily disturbed...". Thus, it is expected that taxol production may be very sensitive to slight varia- tions in culture conditions. Due to this sensitivity cell maintenance conditions, especially initial cell density, length of subculture interval, and temperature, must be maintained as precisely as possible.

As an illustration of the importance of maintaining transfer schedule consider the following observation. The PC2 cell line which was producing approximately 12 mg 1-1 extracellular taxol on day 27 was transferred after 27 days in culture instead of the normal biweekly interval. Following transfer, the taxol yields in several subsequent generations (measured on day 28) were only about 1 mg 1-1. In contrast, cells transferred according to the normal schedule retained their high productivity.

The PC2 culture is an aggregate clone which should minimize genetic diversity in the culture, but the culture contains more intrinsic diversity than a true single cell clone. The fact that the loss of productivity was not irreversible is intriguing and suggests an epigenetic- type of instability. Instability of product formation is well-documented in the literature (Deus-Neumann & Zenk, 1984; Sierra et al., 1992; Schripsema & Verpoorte, 1992). As demonstrated by Schripsema & Verpoorte (1992) changes in productivity in totally independent cultures is reproducible (same value at same time), but productivity can change with time showing multiple valleys and peaks. The time-to-time variation is not necessarily a totally random process, but may reflect underlying intracellular dynamics.

Fructose supplementation in T. baccata suspension cultures

Our line of taxol-producing T. baccata displays a biphasic growth curve. One key time point in the growth curve is at 10 days as the culture enters a secondary lag phase in growth. At this point several adjustments to medium composition were attempted. Since the preferential utilization of different carbo- hydrates may have an effect on cell growth and taxol production, carbohydrate supplementation (sucrose, glucose, or fructose) was investigated. With respect to the shape of the growth curve, the addition of 1% (10 g 1 - I ) fructose at day 10 significantly improved fresh weight levels for the T. baccata line in the later stages of the run (15 days and beyond). For example at 24 days, the control culture had a fresh weight of 10.1 1 - l flask - l . Sucrose, glucose, and fructose treatments (10 1 1 - I added at day 10) led to fresh weight values of 11.5 1 - l flask -1, 8.7 1 - l flask - l , and 15.2 1-1 flask, respectively.

For the production of taxanes, there were marked differences seen with the various carbohydrate treatments. Also, there was a difference in response to the sugar addition, dependent upon the intrinsic taxol producing capability of the culture at the time. Table 1 shows the effect of different carbohydrate treatments on taxane production. For the low and moderate producers, the fructose treatment is advantageous for increasing the level of taxol. However for the high producer, the effect of fructose is slightly negative. Glu- cose addition suppresses taxane production. Table 2 summarizes the effect on taxol productivity to the effect of fructose addition to the cultures at Day 10. We have observed similar enhancement in taxol levels for cultures of Taxus cuspidata by fructose addition and depression of taxol levels in response to glucose. Colleagues working with the same T. cuspidata cell line have also observed a similar response (Ketchum, unpublished; Mirjalili & Linden, 1995).

In addition to increased taxol production, there was a shift in the extracellular taxane spectrum. Tables 1 shows measurements for cephalomannine, baccatin HI, and 10-deacetylbaccatin III. Note that for the low and moderate taxol producers, baccatin III and 10-deacetyl baccatin III are produced in slightly higher amounts when fructose is added but the increase is less than for taxol. However, we do not see a similar effect with the high taxol producer. Also in the low taxol producing case, there was at least one new taxane produced upon fructose addition, which did not correspond to known

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taxanes in bark or needle samples (personal communication, D. Bailey, Hauser Chemical).

These results suggest a limiting step in taxol synthesis which is stimulated by the presence of fructose and inhibited by glucose. When taxol is already produced at a high lever, additional fructose is no longer favorable, which suggests a switch to another step as limiting.

