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Effects of methanol feeding methods on chimeric α-amylase expression in continuous culture of Pichia pastoris
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 101, No. 3, 227–231. 2006 DOI: 10.1263/jbb.101.227
© 2006, The Society for Biotechnology, Japan
Effects of Methanol Feeding Methods on Chimeric α-Amylase Expression in Continuous Culture of Pichia pastoris Atsushi Nakano,1 Chun Yeon Lee,1 Arei Yoshida,1 Takehiro Matsumoto,1 Naofumi Shiomi,2 and Shigeo Katoh1* Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan1 and Department of Biosphere Sciences, Kobe College, 4-1 Okadayama, Nishinomiya 662-8505, Japan2 Received 11 October 2005/Accepted 10 December 2005
The effects of two types of methanol feeding methods in a continuous culture of Pichia system on the cell growth and recombinant protein expression were studied using chimeric α-amylase as a model protein. With the feeding of methanol by a DO-stat method, the α-amylase concentration in the fermentation broth increased with decreasing dilution rate and reached 173 mg/l at a dilution rate of 0.013 h–1, at which the maximum volumetric productivity of α-amylase was obtained. Although almost the same productivity was attained at 0.04 h–1 with continuous methanol feeding, the α-amylase concentration was one third that compared with feeding by the DO-stat method, that is, 55 mg/l. Furthermore, at this dilution rate, the medium volume needed per unit time was three times that required when DO-stat was used. Therefore, continuous culture with methanol feeding by the DO-stat method may be a promising method for the production of recombinant proteins on an industrial scale by Pichia pastoris. [Key words: methylotrophic yeast, induction with methanol, continuous culture, DO-stat, α-amylase]
The methylotrophic yeast Pichia pastoris is a promising candidate as a host for the production of heterologous proteins by secretion. The unique features of this expression system are derived from the promoter which is obtained from the methanol regulated alcohol oxidase I gene (AOX1) of P. pastoris, one of the most efficient and tightly regulated promoters ever discovered (1). This strong promoter, coupled with the high cell density fermentation and induction strategy, has allowed the production of recombinant proteins at high extracellular levels (2, 3). However, this promoter may be repressed by other carbon sources, such as glycerol and ethanol, and thus the production method using P. pastoris involves the cell growth phase with the assimilation of carbon sources other than methanol and the induction phase of heterologous protein production by methanol. Therefore, production is usually operated in fed-batch cultures (4), in which the fermentation conditions change with time and are difficult to precisely control. In continuous culture, on the other hand, cell concentration, specific growth rate, product concentration and other factors are kept constant and are controlled at optimal values for target production. Because the growth of cells is determined from the supply rate and the concentration of a growth-limiting nutrient, the effects of the growth rate of cells and methanol concentration on the production efficiency must be considered in continuous culture (5). In a previous work, using recombinant P. pastoris Mut+ expressing chimeric α-amylase as a model strain, we stud-
ied the effects of regulated glycerol or methanol feeding on the cell growth and recombinant protein expression using the Pichia system with either constant feeding rates or variable feeding rates in DO-stat fed-batch fermentation (6). In this study, two types of methanol feeding methods, the conventional chemostat method and the methanol feeding by the DO-stat method, were applied to the continuous fermentation of the recombinant P. pastoris producing chimeric α-amylase. After cells were grown in fed-batch culture containing glycerol, methanol was continuously supplied to the cells with a medium containing methanol, or was supplied separately by the DO-stat method while the cells receive continuous feeding of medium not containing methanol. The effects of the dilution rate and methanol feeding on the cell growth and protein expression were studied, and the efficiencies of these two continuous culture methods were compared with that of the fed-batch culture method. MATERIALS AND