secondary metabolic activity and intracellular protein and Cellular Adenosine Triphosphate Content ATP levels. Cells of known fresh weight were extracted in 3 mL of ice-cold 90%dimethylsulfoxide (DMSO)by vortexing for MATERIALS AND METHODS 30 s.Extracts were stored at-70C until further analysis Parallel controls revealed that the loss of adenosine triphos- Cell Line phate (ATP)in cell extracts was in the range of 1-7% Lithospermum erythrorhizon cell line transformed with during this period.This difference has been ignored in the Agrobacterium tumefaciens Ti plasmid was obtained from measurements.Prior to measurement,extracts were thawed Dr.Chang Ryul Liu at the Genetic Research Institute, at 4C and centrifuged at 500g for 10 min.The supernatant Taeduck,Korea. was diluted 100-fold in N-(2-hydroxyethyl)piperazine-N'- 2-ethanesulfonic acid (HEPES)buffer (0.5 mM,pH 7.8) Cell Cultivation and 0.1-mL aliquots were used for ATP determination by luminometry.A Turner TD-20e luminometer (Turner De- Cell suspensions were cultivated as described in our earlier paper.16 Thirteen-day-old cells were transferred (20%in- signs,Mountain View,CA)was used to monitor the emitted luminescence of the ATP coupled luciferin-luciferase enzy- oculum v/v)into production medium (M-918 with 5 wt% matic reaction.Lumit-QM luciferin-luciferase and diluent sucrose and without hormones).The culture volume was were obtained from Lumac BV (Lumac BV,Landgraaf, approximately 150 mL after inoculation.The medium was Netherlands).The ATP was quantified by comparing the adjusted to pH 5.9 and overlaid with liquid paraffin 1:6 relative luminescence units (RLUs)measured from cell (v/v)before autoclaving.Liquid paraffin was used to fa- extracts with those produced by known ATP standards cilitate in situ extraction and measurement of product.8 (prepared in HEPES buffer)measured by the same assay. Cultivation was carried out in 500-mL shake flasks with foam-plug closures at 25C in the dark at 125 rpm on a rotary shaker.In this article,control conditions refers to cul- Soluble Phenolics tivation in hormone-free M-9 medium as described above. Total soluble phenolics in the cell-free culture medium were Sucrose-rich (C-rich)and nitrate-rich (N-rich)media measured spectrophotometrically by the method of Swain were prepared as follows.To prepare C-rich medium, and Hillis as reported by Seitz et al.15 Caffeic acid was hormone-free M-9 medium!8(20%concentration without used as the standard. sucrose or nitrate)was prepared as the basal medium and the concentrations of sucrose and nitrate were adjusted to give a final concentration of 30-40 g/L sucrose and RESULTS AND DISCUSSION 15-25 mg/L nitrogen (as nitrate)after addition to the Figure 1 illustrates the growth and production of shikonin culture.The volume of addition was adjusted to compen- derivatives under control conditions (hormone-free M-9 sate for the cumulative sample volume withdrawn from medium,5 wt sucrose).Typically nitrate was consumed the flask.Approximately 5-6-mL samples were taken. by days 6-8 and sucrose by days 12-15.A two-to Hormone-free 2.5-fold concentrated M-9 medium18 was threefold increase in fresh weight was typically observed. designated as N-rich.Upon addition of N-rich medium,the Production of shikonin derivatives started between days 6 resultant concentration of nitrogen was typically between and 8 and the maximum concentration varied between 140 50 and 65 mg/L and sucrose was between 20 and 30 g/L. and 200 mg/L in various experiments.The data shown here It must be mentioned that the C-rich and N-rich definitions represent the mean of duplicate experiments. are based upon the nitrogen content of the medium. Figures 2A-D illustrate the effects of adding C-rich and N-rich media.Soluble phenolics were measured as Measurement of Cell Growth,Product,and an indication of general secondary metabolic activity.All Nutrient Levels experiments were conducted in duplicate.Days 1-13 in Cell growth,sucrose,and nitrate in the medium were Figure 2A represent cultivation under conditions of the con- measured by previously published methods.16 Shikonin trol experiment.A maximum cell yield of about 140 g/L concentration was measured by the method of Heide was obtained.Shikonin production was observed to start on et al. day 5 and a concentration of ~125 mg/L was measured on day 11.The soluble phenolic content of the medium Cellular Protein Content increased from ~20 mg/L on day 5 to ~160 mg/L on day 12.Shikonin and phenolic concentrations did not The cell mass of known fresh weight was homogenized in increase further until day 13.On day 13,C-rich medium 5 mL Tris-HCI (0.2 M,pH 7.1)using a tissue dismember- was added to the culture aseptically.Sucrose and nitrate ator(Fisher Scientific).The homogenate was centrifuged at concentrations following the addition are shown in the 500g for 10 min and the protein content of the supernatant figure.Sharp increases in soluble phenolics and shikonin was measured by the method of Bradford!using bovine derivative yields were observed after a lag of 24-36 h. serum albumin as the standard. Shikonin concentration increased almost twofold over a 794 BIOTECHNOLOGY AND BIOENGINEERING,VOL.42,NO.7,SEPTEMBER 20,1993secondary metabolic activity and intracellular