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Fed-batch culture of recombinant Saccharomyces cerevisiae for glucose 6-phosphate dehydrogenase production
Biochemical Engineering Journal 33 (2007) 248–252
Fed-batch culture of recombinant Saccharomyces cerevisiae for glucose 6-phosphate dehydrogenase production ˆ Angelo Samir Melim Miguel ∗ , Michele Vitolo, Adalberto Pessoa Jr. Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences of University of S˜ao Paulo, Av. Prof. Lineu Prestes, 580, B.16, 05508-900, S˜ao Paulo, SP, Brazil Received 24 February 2006; received in revised form 31 October 2006; accepted 1 November 2006
Abstract We examined glucose 6-phosphate dehydrogenase (G6PD) production by fed-batch cultivation, using a recombinant strain of Saccharomyces cerevisiae W303-181 overexpressing this enzyme. The cultivations were carried out in a 3 L fermenter at pH 5.7, 30 ◦ C, 2.0 vvm aeration, 200 rpm agitation and an inoculum concentration of 1.0 g/L. The volume of the culture medium in the fed-batch process varied from 1.333 to 2.0 L, due to the addition of 15.0 g/L glucose solution during 5 h. Different feeding rates were studied (exponentially increasing and decreasing feeding rates), and the feeding profile was determined by values of the parameter K (time constant), namely: 0.2, 0.5 and 0.8 h−1 . The best enzyme production (847 U/L) was obtained with an exponentially increasing feeding rate and K = 0.2 h−1 . The results attained also showed that this process is promising for G6PD production. © 2006 Elsevier B.V. All rights reserved. Keywords: Fed-batch; Glucose 6-phosphate dehydrogenase; Saccharomyces cerevisiae; Exponential feeding rates
1. Introduction Glucose 6-phosphate dehydrogenase (G6PD) (EC.1.1.1.49), a constitutive enzyme present in almost all animal tissues, plants and microorganisms, is widely used as a reagent in clinical diagnostic and analytical chemistry methods [1]. Saccharomyces cerevisiae is often used for the expression of various genes for protein production. It is generally recognized as safe (GRAS) and is capable of post-translational protein modifications, such as glycosylation and other modifications required for optimal biological activity and stability [2]. Synthesis of a cloned-gene product is influenced by genetic and environmental factors, including plasmid stability and copy number, promoter strength, cell-growth rate and medium composition [3]. Determination of optimal operational conditions for recombinant microorganism cultivation is difficult because of the additional complexity imposed by segregation and structural plasmid instability. The plasmid-free cell formation rate depends on plasmid construction characteristics, medium conditions, and on the rate at which the culture develops [4]. The
∗
Corresponding author. Tel.: +55 11 3091 2376. ˆ E-mail address: [email protected] (A.S.M. Miguel).
1369-703X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2006.11.003
recombinant strain S. cerevisiae W303-181 is auxotrophic for adenine, histidine, uracil and tryptophan; it was modified by Lojudice et al. [5] and carries the plasmid YEpPGK-G6P, containing the G6PD coding sequence, which is under the control of the phosphoglycerate kinase 1 (PGK1) promoter. Lojudice et al. [5] evaluated the growth and G6PD formation capability of this engineered yeast in a batch culture carried out at 35 ◦ C, pH 4.0, 2.3 vvm aeration and in a medium constituted of glucose (20 g/L), peptone (5.0 g/L), yeast extract (3.0 g/L), Na2 HPO4 ·12H2 O (2.4 g/L), MgSO4 ·7H2 O (0.075 g/L), and (NH4 )2 SO4 (5.1 g/L). The G6PD-specific activity attained under these conditions was 8.90 U/mg protein. They directed their efforts preferentially towards retention of the plasmid YEpPGKG6P and not to the formation of G6PD by the modified yeast. However, Das Neves et al. [6] observed that in batch culture G6PD formation (about 300 U/L) was stimulated at glucose concentrations higher than 7.0 g/L in the medium. This result prompted us to make fed-batch tests in order to determine the correlation between G6PD formation and substrate concentration. Fed-batch is defined as a technique in microbial processes in which one or more nutrients are supplied during cultivation and in which the products remain in the bioreactor. Superior to batch, fed-batch is advantageous when changing nutrient concentra-
ˆ A.S.M. Miguel et al. / Biochemical Engineering Journal 33 (2007) 248–252
tions affects yield or productivity [7]. Since both overfeeding and underfeeding of nutrients are detrimental to cell growth and product formation, development of a suitable feeding strategy is critical in fed-batch cultivation. In addition, optimization of fed-batch culture can improve gene expression by the vector [8]. In a previous study [9], we found that S. cerevisiae W303181, grown in a fed-batch process, was a potential source of G6PD, depending on optimization of some culture-condition parameters. We have now evaluated the effect of feeding rate (exponentially increasing and decreasing feeding rates), and feeding profile (time constant (K) = 0.2, 0.5 or 0.8 h−1 ) on G6PD formation by S. cerevisiae W303-181 cultivated in a fed-batch process. 2. Materials and methods 2.1. Material Dimethyl-polysiloxane, aminocaproic acid, ethylenediaminetetraacetic acid (EDTA), uracil, adenine, tryptophan, histidine, NADP, -mercaptoethanol, and glucose 6-phosphate were purchased from SIGMA® (St. Louis, MO, USA). Yeast nitrogen base (YNB) was purchased from DIFCO® (Detroit, MI, USA). Glucose was purchased from MERCK (Darmstad, Germany). 2.2. Microorganism and inoculum preparation A genetically modified strain of S. cerevisiae, W303-181, was cultivated under fed-batch conditions. The stock culture was stored at −70 ◦ C in acetate buffer (0.1 M, pH 5.7) with glycerol (30%, v/v). The medium used in all growth steps of inoculum preparation was composed of acetate/acetic acid buffer (0.1 M, pH 5.7), containing 20 g glucose/L, 7.4 g YNB/L, and 20 mg/L each of adenine, histidine, tryptophan and uracil. For cellular activation, 0.9 mL of a cell suspension was thawed and incubated in 50 mL Erlenmeyer flasks containing 10 mL of culture medium, on a rotary shaker at 100 rpm and 30 ◦ C for 24 h. The contents of were flask were transferred into 500 mL Erlenmeyer flasks containing 90 mL of fresh medium and then incubated on a rotary shaker at 100 rpm, 30 ◦ C for 24 h. Finally, to prepare the inoculum, 10 mL of this pre-inoculum was transferred to a 500 mL Erlenmeyer flask containing 90 mL of culture medium and incubated in a rotary shaker at 100 rpm, at 30 ◦ C for 24 h.