Auxin effects on growth of suspension cultures

Several auxins were compared with a suspension cultures of T. baccata. Cultures were maintained in a medium containing 5 #M NAA and transferred into new medium with the auxins being tested: NAA (control), 2,4,5-T, 2,4-D (2,4-Dichlorophenoxyacetic acid), picloram, and no auxin. Both 5 and 10 pM concentra- tions were tested for each auxin. With the exception of 2,4-D, there was little difference caused by the difference of the amount of auxin. Picloram performed the best of the group, with an average 15% increase of cell fresh weight over the NAA control. No auxin was the worst treatment, with an average 58% decrease in fresh weight. 2,4,5-T led to an average decrease of 42%. 5 #M 2,4-D led to a 49% decrease while 10 #M 2,4-D resulted in a 28% decrease in fresh weight. When picloram was applied to a taxol producing T. cuspidata suspension culture, net fresh weight increase over control was consistently over 25%. After several subsequent transfers, the appearance of the culture improved, becoming an off-white color from the pre- vious light tan color. Unfortunately, although growth with picloram is excellent, the capability for taxol production was sharply curtailed dropping to a level of 0.7 mg 1-1 after 28 days (versus a control value of 6 mg 1-1). A shift away from picloram to the original NAA containing medium did not improve taxol production, but this does not preclude the possibility that a better production medium can be found so that the advantages of picloram can still be used in a growth/maintenance medium.

Response to presumptive precursors and regulators

We examined the addition of selected compounds to the suspension cultures of T. baccata to test their effects on the productivity of taxol production. Several of these compounds were selected due to their possible role in the taxol synthetic pathway. Strobel (1992) found that radiolabeled acetate was incorporated uniformly in labeled taxol; therefore acetate might work such as

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Table 1. Effect of sugar supplementation on taxane production in PC2 suspension cultures in different producing states

Intrinsic productivity of unsupplemented cultures

Supplement Taxane level in medium + (mg 1-1)

Taxol Cephalomannine Baccatin III 10-deacetyl baccatin III

Low None (control) 0.031 N.D ++ 0.013 N.D Low Fructose 0.221 0.061 0.24 0.062 Low Glucose N.D N.D. N.D N.D. Low Sucrose 0.011 0.030 0.015 N.D. Moderate None (control) 3.1 0.83 2.0 N.D Moderate Fructose 8.2 1.2 2.1 0.17 Moderate Glucose 0.50 0.34 0.53 N.D Moderate Sucrose 6.0 0.95 2.2 0.10 High None (control) 13. 0.80 3.6 0.87 High Fructose 10. 1.0 4.0 1.10

*All supplements were added 10 days after inoculation and at the level of 10 g 1- I +Measured 26 days after inoculation ++N.D - not detected (<0.01 mg 1- l )

Table 2. Summary of effects of fructose supplementation at day 10 of batch growth cycle on taxol production in T. baccata culture with different intrinsic productivities.

Taxol producing Unsupplemented Fructose % increase in taxol ability + cultures supplementation production

(10g1-1)

Low 0.03 mg 1- l 0.22 mg I- 1 +703% Moderate 3.1 mg 1- l 8.2 mg l- 1 + 165% High 13 mg 1 - l 10 mg 1-1 -23%

+taxol levels measured on day 26

a precursor in taxol production in suspension culture. In PC2 cultures, acetate was added at 0.1, 1.0, 10, and 50 mM levels at day 18 of culture (so that taxol production would be well under way at the time of acetate addition). 50 mM concentration of acetate was detrimental to the cells and turned the cultures brown. Absolute taxol values were low for this set of cultures (0.082 mg 1-1) for control; 1.0 and 10 mM acetate showed an increase in the level of taxol to 0.17 mg 1-1 and 0.12 mg 1-1, respectively. The 50 mM acetate addition experiment showed an increase in 7-epi taxol and some new peaks with a low retention time. Fett-Neto e t al . (1994b) also explored the use of acids (aromatic carboxylic acids and amino acids) to enhance taxol level. They noted a 5-fold increase in taxol yield.