METHODS Strain Yeast P. pastoris GS115 (his4) was obtained from Invitrogen (Carlsbad, CA, USA). Chimeric α-amylase (Amy1A/3D), which consists of 159aa of one isozyme (Amy1A) of rice -amylase followed by 251aa of another isozyme (Amy3D), was produced from the genes of these two isozymes (7). The cDNA of Amy1A/3D was cloned into the P. pastoris expression vector pPIC9 (Invitrogen). The details of the vector construction and transformation are given elsewhere (8). Continuous fermentation methods The continuous fermentation methods consisted three phases: batch cell growth (batch), glycerol fed-batch (fed-batch) and continuous fermentation with methanol feeding (continuous). The fermentation inoculum was
* Corresponding author. e-mail: [email protected] phone/fax: +81-(0)78-803-6193 227
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prepared from a frozen cell stock vial (1 ml), and cultured for 18 h in 50 ml of YPD medium at 30°C, 200 rpm. In the batch phase, the inoculum was transferred into a 2-l jar fermentor (Mitsuwa KMJ5C; Mitsuwarika, Yamaguchi) with a 1-l working volume containing the basal salts medium (glycerol, 40 g l–1; K2SO4, 18 g l–1; MgSO4 ⋅7H2O, 14.9 g l–1; KOH, 4.13 g l–1; H3PO4, 27 ml l–1; and CaSO4, 0.9 g l–1) plus 4.4 ml l–1 trace metal solution (CuSO4 ⋅5H2O, 6 g l–1; KI, 0.09 g l–1; MnSO4 ⋅ H2O, 3 g l–1; H3BO3, 0.02 g l–1; MoNa2O4 ⋅ 2H2O, 0.24 g l–1; CoCl2, 0.5 g l–1; ZnCl2, 20 g l–1; FeSO4 ⋅ H2O, 65 g l–1; biotin, 0.2 g l–1; and H2SO4, 5.0 ml l–1) (9). The pH of the medium was adjusted to and controlled at 5.0 by adding 30% ammonium hydroxide. The temperature was controlled at 30°C, and the DO level was maintained at more than 10% air saturation by a cascaded control of the agitation rate (400–1000 rpm) and aeration rate (1–1.5 vvm). In the fed-batch phase, the glycerol medium (200 ml of 80% glycerol solution plus 12 ml l–1 the trace metal solution) was fed by the DO-stat method at a predetermined DO set value (50% air saturation), and the glycerol feed rates were decreased in stepwise fashion beginning at 1.6 ml min–1 and decreased by 0.3 ml min–1 after every 6 h (6). In the continuous phase, two different methods of methanol feeding, as shown schematically in Fig. 1, were applied to study the effects on cell growth and α-amylase production. In the first method, the basal salts medium containing 25%(v/v) methanol instead of glycerol was continuously supplied at a constant flow rate using a microtube pump. The dilution rate was calculated on the basis of the flow rate of the medium containing methanol and ranged from 0.01 h–1 to 0.04 h–1. In the second method, the basal salts medium without glycerol was supplied continuously at a constant flow rate, and pure methanol was separately fed at a flow rate
FIG. 1. Two feeding methods of medium and methanol. (A) Medium containing 25% methanol was continuously fed at a constant dilution rate. (B) Medium was continuously fed at a constant rate, and methanol was fed by DO-stat.
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of 1 ml min–1 by the DO-stat method at a predetermined DO set value (50% air saturation). In this case, the dilution rate was determined from the flow rate of the medium plus the average rate of methanol supply and ranged from 0.013 h–1 to 0.046 h–1. Determination of α-amylase activity The activity of recombinant α-amylase was determined by a modified method described by Bernfeld (10). One unit of enzyme activity corresponds to the amount required to liberate 1 µmol of maltose per minute. The amount of α-amylase (mg l–1) was estimated by using the correlation between the α-amylase unit (U) and the protein mass (mg): 1 mg-Amy1A/3D =100 U (11). Determination of cell and methanol concentrations The turbidity of cells in the fermentation broth was measured using a spectrophotometer at 600 nm (UV mini-1240; Shimadzu, Kyoto). The dry cell weight was calculated using the following correlation: Dry cell weight (in g l–1) =0.22 ×OD at 600 nm. In the continuous phase, the residual methanol concentration was determined by gas chromatography (GC) with a flame-ionized detector (Shimadzu GC-14B; Shimadzu). A glass column (3 mm ×2 m) packed with PEG-20M 20% uniport O HP 60/80 was used (Shimadzu). The injection, detector, and column temperatures were 230°C, 250°C, and 130°C, respectively. The gas pressures of nitrogen, hydrogen, and air were 140, 65, and 75 kPa, respectively.