protein and ATP levels. MATERIALS AND METHODS Cell Line Lithosperrnurn erythrorhizon cell line transformed with Agrobacteriurn tumefaciens Ti plasmid was obtained from Dr. Chang Ryul Liu at the Genetic Research Institute, Taeduck, Korea. Cell Cultivation Cell suspensions were cultivated as described in our earlier paper.16 Thirteen-day-old cells were transferred (20% in￾oculum v/v) into production medium (M-9l' with 5 wt % sucrose and without hormones). The culture volume was approximately 150 mL after inoculation. The medium was adjusted to pH 5.9 and overlaid with liquid paraffin 1:6 (v/v) before autoclaving. Liquid paraffin was used to fa￾cilitate in situ extraction and measurement of product.' Cultivation was carried out in 500-mL shake flasks with foam-plug closures at 25°C in the dark at 125 rpm on a rotary shaker. In this article, control conditions refers to cul￾tivation in hormone-free M-9 medium as described above. Sudrose-rich (C-rich) and nitrate-rich (N-rich) media were prepared as follows. To prepare C-rich medium, hormone-free M-9 medium'' (20% concentration without sucrose or nitrate) was prepared as the basal medium and the concentrations of sucrose and nitrate were adjusted to give a final concentration of 30-40 g/L sucrose and 15-25 mg/L nitrogen (as nitrate) after addition to the culture. The volume of addition was adjusted to compen￾sate for the cumulative sample volume withdrawn from the flask. Approximately 5 -6-mL samples were taken. Hormone-free 2.5-fold concentrated M-9 medium" was designated as N-rich. Upon addition of N-rich medium, the resultant concentration of nitrogen was typically between SO and 65 mg/L and sucrose was between 20 and 30 g/L. It must be mentioned that the C-rich and N-rich definitions are based upon the nitrogen content of the medium. Measurement of Cell Growth, Product, and Nutrient Levels Cell growth, sucrose, and nitrate in the medium were measured by previously published methods.16 Shikonin concentration was measured by the method of Heide et a1.8 Cellular Protein Content The cell mass of known fresh weight was homogenized in 5 mL Tris-HC1 (0.2 M, pH 7.1) using a tissue dismember￾ator (Fisher Scientific). The homogenate was centrifuged at 500g for 10 min and the protein content of the supernatant was measured by the method of Bradford' using bovine serum albumin as the standard. Cellular Adenosine Triphosphate Content Cells of known fresh weight were extracted in 3 mL of ice-cold 90% dimethylsulfoxide (DMSO) by vortexing for 30 s. Extracts were stored at -70°C until further analysis. Parallel controls revealed that the loss of adenosine triphos￾phate (ATP) in cell extracts was in the range of 1-7% during this period. This difference has been ignored in the measurements. Prior to measurement, extracts were thawed at 4°C and centrifuged at 500g for 10 min. The supernatant was diluted 100-fold in N-(2-hydroxyethyl)piperazine-N'- 2-ethanesulfonic acid (HEPES) buffer (0.5 mM, pH 7.8) and 0.1-mL aliquots were used for ATP determination by luminometry. A Turner TD-20e luminometer (Turner De￾signs, Mountain View, CA) was used to monitor the emitted luminescence of the ATP coupled luciferin-luciferase enzy￾matic reaction. Lumit-QM luciferin-luciferase and diluent were obtained from Lumac BV (Lumac BV, Landgraaf, Netherlands). The ATP was quantified by comparing the relative luminescence units (RLUs) measured from cell extracts with those produced by known ATP standards (prepared in HEPES buffer) measured by the same assay. Soluble Phenolics Total soluble phenolics in the cell-free culture medium were measured spectrophotometrically by the method of Swain and Hillis as reported by Seitz et al.15 Caffeic acid was used as the standard. RESULTS AND DISCUSSION Figure 1 illustrates the growth and production of shikonin derivatives under control conditions (hormone-free M-9 medium, 5 wt % sucrose). Typically nitrate was consumed by days 6-8 and sucrose by days 12-15. A two- to threefold increase in fresh weight was typically observed. Production of shikonin derivatives started between days 6 and 8 and the maximum concentration varied between 140 and 200 mg/L in various experiments. The data shown here represent the mean of duplicate experiments. Figures 2A-D illustrate the effects of adding C-rich and N-rich media. Soluble phenolics were measured as an indication of general secondary metabolic activity. All experiments were conducted in duplicate. Days 1-13 in Figure 2A represent cultivation under conditions of the con￾trol experiment. A maximum cell yield of about 140 g/L was obtained. Shikonin production was observed to start on day 5 and a concentration of -125 mg/L was measured on day 11. The soluble phenolic content of the medium increased from -20 mg/L on day 5 to -160 mg/L on day 12. Shikonin and phenolic concentrations did not increase further until day 13. On day 13, C-rich medium was added to the culture aseptically. Sucrose and nitrate concentrations following the addition are shown in the figure. Sharp increases in soluble phenolics and shikonin derivative yields were observed after a lag of 24-36 h. Shikonin concentration increased almost twofold over a 794 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 42, NO. 7, SEPTEMBER 20, 1993