Geranylgeranyl pyrophosphate is believed to be a precursor of taxol (Kingston, 1991). However, since geranylgeranyl pyrophosphate and its biogenetic pre-

cursor geranyl pyrophosphate are unstable in culture medium, we considered geraniol as a presumptive precursor. We also considered pinene as a possible regu- lator of monotorpene synthesis. Pinene is an important end-product in monoterpene biosynthesis. If pinene were to feedback inhibit flow of geranyl pyrospho- sphate to monoterpenes, then more substrate might flow to geranylgeranyl pyrophosphate and diterpenoid synthesis. In preliminary experiments using geraniol and pinene, we noted minor enhancements in taxol production and other taxanes. Table 3 summarizes the results of these experiments. In this set, fructose was added at 12 days after transfer, then 10 or 100 mg 1- i of geraniol or pinene was added 7 days later. In terms of enhancement of taxol level, we see an increase of about 2-fold for both geraniol and pinene addition. Because of the potential problems with insolubility of geraniol and pinene in water, the uptake of geraniol and pinene

Table 3. Effect of geraniol and pinene on taxane production in Taxus baccata PC2 suspension cultures*

Supplement T a x o l Cephalomannine Baccatin III Unknown Unknown (mgl - l ) (mgl - I ) (mg1-1) #1(mg1-1) #2(mgl - l )

Control 1.8 0.86 0.97 2.0 0.89 (no addition) Geraniol 3.3 1.1 0.98 1.9 1.2 (10 mg 1- l) Geraniol 2.9 1.2 0.87 2.1 1.4 (100 mg 1-l) Pinene 4.0 1.5 1.1 2.7 2.3 (10 mg I - l ) Pinene 3.5 1.5 1.1 2.5 2.0 (100 mg 1-1)

* In this experimental set 10 g 1- t fructose was added to all cultures on day 12 after inoculation and geraniol or pinene were added on day 19. Taxane levels were measured on day 32 samples.

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is not insured in those experiments. To improve geraniol and pinene solubil i ty they were added as ethanolic solutions. Geraniol and pinene, when administered as ethanolic solutions, did not have a significant effect on taxane product ion compared to a control culture to which an equal volume of ethanol had been added with all cultures displaying about a 50% reduction in taxane production. Ethanol exerts a negative effect on taxol and taxane production even in very small doses (0.1% v/v) but appears to have no significant impact on cell

growth.

Conclus ions

Suspension cultures of Taxus baccata PC2 can produce and release significant amounts of taxol and other taxanes. Levels of up to 13 mg 1-1 extracellular taxol were achieved by extending the culture period but using the same medium as maintenance. The cultures cycle through periods of high and low levels of taxol production during long term culture. Picloram leads to the best cell growth for all of the auxins tested; however, taxol production decreases. Therefore, N A A is the auxin of choice. Addi t ion of fructose improves taxol yield and alters the spectrum of taxanes produced by low and moderate producers o f taxol but does not improve taxol production in high producing cultures. Addit ion o f geraniol and pinene to moderate producing cultures enhances taxol production by about two-fold. I f ethanol was used as a carrier for geraniol and pinene addition to a high producing cell line, taxane produc-

tion fell, but the decrease was the same as caused by ethanol addition alone.

A c k n o w l e d g e m e n t s

The work was supported in part by NCI Grant Num- ber R01 CA 556138-01 and USDA. We thank Sue Moser, Diana Willard, Mike Kennedy, and Paul Men- sah for their technical assistance. Helpful discussions with Donna Gibson, Ray Ketchum, Dave Bailey, Steve Richheimer, Jim Linden, Noushin Mirjalil i , Eugene Kane, and Bobby Bringi are gratefully acknowledged.

References

Christen AA, Gibson DM & Bland J (1991) Production of taxol or taxol-like compounds in cell culture. U.S. Pat. 5,019,504

Cmgg GM, Schepartz SA, Suffness M & Grever MR (1993) The taxol supply crisis. New NCI policies for handling the large- scale production of novel natural product anticancer and anti-HIV agents. J. Nat. Prod. 56:1657-1668

Dcus-Neumann G & Zenk MH (1984) Instability of indole alkaloid production in Catharanthus roseus cell suspension cultures. Planta Med. 50:427-431