RESULTS AND DISCUSSION Time courses of batch, fed-batch and continuous phases The typical time courses of the cell growth and α-amylase expression are shown in Fig. 2. Upon the depletion of glycerol, which was indicated by a sharp increase in DO level in the fermentation broth after growth in the batch phase for approximately 18 h, the fed-batch feeding of glycerol in three-stepwise decreasing feed rates was started. After feeding 200 ml of 80% glycerol solution plus 12 ml l–1 trace metal solution, the continuous phase was started using one of the two methanol feeding methods, and α-amylase expression was induced. After approximately 60 h from the start of the continuous phase, the cell weight and α-amylase concentration reached a steady state. From these values at the steady state and the average amount of methanol supplied, cell and α-amylase yields, specific methanol consumption rate (g-MeOH g-DCW–1 h–1) and specific α-amy-
FIG. 2. Time courses of cell growth and α-amylase expression. Phase 1, Glycerol batch phase; phase 2, glycerol fed-batch phase; phase 3, continuous phase; continuous supply of medium containing methanol at dilution rate of 0.03 h–1. Squares, DCW (g/l); circles, α-amylase (mg/l).
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FIG. 3. Dry cell weight and α-amylase concentration in continuous culture. Open squares, DCW (g/l); solid squares, DCW (DO-stat) (g/l); open circles, α-amylase (mg/l); solid circles, α-amylase (DOstat) (mg/l).
lase production rate (mg-α-amylase g-DCW–1 h–1) were calculated. Effects of methanol feeding methods on cell growth and α-amylase expression Figure 3 shows the dry cell weight and α-amylase concentration in continuous cultures using two different methanol feeding methods. In the first method, in which the medium containing 25%(v/v) methanol was continuously supplied at a constant flow rate, the dry cell weight was independent of the dilution rate in the range from 0.01 h–1 to 0.04 h–1, as expected from the characteristics of the chemostat. In this range, the cell yield (gDCW/g-methanol) was constant at approximately 0.4, and the methanol concentration in the medium was below the detection level (< 0.002 v/v%). Thus, the specific methanol consumption rate (g-MeOH g-DCW–1 h–1) shown in Fig. 4 increased linearly with the dilution rate, as well as the methanol feeding rate (g-MeOH h–1, data not shown). The washout of cells occurred above the dilution rate of 0.05 h–1. With the feeding of methanol by the DO-stat method, the dry cell weight decreased above the dilution rate of 0.025 h–1, whereas the specific methanol consumption rate and the cell yield were the same as those in the first method. The methanol feeding rate was almost constant above this dilution rate with the feeding of methanol by the DO-stat method, which means that the specific growth rate was almost constant in this range. The α-amylase concentration in the fermentation broth increased with decreasing dilution rate, as shown in Fig. 3, and at lower dilution rates with the feeding of methanol by the DO-stat method, the concentration became much higher than that observed with continuous methanol feeding. At higher dilution rates, however, the α-amylase concentration decreased rapidly with the feeding of methanol by the DOstat method, because the dry cell weight decreased in this
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FIG. 4. Specific methanol consumption rate and specific α-amylase production rate in continuous culture. Open squares, Specific MeOH consumption (g/[DCW h]); solid squares, specific MeOH consumption (DO-stat) (g/[DCW h]); open circles, specific α-amyl production (mg/[DCW h]); solid circles, specific α-amyl production (DO-stat) (mg/[DCW h]).
range. As shown in Fig. 4, the specific α-amylase production rate was constant at all dilution rates with the feeding of methanol by the DO-stat method, whereas it decreased with decreasing dilution rate in this range with continuous methanol feeding. In the dilution rate range from 0.01 h–1 to 0.025 h–1, the cell concentrations and specific methanol consumption rates coincided in the two methanol feeding methods, as shown in Figs. 3 and 4. With continuous methanol feeding, however, the specific α-amylase production rate decreased with decreasing dilution rate in this range. The reason for this decrease is unknown, but in these two feeding methods the periodical pattern of the concentration of methanol in the fermentation broth was different. With continuous feeding, the methanol concentration was below the detection level (<0.002 v/v%). On the other hand, with methanol feeding by the DO-stat method, the concentration fluctuated with repeated start and stop methanol feeding, as shown in Fig. 5. It reached a maximum value of 0.04–0.05 v/v% immediately after stoping methanol feeding and had become zero before the start of methanol feeding. This fluctuation, which continued over the same range during the continuous culture for 40 h, above a specific concentration of methanol with repeated methanol feeding by the DO-stat method may have an enhancing effect on the induction of α-amylase production by the AOX1 promoter. Comparison of α-amylase productivity using fed-batch and two continuous culture methods Volumetric productivity is of major interest in fermentation processes, because it describes the rate of production of a target protein per unit of fermentor capacity. The volumetric productivity in the continuous culture using different methanol feeding
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FIG. 5. Variations in DO and methanol concentration during one cycle of DO stat. Dilution rate, 0.025 h–1; solid line, DO; broken line, methanol (v/v%).