249
Fig. 1. Volume of glucose solution added into biorreactor as a function of the cultivation time for different feeding rates. Fed-batch cultivations were carried out in an exponentially increasing feeding rate with K (h−1 ) = 0.2 (), and exponentially decreasing exponential feeding rates with K = 0.2 (), K = 0.5 (), K = 0.8 (♦).
gen mass transfer coefficient (KLa ) was 42 h−1 . Airflow rate was measured with an on-line rotameter and was set with a needle valve to keep KLa constant during the feeding period. The pH of the medium in the aerobic fed-batch was measured with an electrode (Ingold, Woburn, MA, USA) and kept at the desired value (pH 5.7) by automatic addition of 0.5 M NaOH or 0.5 M H2 SO4 solutions. After inoculum addition, the impeller speed, aeration rate, and temperature were adjusted in the fermenter. The glucose solution (15.0 g/L) was then fed into the fermenter, in pulses at intervals of time (t) of 30 min, increasing the initial volume (V0 ) of 1.333 L up to a final volume (Vf ) of 2 L in exponentially increasing or decreasing mode at time constants (K) of 0.2, 0.5 or 0.8 h−1 . In all experiments, the fermenter filling-up-time (T) was set at 5 h. Aliquots of 15 mL of the culture medium were collected hourly for analyses. After sampling, 15.0 mL of sterile water were added back to the fermenter. The volume added (V) was calculated through Eqs. (1) (exponentially increasing feeding rate) and (2) (exponentially decreasing feeding rate): (V − V0 ) =
(Vf − V0 )(eKt − 1) (eKT − 1)
(1)
(V − V0 ) =
(Vf − V0 )(e−Kt − 1) (e−KT − 1)
(2)
Fig. 1 shows the volume of glucose solution (15.0 g/L) added into the fermenter during the fed-batch phase. 2.4. Cell disruption
2.3. Fed-batch cultivation An aliquot of 0.10 L of inoculum (total cell mass (CM) of 1.33 g on a dry basis) was transferred to a 3 L fermenter (BIOFLO 110, New Brunswick Scientific CO., Madison, NJ, USA) containing 0.266 L of the following culture medium: 14.0 g yeast nitrogen base/L, 522.3 mg adenine/L, 601.5 mg tryptophan/L, 353.4 mg uracil/L, and 496.2 mg histidine/L. Total cultivation time was about 14 h; the glucose concentration in this broth was negligible. Foaming was controlled, whenever needed, by the addition of 0.5 mL dimethylpolysiloxane. Agitation and aeration were 200 rpm and 2.0 vvm, respectively, and the oxy-
Hourly, a 5 mL sample of the fermenting broth was centrifuged (3025 × g; 20 min, 6 ◦ C) (BR4i, Jouan, St. Herblain, France); the resulting pellet was suspended in 50 mM TRIS–HCl buffer (pH 7.5), 5.0 mM MgCl2 , 0.2 mM EDTA, 10.0 mM -mercaptoethanol, and 2.0 mM aminocaproic acid. The suspended cells were then disrupted in a vortex for 12 min, using 7.45 g of glass beads (0.5 mm diameter). The mixture was maintained below 10 ◦ C throughout the process. Cell debris and the glass beads were removed by centrifugation (3025 × g; 20 min, 6 ◦ C). The supernatant was used for the determinations of protein concentration and enzyme activity.
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250
2.5. Analytical procedures Cell concentration was estimated by measuring the optical density (OD) of samples at 600 nm, utilizing a standard curve of cell mass as a function of OD (Eq. (3)). Deionized water was used as a blank for this analysis. Total reducing sugar concentration (TRS) was measured according to Somogyi [10], G6PD activity according to Bergmeyer [11], and total soluble protein according to Lowry et al. [12]. A DU640 spectrophotometer (Beckman, Fullerton, CA, USA) was used for all spectrophotometric analyses. CM = 0.68644 × OD − 0.01372
(3)
Fig. 2. Cell concentration (X) as a function of the cultivation time for different feeding rates. Fed-batch cultivations were carried out in an exponentially increasing feeding rate with K (h−1 ) = 0.2 (), and exponentially decreasing feeding rates with K = 0.2 (), K = 0.5 (), K = 0.8 (♦).