Evans DA, Sharp WR, Ammirato PV & Yamada Y (1983) Handbook of Plant Cell Culture, Vol 1. Techniques for Propagation and Breeding. Macmillan, New York

Fett-Neto AG, DiCosmo F, Reynolds WF & Sakata K (1992) Cell culture of Taxus as a source of the antineoplastic drug taxol and related taxanes. Bio/Technology 10:1572-1575

Fett-Neto AG, Melanson SJ, Sakata K & DiCosmo F (1993) Improved growth and taxol yield in developing calli of Taxus cuspidata by medium composition modification. Bio/Technology 11: 731-734

102

Fett-Neto AG, Zhang WY & DiCosmo F (1994a) Kinetics of taxol production, growth, and nutrient uptake in cell suspensions of Taxus cuspidata. Biotechnol. Bioeng. 44:205-210

Fett-Neto AG, Melanson S J, Nicholson SA, Pennington JJ & DiCos- mo F (1994b) Improved taxol yield by aromatic carboxylic acid and amino acid feeding to cell cultures of Taxus cuspidata. Biotechnol. Bioeng. 44:967-971

Gamborg OL, Miller RA & Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 148-151

Holmes, FA, Kudelka AP, Kavanagh JJ, Huber MH, Ajani JA &: Valero V (1995) Current status of clinical trials with paclitaxel and docetaxel. In: Georg GI, Chen JT Ojima I & Vyas DM Taxane Anticancer Agents: Basic Science and Current Status. ACS Symp. Ser. 583, ACS, Washington, DC

Ketchum REB & Gibson DM (1993) Rapid isocratic reversed phase HPLC of taxanes on new columns developed specifically for taxol analysis. J. Liq. Chromatog. 16:2519-2530

Kleinig H (1989) The role of plastids in isoprenoid biosynthesis. Annu Rev Plant Physiol. Plant Mol. Biol. 40:39-59

Kingston DGI (1994) Taxol: the chemistry and structure-activity relationships of a novel anticancer agent. Trends Biotechnol. 12: 222-227

Mirjalili N & Linden JC (1995) Gas phase composition effects on suspension cultures of Taxus cuspidata. Biotechnol. Bioeng. (In Press)

Pouvelle B, Farley P J, Long CA & Taraschi TF (1994) Taxol arrests the development of blood-stage Plasmodium falciparum in vitro and Plasmodium chabaudi adami in malaria-infected mice. Journal of Clinical Investigation 94(1): 413-417

Richheimer SL, Tinnermeier DM & Timmons DW (1992) High per- formance liquid chromatographic assay of taxol. Analyt. Chem. 64:2323-2326

Schripsema J & Verpoorte R (1992) Search for factors related to the indole alkaloid production in cell suspension cultures of Taber- naemontana divaricata. Planta Med 58: 245-249.

Shuler ML (1994) B ioreactor engineering as an enabling technology to tap biodiversity: The case of taxol. Ann. N.Y. Acad. Sci. 745: 455-461.

Sierra MI, van der Heijden R, van der Leer T & Verpoorte R (1992) Stability of alkaloid production in cell suspension cultures of Tabernaemontana divavicata during long-term subculture. Plant Cell, Tiss. Org. Cult. 28 :59~8

Srinivasan V, Pestchanker L, Moser S, Hirasuna TJ, Taticek RA & Shuler ML (1995) Taxol production in bioreactors: Kinetics of biomass accumulation, nutrient uptake and taxol production by cell suspensions of Taxus baccata. Biotechnol. Bioeng. (in press)

Strobel GA, Stierle A & van Kuijk FJGM (1992) Factors influencing the in vitro production of radiolabeled taxoi by Pacific yew, Taxus brevifolia. Plant Sci. 84P: 65-74

Wickremesinhe ERM & Arteca RN (1993) Establishment of Taxus callus cultures, optimization of growth and confirmation of taxol production. Plant Cell Tiss. Org. Cult. 35:181-193

Wickremesinhe ERM & Arteca RN (1994) Taxus cell suspension cultures: Optimizing growth and production of taxol. J. Plant Physiol. 144:183-188

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Taxol production in suspension cultures of Taxus baccata