get protein and the reduction in medium volume supplied can reduce the costs of medium and downstream processes, the continuous culture of P. pastoris with methanol feeding by the DO-stat method should have a high potential for the production of recombinant proteins on an industrial scale. The volumetric productivity in fed-batch culture, reported in a previous work (6), was 4.4–4.7 mg-amylase l–1 h–1 over the net fermentation time (batch + glycerol fed-batch + methanol fed-batch, 72–80 h). It is necessary, however, to consider vollumetric productivity over the total processing time, which includes the time required for harvesting, washing and the reassembly of equipment and the charging and sterilization of medium, rather than just considering the net fermentation time. If we assume that the total processing time for one cycle of fed-batch fermentation is 120 h, the volumetric productivity of fed-batch fermentation becomes 2.6– 2.8 mg-amylase l–1 h–1, which is only slightly higher than that obtained in continuous culture. In continuous culture, a steady state in the cell and product concentrations, growth rate and other fermentation conditions can be maintained, and thus it is possible to continuously produce cells or other products with specific characteristics under optimal conditions. This point can also be an advantage of continuous culture in industrial production. The α-amylase production in a continuous culture of P. pastoris using two different methods of methanol feeding was compared. With methanol feeding by the DO-stat method, high volumetric productivity and α-amylase concentrations were obtained at low dilution rates. This method may reduce the consumption of medium and the cost of downstream processes, and showed a comparable performance with the fed-batch culture method. Although further comparison with other methanol feeding methods including repeated fed-batch is necessary, methanol feeding by the DO-stat method has merits in the continuous industrial scale production of recombinant proteins by P. pastoris. ACKNOWLEDGMENTS
FIG. 6. Comparison of volumetric productivity of α-amylase. α-Amylase productivity: open triangles, continuous feeding; solid triangles, DO-stat; cross, fed-batch.
methods is shown in Fig. 6. It increased at high dilution rates with continuous methanol feeding, again as expected from the characteristics of the chemostat, and reached 2.3 mg-amylase l–1 h–1 at the dilution rate of 0.04 h–1, where the α-amylase concentration was at the lowest value of 55 mg/l. On the other hand, the volumetric productivity increased with decreasing dilution rate with methanol feeding by the DOstat method and reached 2.2 mg-amylase l–1 h–1 at the dilution rate of 0.013 h–1, and the highest α-amylase concentration of 173 mg/l was attained at this dilution rate. Although the maximum productivity was almost the same for these two methanol feeding methods, the α-amylase concentration was three times higher with methanol feeding by the DO-stat method and the medium volume needed per unit time was one third. Because the high concentration of a tar-
The partial support of a Grant-in-Aid for Scientific Research (B) (no. 15360442) from the Japan Society for Promotion of Science is gratefully acknowledged. We would also like to thank Dr. R.L. Rodriguez for kindly providing two isozyme genes of α-amylase.
REFERENCES 1. Cregg, J. M., Vedvick, T. S., and Raschke, W. C.: Recent advances in the expression of foreign genes in Pichia pastoris. Bio/Technology, 11, 905–910 (1993). 2. Romanos, M.: Advances in the use of Pichia pastoris for high-level gene expression. Curr. Opin. Biotechnol., 6, 527– 533 (1995). 3. Zhang, W. H., Bevins, M. A., Plantz, B. A., Smith, L. A., and Meagher, M. M.: Modeling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of botulinum neurotoxin, serotype A. Biotechnol. Bioeng., 70, 1–8 (2000). 4. Bushell, M. E., Rowe, M., Avignone-Rossa, C. A., and Wardell, J. N.: Cyclic fed-batch culture for production of human serum albumin in Pichia pastoris. Biotechnol. Bioeng., 82, 678–683 (2003).