Overall enzyme productivity (Prenz ), overall cell productivity (Prx ), cell production (Xprod ), enzyme production (G6PDprod ), specific cell-growth rate (μx ) and specific G6PD production rate (μG6PD ) were calculated according to the following equations:
In all experiments, the final cell concentration was about 2.3 g/L (Fig. 2); but maximum specific growth rate (μmax ) and cell productivity (Prx ) varied with feeding strategy and time constant (Table 1). These parameters lead to different amounts of glucose in the broth available to the yeast, because the volume of glucose solution varied with feeding strategy (Fig. 1). In addition, as K decreases, the substrate is more slowly furnished to the yeast. The exponentially decreasing feeding strategy carried out at K equal to 0.2, 0.5 or 0.8 h−1 led to a μmax decrease of about 9% as K increased (Table 1). However, the culture fed in the exponentially increasing mode with K = 0.2 h−1 resulted in a μmax about 15% higher than that of the culture fed in the exponentially decreasing mode with K = 0.2 h−1 (Table 1). In terms of cell productivity, the cultures fed with substrate using the exponentially increasing (K = 0.2 h−1 ) or decreasing (K = 0.2 or 0.5 h−1 ) strategy had an average Prx of about 0.11 g/L h, which is about 22% higher than that observed for the cultures fed at the exponentially decreasing strategy rate with K = 0.8 h−1 (Table 1). Undoubtedly, a compromise between glucose provision and cell growth must be made, in order to achieve improvement of G6PD formation. The G6PD formation during fermentation depended on the glucose feeding strategy and time constant (K) employed (Fig. 4). Similar to what was found with cell growth, a decrease in G6PD activity was observed during the first 2 h of cultivation,
2.6. Cultivation parameters
Prenz = Prx =
Umax t
X t
(4) (5)
Xprod = X
(6)
G6PDprod = U dMx 1 μx = Mx dt dU 1 μG6PD = Mx dt
(7) (8) (9)
where: Umax = (Umax − Ui ), t = (tf − ti ), X = (Xf − Xi ), U = (Uf − Ui ), Mx = cell mass (g), Uf = final G6PD activity per liter (U/L), Ui = initial G6PD activity per liter (U/L), ti = initial fermentation time (h), tf = time corresponding to the end of the fermentation (h), Xf = final cell concentration (g/L), Xi = initial cell concentration (g/L), and Umax = highest G6PD activity per liter (U/L). 3. Results and discussion When an engineered cell is utilized, as is the case for the S. cerevisiae W303-181 strain that we used, unpredictable behavior can arise during cultivation. A measure of the normal or abnormal behavior might be taken by following growth under specified conditions. In all experiments cell growth continued beyond t = 6 h, although the glucose had been completely consumed at t = 6 h (Figs. 2 and 3). The diauxic growth behavior presented by S. cerevisiae W303-181 is typical for a Crabtreepositive yeast, in which the ethanol produced under aerobic conditions is utilized as a carbon source for growth [13–14]. The decrease in cell concentration up to 2 h of cultivation observed in all experiments (Fig. 2) might be due to cell dilution soon after the feeding phase began.
Fig. 3. Glucose concentration (S) as a function of the cultivation time for different feeding rates. Fed-batch cultivations were carried out in an exponentially increasing feeding rate with K (h−1 ) = 0.2 (), and exponentially decreasing feeding rates with K = 0.2 (), K = 0.5 (), K = 0.8 (♦).
ˆ A.S.M. Miguel et al. / Biochemical Engineering Journal 33 (2007) 248–252
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Table 1 Parameters related to the fed-batch cultivation of Saccharomyces cerevisiae W303-181 for the production of glucose 6-phosphate dehydrogenase Parameters
Feeding modes Increasing
Xi (g/L) Xf (g/L) Xprod (g/L) μx,max (h−1 ) Prx (g/L h) (Ui /L) (Uf /L) G6PDprod (U/L) μG6PDmax (h−1 ) PrG6PD (U/L h) (U/mgprotein ) (U/gcell )
Decreasing
0.2aa
0.2bb
0.2
0.5
0.8
1.28 2.10 0.82 0.16 0.058 527 1085 558 60 53 2.87 517
0.94 2.45 1.51 0.25 0.11 774 1621 847 119 61 3.04 661
0.78 2.31 1.53 0.21 0.11 710 1267 557 125 40 2.53 564
0.91 2.43 1.50 0.20 0.10 1075 1475 400 88 29 2.81 675
1.02 2.22 1.20 0.19 0.086 951 1081 130 73 9 1.88 448
The carbon source was added into the fermenter through increasing and decreasing exponential modes. a 0.2a: the fed-batch cultivation was carried out with the medium composed of 5.0 g/L glucose, 1.85 g/L YNB, 8.0 mg/L adenine, 8.0 mg/L histidine, 8.0 mg/L tryptophan and 8.0 mg/L uracil [9]. b 0.2b: the fed-batch cultivation was carried out with the medium described in the present work.
as a result of the dilution associated with the feeding process. After the feeding period, G6PD activity increased until the end of the fermentation (Fig. 4). The fermentation conducted with the exponentially increasing feeding rate and K = 0.2 h−1 gave the highest enzyme production (847 U/L) and enzyme productivity (61 U/L h). In the cultures conducted with exponentially decreasing feeding rates, both enzyme production and productivity decreased as the time constant increased (Table 1). We found that G6PD formation is clearly associated with growth, since μx and μG6PD had similar behaviors (Fig. 5), mainly in the fed-batch fermentations done with exponentially decreasing feeding rates, with the parameters K = 0.5 and 0.8 h−1 (Fig. 5C and D). Silva et al. [1] also found that G6PD formation and growth are coupled events in batch culture. We can see that, for the fermentation conducted with an exponentially increasing glucose feeding rate and K = 0.2 h−1
Fig. 5. Specific growth rate (μx ) (—) and specific production rate (μG6PDH ) (- - -) as a function of the cultivation time for different feeding rates. (A) Fedbatch cultivation carried out in a exponentially increasing feeding rate with K = 0.2 h−1 ; (B) fed-batch cultivation carried out in a exponentially decreasing feeding rate with K = 0.2 h−1 ; (C) fed-batch cultivation carried out in a exponentially decreasing feeding rate with K = 0.5 h−1 ; and (D) fed-batch cultivation carried out in a exponentially decreasing feeding rate with K = 0.8 h−1 .
Fig. 4. Glucose-6-phosphate dehydrogendase activity (G6PD) as a function of the cultivation time for different feeding rates. Fed-batch cultivations were carried out in an exponentially increasing feeding rate with K (h−1 ) = 0.2 (), and exponentially decreasing feeding rates with K = 0.2 (), K = 0.5 (), K = 0.8 (♦).
