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 .

The kinetics of biomass accumulation, nutrient uptake and taxol production of Taxus baccata cell suspensions were examined in three bioreactor configurations, viz. 250-mL Erienmeyerflasks, 1-L working volume pneumatically mixed (PMB), and stirred tank (STB) bioreactors. Qualitatively similar kinetics were observed in all three bioreactor types. Biomass accumulation and specific nutrient uptake rates exhibited biphasic characteristics. Carbohydrate uptake and biomass accumulation substantially ceased when phosphate was depleted from the medium. Phosphate was identified as a possible growth-limiting nutrient. Taxol accumulated exclusively in the second phase of growth. A maximum taxol concentration of 1.5 mg/L was obtained in the PMB which was fivefold greater than that obtained in the Erienmeyer flasks and the STB, but the relative kinetics of taxol production was the same in all three reactor types. Biomass yields were calculated from the kinetic data and a stoichiometry for biomass formation was evaluated. The similarity of kinetics in the three bioreactor configurations suggests that taxol production by T. baccata cell suspensions is amenable to scateup. (c) 1995 John Wiley & Sons, Inc.

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