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5. d’Anjou, M. C. and Daugulis, A. J.: A rational approach to improving productivity in recombinant Pichia pastoris fermentation. Biotechnol. Bioeng., 72, 1–11 (2001). 6. Lee, C. Y., Nakano, A., Shiomi, N., Lee, E. K., and Katoh, S.: Effects of substrate feed rate on heterologous protein expression by Pichia pastoris in DO-stat fed-batch fermentation. Enzyme Microb. Technol., 33, 358–365 (2003). 7. Kumagai, M. H., Shaw, M., Terashima, M., Vrkljan, Z., Whitaker, J. R., and Rodriguez, R. L.: Expression and secretion of rice α-amylase by Saccharomyces cerevisiae. Gene, 94, 209–216 (1990). 8. Kurokawa, T., Lee, C. Y., Shiomi, N., Nakano, A., and Katoh, S.: Secretion of α-amylase from Saccharomyces cere-
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visiae and Pichia pastoris and characterization of its C-terminus with an anti-peptide antibody. J. Chem. Eng. Jpn., 35, 1277–1281 (2002). 9. Lee, C. Y., Lee, S. J., Jung, K. H., Katoh, S., and Lee, E. K.: High dissolved oxygen tension enhances heterologous protein expression by recombinant Pichia pastoris. Process Biochem., 38, 1147–1154 (2003). 10. Bernfeld, B.: Amylases α and α. Methods Enzymol., 1, 149– 158 (1955). 11. Katoh, S., Terashima, M., and Miyaoku, K.: Purification of α-amylase by specific elution from anti-peptide antibodies. Appl. Microbol. Biotechnol., 47, 521–524 (1997).
© 2006, The Society for Biotechnology, Japan
Effects of Methanol Feeding Methods on Chimeric α-Amylase Expression in Continuous Culture of Pichia pastoris Atsushi Nakano,1 Chun Yeon Lee,1 Arei Yoshida,1 Takehiro Matsumoto,1 Naofumi Shiomi,2 and Shigeo Katoh1* Graduate School of Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan1 and Department of Biosphere Sciences, Kobe College, 4-1 Okadayama, Nishinomiya 662-8505, Japan2 Received 11 October 2005/Accepted 10 December 2005
The effects of two types of methanol feeding methods in a continuous culture of Pichia system on the cell growth and recombinant protein expression were studied using chimeric α-amylase as a model protein. With the feeding of methanol by a DO-stat method, the α-amylase concentration in the fermentation broth increased with decreasing dilution rate and reached 173 mg/l at a dilution rate of 0.013 h–1, at which the maximum volumetric productivity of α-amylase was obtained. Although almost the same productivity was attained at 0.04 h–1 with continuous methanol feeding, the α-amylase concentration was one third that compared with feeding by the DO-stat method, that is, 55 mg/l. Furthermore, at this dilution rate, the medium volume needed per unit time was three times that required when DO-stat was used. Therefore, continuous culture with methanol feeding by the DO-stat method may be a promising method for the production of recombinant proteins on an industrial scale by Pichia pastoris. [Key words: methylotrophic yeast, induction with methanol, continuous culture, DO-stat, α-amylase]
The methylotrophic yeast Pichia pastoris is a promising candidate as a host for the production of heterologous proteins by secretion. The unique features of this expression system are derived from the promoter which is obtained from the methanol regulated alcohol oxidase I gene (AOX1) of P. pastoris, one of the most efficient and tightly regulated promoters ever discovered (1). This strong promoter, coupled with the high cell density fermentation and induction strategy, has allowed the production of recombinant proteins at high extracellular levels (2, 3). However, this promoter may be repressed by other carbon sources, such as glycerol and ethanol, and thus the production method using P. pastoris involves the cell growth phase with the assimilation of carbon sources other than methanol and the induction phase of heterologous protein production by methanol. Therefore, production is usually operated in fed-batch cultures (4), in which the fermentation conditions change with time and are difficult to precisely control. In continuous culture, on the other hand, cell concentration, specific growth rate, product concentration and other factors are kept constant and are controlled at optimal values for target production. Because the growth of cells is determined from the supply rate and the concentration of a growth-limiting nutrient, the effects of the growth rate of cells and methanol concentration on the production efficiency must be considered in continuous culture (5). In a previous work, using recombinant P. pastoris Mut+ expressing chimeric α-amylase as a model strain, we stud-