(Fig. 5A), in which the highest G6PD production occurred (847 U/L)—the profile of the curve μG6PD versus time shifted about 2 h ahead in comparison to the curve of μx versus time. This might be an indication, even if indirectly, of overexpression of the G6PD gene inserted in the plasmid or of combined expression of the two G6PD genes of S. cerevisiae W303-181
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(one located in the chromosome and the other in the plasmid). Derepression of the constitutive gene is another possibility. A similar behavior, although less conspicuous (shift forward about 1 h), was noted when the culture was made with an exponentially decreasing feeding strategy and K = 0.2 h−1 (Fig. 5B). Therefore, it seems that the high rate of G6PD formation by S. cerevisiae W303-181 is favored by culture conditions under which the difference between the times of μG6PD,max and μx,max is as large as possible. We had previously cultivated S. cerevisiae W303-181 in medium composed of 0.74 g YNB/L, and 8 mg micronutrients/L (adenine, histidine, uracil and tryptophan), with the glucose solution (5.0 g/L) added according to an exponentially increasing feeding strategy at K values of 0.2, 0.3, 0.5, 0.7 and 0.8 h−1 [9]. The best results, which occurred at K = 0.2 h−1 , are summarized in Table 1, for comparison. As can be seen, G6PD production in experiment 0.2a (558 U/L) was about 34% lower than in experiment 0.2b (Table 1). Those results clearly indicate that, to optimize G6PD production, the nitrogen source and micronutrients should not be offered at limiting amounts, especially if we consider that the S. cerevisiae W303-181 has two genes that can be expressed. Although the best fed-batch culture conditions for G6PD production by S. cerevisiae W303-181 still need to be established, the improvement achieved until now is remarkable. We obtained approximately 4.4 and 1.2 times higher G6PD productivity when compared with the rates obtained by Das Neves et al. [6] and Miguel et al. [9], respectively. Moreover, the G6PD activity of 847 U/L is comparable with those of currently-marketed enzyme preparations [6]. Finally, having “in house” alternatives for G6PD production is quite important for an enzyme-importing country such as Brazil, because this enzyme is widely used in diagnostic enzyme-immunoassay tests. 4. Conclusions The data lead us to conclude that the yeast strain S. cerevisiae W303-181 has a growth pattern similar to that of other yeasts of the genus Saccharomyces. The G6PD formation is coupled with growth to some extent. To enhance G6PD production, (Eq. (1)) culture conditions should be set such that μG6PDmax and μx,max are separated by an as large as possible time interval. The G6PD production depends on the feeding rate and time constant employed. Best results were obtained with the exponentially increasing feeding rate and a time constant of 0.2 h−1 . The availability of glucose, nitrogen source and micronutrients to the yeast affected G6PD formation; the cell-free extract gave a G6PD activity (847 U/L) comparable with those described in commercial G6PD preparation.
Acknowledgements The authors acknowledge fellowships and financial support from FAPESP (Fundac¸a˜ o de Amparo a` Pesquisa do Estado de S˜ao Paulo), CAPES (Coordenac¸a˜ o de Aperfeic¸oamento de Pessoal de N´ıvel Superior), and CNPq (Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico)/Brazil. The authors thank Dr. Dante Augusto Moraes and Prof. Jo˜ao Carlos M. Carvalho for their assistance with some of the experiments as well as Prof. Carla Columbano Oliveira, who provided the genetically modified yeast. We also acknowledge Prof. Carlota O. Rangel-Yagui for her valuable technical suggestions and English revision. References [1] D.P. Silva, A. Pessoa Jr., I.C. Roberto, M. Vitolo, Effect of KL a on the production of glucose-6-phosphate dehydrogenase from Saccharomyces cerevisiae grown by fermentation process, Appl. Biochem. Biotechnol. 98–100 (2002) 205–213. [2] M.C.M. Hensing, R.J. Rouwenhorst, J.P. van Dijken, J.T. Pronk, Physiological and technological aspects of large-scale heterologous-protein production with yeasts, Ant. V. Leeuvenhoek 67 (1995) 271–279. [3] V. Chiruvolu, J.M. Stratton, T.L. Ott, F.W. Bazer, M.M. Meagher, Effect of media composition on growth, plasmid stability and ovine interferon- production in Saccharomyces cerevisiae, J. Ferment. Bioeng. 82 (1996) 565–569. [4] C. Shene, N. Mir, B.A. Andrews, J. Asenjo, Effect of the growth conditions on the synthesis of a recombinant -1,4-endoglucanase in continuous and fed-batch cultures, Enzymol. Microb. Technol. 27 (2000) 248–253. [5] F.H. Lojudice, D.P. Silva, N.I.T. Zanchin, O.C. Oliveira, A. Pessoa Jr., Overexpression of glucose-6-phosphate dehydrogenase (G6PDH) in genetically modified Saccharomyces cerevisiae, Appl. Biochem. Biotechnol. 91–93 (2001) 161–169. [6] L.C.M. Das Neves, A. Pessoa Jr., M. Vitolo, Production of glucose 6-phosphate dehydrogenase from genetically modified Saccharomyces cerevisiae grown by batch fermentation process, Biotechnol. Prog. 21 (2003) 1136–1139. [7] T. Yamane, S. Shimizu, Fed-batch in microbial process, Adv. Biochem. Eng. 30 (1984) 147–194. [8] C.H. Kim, K.J. Rao, D.J. Youn, S.K. Rhee, Scale-up of recombinant hirudin production from Saccharomyces cerevisiae, Biotechnol. Bioprocess Eng. 8 (2003) 303–305. [9] A.S.M. Miguel, L.C.M. Neves, M. Vitolo, A. Pessoa Jr., Effect of flow rate pattern on glucose-6-phosphate dehydrogenase synthesis in fed-batch culture of recombinant Saccharomyces cerevisiae, Biotechnol. Prog. 19 (2003) 320–324. [10] M. Somogyi, Notes on sugar determination, J. Biol. Chem. 195 (1952) 19–23. [11] H.U. Bergmeyer, Methods of Enzymatic Analysis, Verlag Chemie, Weinheim, Germany, 1984. [12] O.H. Lowry, N.J. Rosebough, A.L. Farr, R.J. Randall, Protein measurement with the folin-phenol-reagent, J. Biol. Chem. 193 (1951) 265–275. [13] A.H. Rose, J.S. Harrison, The Yeast, Academic Press, New York, 1999. [14] G.M. Walker, Yeast Physiology and Biotechnology, John Wiley & Sons, 1998.