ied the effects of regulated glycerol or methanol feeding on the cell growth and recombinant protein expression using the Pichia system with either constant feeding rates or variable feeding rates in DO-stat fed-batch fermentation (6). In this study, two types of methanol feeding methods, the conventional chemostat method and the methanol feeding by the DO-stat method, were applied to the continuous fermentation of the recombinant P. pastoris producing chimeric α-amylase. After cells were grown in fed-batch culture containing glycerol, methanol was continuously supplied to the cells with a medium containing methanol, or was supplied separately by the DO-stat method while the cells receive continuous feeding of medium not containing methanol. The effects of the dilution rate and methanol feeding on the cell growth and protein expression were studied, and the efficiencies of these two continuous culture methods were compared with that of the fed-batch culture method. MATERIALS AND METHODS Strain Yeast P. pastoris GS115 (his4) was obtained from Invitrogen (Carlsbad, CA, USA). Chimeric α-amylase (Amy1A/3D), which consists of 159aa of one isozyme (Amy1A) of rice -amylase followed by 251aa of another isozyme (Amy3D), was produced from the genes of these two isozymes (7). The cDNA of Amy1A/3D was cloned into the P. pastoris expression vector pPIC9 (Invitrogen). The details of the vector construction and transformation are given elsewhere (8). Continuous fermentation methods The continuous fermentation methods consisted three phases: batch cell growth (batch), glycerol fed-batch (fed-batch) and continuous fermentation with methanol feeding (continuous). The fermentation inoculum was
* Corresponding author. e-mail: [email protected] phone/fax: +81-(0)78-803-6193 227
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prepared from a frozen cell stock vial (1 ml), and cultured for 18 h in 50 ml of YPD medium at 30°C, 200 rpm. In the batch phase, the inoculum was transferred into a 2-l jar fermentor (Mitsuwa KMJ5C; Mitsuwarika, Yamaguchi) with a 1-l working volume containing the basal salts medium (glycerol, 40 g l–1; K2SO4, 18 g l–1; MgSO4 ⋅7H2O, 14.9 g l–1; KOH, 4.13 g l–1; H3PO4, 27 ml l–1; and CaSO4, 0.9 g l–1) plus 4.4 ml l–1 trace metal solution (CuSO4 ⋅5H2O, 6 g l–1; KI, 0.09 g l–1; MnSO4 ⋅ H2O, 3 g l–1; H3BO3, 0.02 g l–1; MoNa2O4 ⋅ 2H2O, 0.24 g l–1; CoCl2, 0.5 g l–1; ZnCl2, 20 g l–1; FeSO4 ⋅ H2O, 65 g l–1; biotin, 0.2 g l–1; and H2SO4, 5.0 ml l–1) (9). The pH of the medium was adjusted to and controlled at 5.0 by adding 30% ammonium hydroxide. The temperature was controlled at 30°C, and the DO level was maintained at more than 10% air saturation by a cascaded control of the agitation rate (400–1000 rpm) and aeration rate (1–1.5 vvm). In the fed-batch phase, the glycerol medium (200 ml of 80% glycerol solution plus 12 ml l–1 the trace metal solution) was fed by the DO-stat method at a predetermined DO set value (50% air saturation), and the glycerol feed rates were decreased in stepwise fashion beginning at 1.6 ml min–1 and decreased by 0.3 ml min–1 after every 6 h (6). In the continuous phase, two different methods of methanol feeding, as shown schematically in Fig. 1, were applied to study the effects on cell growth and α-amylase production. In the first method, the basal salts medium containing 25%(v/v) methanol instead of glycerol was continuously supplied at a constant flow rate using a microtube pump. The dilution rate was calculated on the basis of the flow rate of the medium containing methanol and ranged from 0.01 h–1 to 0.04 h–1. In the second method, the basal salts medium without glycerol was supplied continuously at a constant flow rate, and pure methanol was separately fed at a flow rate
FIG. 1. Two feeding methods of medium and methanol. (A) Medium containing 25% methanol was continuously fed at a constant dilution rate. (B) Medium was continuously fed at a constant rate, and methanol was fed by DO-stat.