Fed-batch culture of recombinant Saccharomyces cerevisiae for glucose 6-phosphate dehydrogenase production ˆ Angelo Samir Melim Miguel ∗ , Michele Vitolo, Adalberto Pessoa Jr. Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences of University of S˜ao Paulo, Av. Prof. Lineu Prestes, 580, B.16, 05508-900, S˜ao Paulo, SP, Brazil Received 24 February 2006; received in revised form 31 October 2006; accepted 1 November 2006
Abstract We examined glucose 6-phosphate dehydrogenase (G6PD) production by fed-batch cultivation, using a recombinant strain of Saccharomyces cerevisiae W303-181 overexpressing this enzyme. The cultivations were carried out in a 3 L fermenter at pH 5.7, 30 ◦ C, 2.0 vvm aeration, 200 rpm agitation and an inoculum concentration of 1.0 g/L. The volume of the culture medium in the fed-batch process varied from 1.333 to 2.0 L, due to the addition of 15.0 g/L glucose solution during 5 h. Different feeding rates were studied (exponentially increasing and decreasing feeding rates), and the feeding profile was determined by values of the parameter K (time constant), namely: 0.2, 0.5 and 0.8 h−1 . The best enzyme production (847 U/L) was obtained with an exponentially increasing feeding rate and K = 0.2 h−1 . The results attained also showed that this process is promising for G6PD production. © 2006 Elsevier B.V. All rights reserved. Keywords: Fed-batch; Glucose 6-phosphate dehydrogenase; Saccharomyces cerevisiae; Exponential feeding rates
1. Introduction Glucose 6-phosphate dehydrogenase (G6PD) (EC.1.1.1.49), a constitutive enzyme present in almost all animal tissues, plants and microorganisms, is widely used as a reagent in clinical diagnostic and analytical chemistry methods [1]. Saccharomyces cerevisiae is often used for the expression of various genes for protein production. It is generally recognized as safe (GRAS) and is capable of post-translational protein modifications, such as glycosylation and other modifications required for optimal biological activity and stability [2]. Synthesis of a cloned-gene product is influenced by genetic and environmental factors, including plasmid stability and copy number, promoter strength, cell-growth rate and medium composition [3]. Determination of optimal operational conditions for recombinant microorganism cultivation is difficult because of the additional complexity imposed by segregation and structural plasmid instability. The plasmid-free cell formation rate depends on plasmid construction characteristics, medium conditions, and on the rate at which the culture develops [4]. The
∗
Corresponding author. Tel.: +55 11 3091 2376. ˆ E-mail address: [email protected] (A.S.M. Miguel).
1369-703X/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2006.11.003
recombinant strain S. cerevisiae W303-181 is auxotrophic for adenine, histidine, uracil and tryptophan; it was modified by Lojudice et al. [5] and carries the plasmid YEpPGK-G6P, containing the G6PD coding sequence, which is under the control of the phosphoglycerate kinase 1 (PGK1) promoter. Lojudice et al. [5] evaluated the growth and G6PD formation capability of this engineered yeast in a batch culture carried out at 35 ◦ C, pH 4.0, 2.3 vvm aeration and in a medium constituted of glucose (20 g/L), peptone (5.0 g/L), yeast extract (3.0 g/L), Na2 HPO4 ·12H2 O (2.4 g/L), MgSO4 ·7H2 O (0.075 g/L), and (NH4 )2 SO4 (5.1 g/L). The G6PD-specific activity attained under these conditions was 8.90 U/mg protein. They directed their efforts preferentially towards retention of the plasmid YEpPGKG6P and not to the formation of G6PD by the modified yeast. However, Das Neves et al. [6] observed that in batch culture G6PD formation (about 300 U/L) was stimulated at glucose concentrations higher than 7.0 g/L in the medium. This result prompted us to make fed-batch tests in order to determine the correlation between G6PD formation and substrate concentration. Fed-batch is defined as a technique in microbial processes in which one or more nutrients are supplied during cultivation and in which the products remain in the bioreactor. Superior to batch, fed-batch is advantageous when changing nutrient concentra-
ˆ A.S.M. Miguel et al. / Biochemical Engineering Journal 33 (2007) 248–252
tions affects yield or productivity [7]. Since both overfeeding and underfeeding of nutrients are detrimental to cell growth and product formation, development of a suitable feeding strategy is critical in fed-batch cultivation. In addition, optimization of fed-batch culture can improve gene expression by the vector [8]. In a previous study [9], we found that S. cerevisiae W303181, grown in a fed-batch process, was a potential source of G6PD, depending on optimization of some culture-condition parameters. We have now evaluated the effect of feeding rate (exponentially increasing and decreasing feeding rates), and feeding profile (time constant (K) = 0.2, 0.5 or 0.8 h−1 ) on G6PD formation by S. cerevisiae W303-181 cultivated in a fed-batch process. 2. Materials and methods 2.1. Material Dimethyl-polysiloxane, aminocaproic acid, ethylenediaminetetraacetic acid (EDTA), uracil, adenine, tryptophan, histidine, NADP, -mercaptoethanol, and glucose 6-phosphate were purchased from SIGMA® (St. Louis, MO, USA). Yeast nitrogen base (YNB) was purchased from DIFCO® (Detroit, MI, USA). Glucose was purchased from MERCK (Darmstad, Germany). 2.2. Microorganism and inoculum preparation A genetically modified strain of S. cerevisiae, W303-181, was cultivated under fed-batch conditions. The stock culture was stored at −70 ◦ C in acetate buffer (0.1 M, pH 5.7) with glycerol (30%, v/v). The medium used in all growth steps of inoculum preparation was composed of acetate/acetic acid buffer (0.1 M, pH 5.7), containing 20 g glucose/L, 7.4 g YNB/L, and 20 mg/L each of adenine, histidine, tryptophan and uracil. For cellular activation, 0.9 mL of a cell suspension was thawed and incubated in 50 mL Erlenmeyer flasks containing 10 mL of culture medium, on a rotary shaker at 100 rpm and 30 ◦ C for 24 h. The contents of were flask were transferred into 500 mL Erlenmeyer flasks containing 90 mL of fresh medium and then incubated on a rotary shaker at 100 rpm, 30 ◦ C for 24 h. Finally, to prepare the inoculum, 10 mL of this pre-inoculum was transferred to a 500 mL Erlenmeyer flask containing 90 mL of culture medium and incubated in a rotary shaker at 100 rpm, at 30 ◦ C for 24 h.
249
Fig. 1. Volume of glucose solution added into biorreactor as a function of the cultivation time for different feeding rates. Fed-batch cultivations were carried out in an exponentially increasing feeding rate with K (h−1 ) = 0.2 (), and exponentially decreasing exponential feeding rates with K = 0.2 (), K = 0.5 (), K = 0.8 (♦).
gen mass transfer coefficient (KLa ) was 42 h−1 . Airflow rate was measured with an on-line rotameter and was set with a needle valve to keep KLa constant during the feeding period. The pH of the medium in the aerobic fed-batch was measured with an electrode (Ingold, Woburn, MA, USA) and kept at the desired value (pH 5.7) by automatic addition of 0.5 M NaOH or 0.5 M H2 SO4 solutions. After inoculum addition, the impeller speed, aeration rate, and temperature were adjusted in the fermenter. The glucose solution (15.0 g/L) was then fed into the fermenter, in pulses at intervals of time (t) of 30 min, increasing the initial volume (V0 ) of 1.333 L up to a final volume (Vf ) of 2 L in exponentially increasing or decreasing mode at time constants (K) of 0.2, 0.5 or 0.8 h−1 . In all experiments, the fermenter filling-up-time (T) was set at 5 h. Aliquots of 15 mL of the culture medium were collected hourly for analyses. After sampling, 15.0 mL of sterile water were added back to the fermenter. The volume added (V) was calculated through Eqs. (1) (exponentially increasing feeding rate) and (2) (exponentially decreasing feeding rate): (V − V0 ) =
(Vf − V0 )(eKt − 1) (eKT − 1)
(1)
(V − V0 ) =
(Vf − V0 )(e−Kt − 1) (e−KT − 1)
(2)
Fig. 1 shows the volume of glucose solution (15.0 g/L) added into the fermenter during the fed-batch phase. 2.4. Cell disruption
2.3. Fed-batch cultivation An aliquot of 0.10 L of inoculum (total cell mass (CM) of 1.33 g on a dry basis) was transferred to a 3 L fermenter (BIOFLO 110, New Brunswick Scientific CO., Madison, NJ, USA) containing 0.266 L of the following culture medium: 14.0 g yeast nitrogen base/L, 522.3 mg adenine/L, 601.5 mg tryptophan/L, 353.4 mg uracil/L, and 496.2 mg histidine/L. Total cultivation time was about 14 h; the glucose concentration in this broth was negligible. Foaming was controlled, whenever needed, by the addition of 0.5 mL dimethylpolysiloxane. Agitation and aeration were 200 rpm and 2.0 vvm, respectively, and the oxy-
Hourly, a 5 mL sample of the fermenting broth was centrifuged (3025 × g; 20 min, 6 ◦ C) (BR4i, Jouan, St. Herblain, France); the resulting pellet was suspended in 50 mM TRIS–HCl buffer (pH 7.5), 5.0 mM MgCl2 , 0.2 mM EDTA, 10.0 mM -mercaptoethanol, and 2.0 mM aminocaproic acid. The suspended cells were then disrupted in a vortex for 12 min, using 7.45 g of glass beads (0.5 mm diameter). The mixture was maintained below 10 ◦ C throughout the process. Cell debris and the glass beads were removed by centrifugation (3025 × g; 20 min, 6 ◦ C). The supernatant was used for the determinations of protein concentration and enzyme activity.
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2.5. Analytical procedures Cell concentration was estimated by measuring the optical density (OD) of samples at 600 nm, utilizing a standard curve of cell mass as a function of OD (Eq. (3)). Deionized water was used as a blank for this analysis. Total reducing sugar concentration (TRS) was measured according to Somogyi [10], G6PD activity according to Bergmeyer [11], and total soluble protein according to Lowry et al. [12]. A DU640 spectrophotometer (Beckman, Fullerton, CA, USA) was used for all spectrophotometric analyses. CM = 0.68644 × OD − 0.01372
(3)
Fig. 2. Cell concentration (X) as a function of the cultivation time for different feeding rates. Fed-batch cultivations were carried out in an exponentially increasing feeding rate with K (h−1 ) = 0.2 (), and exponentially decreasing feeding rates with K = 0.2 (), K = 0.5 (), K = 0.8 (♦).