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of 1 ml min–1 by the DO-stat method at a predetermined DO set value (50% air saturation). In this case, the dilution rate was determined from the flow rate of the medium plus the average rate of methanol supply and ranged from 0.013 h–1 to 0.046 h–1. Determination of α-amylase activity The activity of recombinant α-amylase was determined by a modified method described by Bernfeld (10). One unit of enzyme activity corresponds to the amount required to liberate 1 µmol of maltose per minute. The amount of α-amylase (mg l–1) was estimated by using the correlation between the α-amylase unit (U) and the protein mass (mg): 1 mg-Amy1A/3D =100 U (11). Determination of cell and methanol concentrations The turbidity of cells in the fermentation broth was measured using a spectrophotometer at 600 nm (UV mini-1240; Shimadzu, Kyoto). The dry cell weight was calculated using the following correlation: Dry cell weight (in g l–1) =0.22 ×OD at 600 nm. In the continuous phase, the residual methanol concentration was determined by gas chromatography (GC) with a flame-ionized detector (Shimadzu GC-14B; Shimadzu). A glass column (3 mm ×2 m) packed with PEG-20M 20% uniport O HP 60/80 was used (Shimadzu). The injection, detector, and column temperatures were 230°C, 250°C, and 130°C, respectively. The gas pressures of nitrogen, hydrogen, and air were 140, 65, and 75 kPa, respectively.
RESULTS AND DISCUSSION Time courses of batch, fed-batch and continuous phases The typical time courses of the cell growth and α-amylase expression are shown in Fig. 2. Upon the depletion of glycerol, which was indicated by a sharp increase in DO level in the fermentation broth after growth in the batch phase for approximately 18 h, the fed-batch feeding of glycerol in three-stepwise decreasing feed rates was started. After feeding 200 ml of 80% glycerol solution plus 12 ml l–1 trace metal solution, the continuous phase was started using one of the two methanol feeding methods, and α-amylase expression was induced. After approximately 60 h from the start of the continuous phase, the cell weight and α-amylase concentration reached a steady state. From these values at the steady state and the average amount of methanol supplied, cell and α-amylase yields, specific methanol consumption rate (g-MeOH g-DCW–1 h–1) and specific α-amy-
FIG. 2. Time courses of cell growth and α-amylase expression. Phase 1, Glycerol batch phase; phase 2, glycerol fed-batch phase; phase 3, continuous phase; continuous supply of medium containing methanol at dilution rate of 0.03 h–1. Squares, DCW (g/l); circles, α-amylase (mg/l).
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FIG. 3. Dry cell weight and α-amylase concentration in continuous culture. Open squares, DCW (g/l); solid squares, DCW (DO-stat) (g/l); open circles, α-amylase (mg/l); solid circles, α-amylase (DOstat) (mg/l).
lase production rate (mg-α-amylase g-DCW–1 h–1) were calculated. Effects of methanol feeding methods on cell growth and α-amylase expression Figure 3 shows the dry cell weight and α-amylase concentration in continuous cultures using two different methanol feeding methods. In the first method, in which the medium containing 25%(v/v) methanol was continuously supplied at a constant flow rate, the dry cell weight was independent of the dilution rate in the range from 0.01 h–1 to 0.04 h–1, as expected from the characteristics of the chemostat. In this range, the cell yield (gDCW/g-methanol) was constant at approximately 0.4, and the methanol concentration in the medium was below the detection level (< 0.002 v/v%). Thus, the specific methanol consumption rate (g-MeOH g-DCW–1 h–1) shown in Fig. 4 increased linearly with the dilution rate, as well as the methanol feeding rate (g-MeOH h–1, data not shown). The washout of cells occurred above the dilution rate of 0.05 h–1. With the feeding of methanol by the DO-stat method, the dry cell weight decreased above the dilution rate of 0.025 h–1, whereas the specific methanol consumption rate and the cell yield were the same as those in the first method. The methanol feeding rate was almost constant above this dilution rate with the feeding of methanol by the DO-stat method, which means that the specific growth rate was almost constant in this range. The α-amylase concentration in the fermentation broth increased with decreasing dilution rate, as shown in Fig. 3, and at lower dilution rates with the feeding of methanol by the DO-stat method, the concentration became much higher than that observed with continuous methanol feeding. At higher dilution rates, however, the α-amylase concentration decreased rapidly with the feeding of methanol by the DOstat method, because the dry cell weight decreased in this
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FIG. 4. Specific methanol consumption rate and specific α-amylase production rate in continuous culture. Open squares, Specific MeOH consumption (g/[DCW h]); solid squares, specific MeOH consumption (DO-stat) (g/[DCW h]); open circles, specific α-amyl production (mg/[DCW h]); solid circles, specific α-amyl production (DO-stat) (mg/[DCW h]).