Overall enzyme productivity (Prenz ), overall cell productivity (Prx ), cell production (Xprod ), enzyme production (G6PDprod ), specific cell-growth rate (μx ) and specific G6PD production rate (μG6PD ) were calculated according to the following equations:
In all experiments, the final cell concentration was about 2.3 g/L (Fig. 2); but maximum specific growth rate (μmax ) and cell productivity (Prx ) varied with feeding strategy and time constant (Table 1). These parameters lead to different amounts of glucose in the broth available to the yeast, because the volume of glucose solution varied with feeding strategy (Fig. 1). In addition, as K decreases, the substrate is more slowly furnished to the yeast. The exponentially decreasing feeding strategy carried out at K equal to 0.2, 0.5 or 0.8 h−1 led to a μmax decrease of about 9% as K increased (Table 1). However, the culture fed in the exponentially increasing mode with K = 0.2 h−1 resulted in a μmax about 15% higher than that of the culture fed in the exponentially decreasing mode with K = 0.2 h−1 (Table 1). In terms of cell productivity, the cultures fed with substrate using the exponentially increasing (K = 0.2 h−1 ) or decreasing (K = 0.2 or 0.5 h−1 ) strategy had an average Prx of about 0.11 g/L h, which is about 22% higher than that observed for the cultures fed at the exponentially decreasing strategy rate with K = 0.8 h−1 (Table 1). Undoubtedly, a compromise between glucose provision and cell growth must be made, in order to achieve improvement of G6PD formation. The G6PD formation during fermentation depended on the glucose feeding strategy and time constant (K) employed (Fig. 4). Similar to what was found with cell growth, a decrease in G6PD activity was observed during the first 2 h of cultivation,
2.6. Cultivation parameters
Prenz = Prx =
Umax t
X t
(4) (5)
Xprod = X
(6)
G6PDprod = U dMx 1 μx = Mx dt dU 1 μG6PD = Mx dt
(7) (8) (9)
where: Umax = (Umax − Ui ), t = (tf − ti ), X = (Xf − Xi ), U = (Uf − Ui ), Mx = cell mass (g), Uf = final G6PD activity per liter (U/L), Ui = initial G6PD activity per liter (U/L), ti = initial fermentation time (h), tf = time corresponding to the end of the fermentation (h), Xf = final cell concentration (g/L), Xi = initial cell concentration (g/L), and Umax = highest G6PD activity per liter (U/L). 3. Results and discussion When an engineered cell is utilized, as is the case for the S. cerevisiae W303-181 strain that we used, unpredictable behavior can arise during cultivation. A measure of the normal or abnormal behavior might be taken by following growth under specified conditions. In all experiments cell growth continued beyond t = 6 h, although the glucose had been completely consumed at t = 6 h (Figs. 2 and 3). The diauxic growth behavior presented by S. cerevisiae W303-181 is typical for a Crabtreepositive yeast, in which the ethanol produced under aerobic conditions is utilized as a carbon source for growth [13–14]. The decrease in cell concentration up to 2 h of cultivation observed in all experiments (Fig. 2) might be due to cell dilution soon after the feeding phase began.
Fig. 3. Glucose concentration (S) as a function of the cultivation time for different feeding rates. Fed-batch cultivations were carried out in an exponentially increasing feeding rate with K (h−1 ) = 0.2 (), and exponentially decreasing feeding rates with K = 0.2 (), K = 0.5 (), K = 0.8 (♦).
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Table 1 Parameters related to the fed-batch cultivation of Saccharomyces cerevisiae W303-181 for the production of glucose 6-phosphate dehydrogenase Parameters
Feeding modes Increasing
Xi (g/L) Xf (g/L) Xprod (g/L) μx,max (h−1 ) Prx (g/L h) (Ui /L) (Uf /L) G6PDprod (U/L) μG6PDmax (h−1 ) PrG6PD (U/L h) (U/mgprotein ) (U/gcell )
Decreasing
0.2aa
0.2bb
0.2
0.5
0.8
1.28 2.10 0.82 0.16 0.058 527 1085 558 60 53 2.87 517
0.94 2.45 1.51 0.25 0.11 774 1621 847 119 61 3.04 661
0.78 2.31 1.53 0.21 0.11 710 1267 557 125 40 2.53 564
0.91 2.43 1.50 0.20 0.10 1075 1475 400 88 29 2.81 675
1.02 2.22 1.20 0.19 0.086 951 1081 130 73 9 1.88 448
The carbon source was added into the fermenter through increasing and decreasing exponential modes. a 0.2a: the fed-batch cultivation was carried out with the medium composed of 5.0 g/L glucose, 1.85 g/L YNB, 8.0 mg/L adenine, 8.0 mg/L histidine, 8.0 mg/L tryptophan and 8.0 mg/L uracil [9]. b 0.2b: the fed-batch cultivation was carried out with the medium described in the present work.
as a result of the dilution associated with the feeding process. After the feeding period, G6PD activity increased until the end of the fermentation (Fig. 4). The fermentation conducted with the exponentially increasing feeding rate and K = 0.2 h−1 gave the highest enzyme production (847 U/L) and enzyme productivity (61 U/L h). In the cultures conducted with exponentially decreasing feeding rates, both enzyme production and productivity decreased as the time constant increased (Table 1). We found that G6PD formation is clearly associated with growth, since μx and μG6PD had similar behaviors (Fig. 5), mainly in the fed-batch fermentations done with exponentially decreasing feeding rates, with the parameters K = 0.5 and 0.8 h−1 (Fig. 5C and D). Silva et al. [1] also found that G6PD formation and growth are coupled events in batch culture. We can see that, for the fermentation conducted with an exponentially increasing glucose feeding rate and K = 0.2 h−1
Fig. 5. Specific growth rate (μx ) (—) and specific production rate (μG6PDH ) (- - -) as a function of the cultivation time for different feeding rates. (A) Fedbatch cultivation carried out in a exponentially increasing feeding rate with K = 0.2 h−1 ; (B) fed-batch cultivation carried out in a exponentially decreasing feeding rate with K = 0.2 h−1 ; (C) fed-batch cultivation carried out in a exponentially decreasing feeding rate with K = 0.5 h−1 ; and (D) fed-batch cultivation carried out in a exponentially decreasing feeding rate with K = 0.8 h−1 .
Fig. 4. Glucose-6-phosphate dehydrogendase activity (G6PD) as a function of the cultivation time for different feeding rates. Fed-batch cultivations were carried out in an exponentially increasing feeding rate with K (h−1 ) = 0.2 (), and exponentially decreasing feeding rates with K = 0.2 (), K = 0.5 (), K = 0.8 (♦).
(Fig. 5A), in which the highest G6PD production occurred (847 U/L)—the profile of the curve μG6PD versus time shifted about 2 h ahead in comparison to the curve of μx versus time. This might be an indication, even if indirectly, of overexpression of the G6PD gene inserted in the plasmid or of combined expression of the two G6PD genes of S. cerevisiae W303-181
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(one located in the chromosome and the other in the plasmid). Derepression of the constitutive gene is another possibility. A similar behavior, although less conspicuous (shift forward about 1 h), was noted when the culture was made with an exponentially decreasing feeding strategy and K = 0.2 h−1 (Fig. 5B). Therefore, it seems that the high rate of G6PD formation by S. cerevisiae W303-181 is favored by culture conditions under which the difference between the times of μG6PD,max and μx,max is as large as possible. We had previously cultivated S. cerevisiae W303-181 in medium composed of 0.74 g YNB/L, and 8 mg micronutrients/L (adenine, histidine, uracil and tryptophan), with the glucose solution (5.0 g/L) added according to an exponentially increasing feeding strategy at K values of 0.2, 0.3, 0.5, 0.7 and 0.8 h−1 [9]. The best results, which occurred at K = 0.2 h−1 , are summarized in Table 1, for comparison. As can be seen, G6PD production in experiment 0.2a (558 U/L) was about 34% lower than in experiment 0.2b (Table 1). Those results clearly indicate that, to optimize G6PD production, the nitrogen source and micronutrients should not be offered at limiting amounts, especially if we consider that the S. cerevisiae W303-181 has two genes that can be expressed. Although the best fed-batch culture conditions for G6PD production by S. cerevisiae W303-181 still need to be established, the improvement achieved until now is remarkable. We obtained approximately 4.4 and 1.2 times higher G6PD productivity when compared with the rates obtained by Das Neves et al. [6] and Miguel et al. [9], respectively. Moreover, the G6PD activity of 847 U/L is comparable with those of currently-marketed enzyme preparations [6]. Finally, having “in house” alternatives for G6PD production is quite important for an enzyme-importing country such as Brazil, because this enzyme is widely used in diagnostic enzyme-immunoassay tests. 4. Conclusions The data lead us to conclude that the yeast strain S. cerevisiae W303-181 has a growth pattern similar to that of other yeasts of the genus Saccharomyces. The G6PD formation is coupled with growth to some extent. To enhance G6PD production, (Eq. (1)) culture conditions should be set such that μG6PDmax and μx,max are separated by an as large as possible time interval. The G6PD production depends on the feeding rate and time constant employed. Best results were obtained with the exponentially increasing feeding rate and a time constant of 0.2 h−1 . The availability of glucose, nitrogen source and micronutrients to the yeast affected G6PD formation; the cell-free extract gave a G6PD activity (847 U/L) comparable with those described in commercial G6PD preparation.
Acknowledgements The authors acknowledge fellowships and financial support from FAPESP (Fundac¸a˜ o de Amparo a` Pesquisa do Estado de S˜ao Paulo), CAPES (Coordenac¸a˜ o de Aperfeic¸oamento de Pessoal de N´ıvel Superior), and CNPq (Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico)/Brazil. The authors thank Dr. Dante Augusto Moraes and Prof. Jo˜ao Carlos M. Carvalho for their assistance with some of the experiments as well as Prof. Carla Columbano Oliveira, who provided the genetically modified yeast. We also acknowledge Prof. Carlota O. Rangel-Yagui for her valuable technical suggestions and English revision. References [1] D.P. Silva, A. Pessoa Jr., I.C. Roberto, M. Vitolo, Effect of KL a on the production of glucose-6-phosphate dehydrogenase from Saccharomyces cerevisiae grown by fermentation process, Appl. Biochem. Biotechnol. 98–100 (2002) 205–213. [2] M.C.M. Hensing, R.J. Rouwenhorst, J.P. van Dijken, J.T. Pronk, Physiological and technological aspects of large-scale heterologous-protein production with yeasts, Ant. V. Leeuvenhoek 67 (1995) 271–279. [3] V. Chiruvolu, J.M. Stratton, T.L. Ott, F.W. Bazer, M.M. Meagher, Effect of media composition on growth, plasmid stability and ovine interferon- production in Saccharomyces cerevisiae, J. Ferment. Bioeng. 82 (1996) 565–569. [4] C. Shene, N. Mir, B.A. Andrews, J. Asenjo, Effect of the growth conditions on the synthesis of a recombinant -1,4-endoglucanase in continuous and fed-batch cultures, Enzymol. Microb. Technol. 27 (2000) 248–253. [5] F.H. Lojudice, D.P. Silva, N.I.T. Zanchin, O.C. Oliveira, A. Pessoa Jr., Overexpression of glucose-6-phosphate dehydrogenase (G6PDH) in genetically modified Saccharomyces cerevisiae, Appl. Biochem. Biotechnol. 91–93 (2001) 161–169. [6] L.C.M. Das Neves, A. Pessoa Jr., M. Vitolo, Production of glucose 6-phosphate dehydrogenase from genetically modified Saccharomyces cerevisiae grown by batch fermentation process, Biotechnol. Prog. 21 (2003) 1136–1139. [7] T. Yamane, S. Shimizu, Fed-batch in microbial process, Adv. Biochem. Eng. 30 (1984) 147–194. [8] C.H. Kim, K.J. Rao, D.J. Youn, S.K. Rhee, Scale-up of recombinant hirudin production from Saccharomyces cerevisiae, Biotechnol. Bioprocess Eng. 8 (2003) 303–305. [9] A.S.M. Miguel, L.C.M. Neves, M. Vitolo, A. Pessoa Jr., Effect of flow rate pattern on glucose-6-phosphate dehydrogenase synthesis in fed-batch culture of recombinant Saccharomyces cerevisiae, Biotechnol. Prog. 19 (2003) 320–324. [10] M. Somogyi, Notes on sugar determination, J. Biol. Chem. 195 (1952) 19–23. [11] H.U. Bergmeyer, Methods of Enzymatic Analysis, Verlag Chemie, Weinheim, Germany, 1984. [12] O.H. Lowry, N.J. Rosebough, A.L. Farr, R.J. Randall, Protein measurement with the folin-phenol-reagent, J. Biol. Chem. 193 (1951) 265–275. [13] A.H. Rose, J.S. Harrison, The Yeast, Academic Press, New York, 1999. [14] G.M. Walker, Yeast Physiology and Biotechnology, John Wiley & Sons, 1998.