range. As shown in Fig. 4, the specific α-amylase production rate was constant at all dilution rates with the feeding of methanol by the DO-stat method, whereas it decreased with decreasing dilution rate in this range with continuous methanol feeding. In the dilution rate range from 0.01 h–1 to 0.025 h–1, the cell concentrations and specific methanol consumption rates coincided in the two methanol feeding methods, as shown in Figs. 3 and 4. With continuous methanol feeding, however, the specific α-amylase production rate decreased with decreasing dilution rate in this range. The reason for this decrease is unknown, but in these two feeding methods the periodical pattern of the concentration of methanol in the fermentation broth was different. With continuous feeding, the methanol concentration was below the detection level (<0.002 v/v%). On the other hand, with methanol feeding by the DO-stat method, the concentration fluctuated with repeated start and stop methanol feeding, as shown in Fig. 5. It reached a maximum value of 0.04–0.05 v/v% immediately after stoping methanol feeding and had become zero before the start of methanol feeding. This fluctuation, which continued over the same range during the continuous culture for 40 h, above a specific concentration of methanol with repeated methanol feeding by the DO-stat method may have an enhancing effect on the induction of α-amylase production by the AOX1 promoter. Comparison of α-amylase productivity using fed-batch and two continuous culture methods Volumetric productivity is of major interest in fermentation processes, because it describes the rate of production of a target protein per unit of fermentor capacity. The volumetric productivity in the continuous culture using different methanol feeding
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FIG. 5. Variations in DO and methanol concentration during one cycle of DO stat. Dilution rate, 0.025 h–1; solid line, DO; broken line, methanol (v/v%).
get protein and the reduction in medium volume supplied can reduce the costs of medium and downstream processes, the continuous culture of P. pastoris with methanol feeding by the DO-stat method should have a high potential for the production of recombinant proteins on an industrial scale. The volumetric productivity in fed-batch culture, reported in a previous work (6), was 4.4–4.7 mg-amylase l–1 h–1 over the net fermentation time (batch + glycerol fed-batch + methanol fed-batch, 72–80 h). It is necessary, however, to consider vollumetric productivity over the total processing time, which includes the time required for harvesting, washing and the reassembly of equipment and the charging and sterilization of medium, rather than just considering the net fermentation time. If we assume that the total processing time for one cycle of fed-batch fermentation is 120 h, the volumetric productivity of fed-batch fermentation becomes 2.6– 2.8 mg-amylase l–1 h–1, which is only slightly higher than that obtained in continuous culture. In continuous culture, a steady state in the cell and product concentrations, growth rate and other fermentation conditions can be maintained, and thus it is possible to continuously produce cells or other products with specific characteristics under optimal conditions. This point can also be an advantage of continuous culture in industrial production. The α-amylase production in a continuous culture of P. pastoris using two different methods of methanol feeding was compared. With methanol feeding by the DO-stat method, high volumetric productivity and α-amylase concentrations were obtained at low dilution rates. This method may reduce the consumption of medium and the cost of downstream processes, and showed a comparable performance with the fed-batch culture method. Although further comparison with other methanol feeding methods including repeated fed-batch is necessary, methanol feeding by the DO-stat method has merits in the continuous industrial scale production of recombinant proteins by P. pastoris. ACKNOWLEDGMENTS
FIG. 6. Comparison of volumetric productivity of α-amylase. α-Amylase productivity: open triangles, continuous feeding; solid triangles, DO-stat; cross, fed-batch.
methods is shown in Fig. 6. It increased at high dilution rates with continuous methanol feeding, again as expected from the characteristics of the chemostat, and reached 2.3 mg-amylase l–1 h–1 at the dilution rate of 0.04 h–1, where the α-amylase concentration was at the lowest value of 55 mg/l. On the other hand, the volumetric productivity increased with decreasing dilution rate with methanol feeding by the DOstat method and reached 2.2 mg-amylase l–1 h–1 at the dilution rate of 0.013 h–1, and the highest α-amylase concentration of 173 mg/l was attained at this dilution rate. Although the maximum productivity was almost the same for these two methanol feeding methods, the α-amylase concentration was three times higher with methanol feeding by the DO-stat method and the medium volume needed per unit time was one third. Because the high concentration of a tar-
The partial support of a Grant-in-Aid for Scientific Research (B) (no. 15360442) from the Japan Society for Promotion of Science is gratefully acknowledged. We would also like to thank Dr. R.L. Rodriguez for kindly providing two isozyme genes of α-amylase.
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