Effects of dissolved oxygen and oxygen mass transfer ... - Science Direct

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Effects of dissolved oxygen and oxygen mass transfer on overexpression of target gene in recombinant E. coli Sanjoy K. Bhattacharya and Ashok K. Dubey Department of Biochemical Engineering & Biotechnology, Indian Institute of Technology. New Delhi, India Overexpression of target gene (Mspl methylase) in recombinant Escherichia coli has been studied in response to dissolved oxygen and oxygen mass transfer. The rate of target gene expression attained its maximum value (2.67 U mg-‘p-‘h-‘) when the culture was induced with 1 llzMisopropyl+-o-thiogalactopyranoside (IPTG) in the mid-exponential phase of growth. The maximum value of 6.7 x ld U mg-‘p-’ for the target gene expression was attained in about 3 h ofpostinduction cultivation. An oxygen-suficient condition (0.22 mmoles I-‘) was necessary to obtain optimal expression and cell productivity. Expression of the target gene upon induction was accompanied by an increase in oxygen uptake rate (OUR) of the cells and decrease in dissolved oxygen (DO) concentration in the cultivation broth. Volumetric oxygen transfer coeficient (K,a) of 61 h-’ was optimum for cell productivity of uninduced culture while that for induced culture was 84 h-’ which was optimum for foreign protein synthesis also. 0 1997 by Elsevier Science Inc. Keywords: cloned gene

Recombinant E. coli; IFTG induction: bioreactor; dissolved oxygen level: oxygen mass transfer; expression of

Introduction One of the significant achievements of modem biotechnological research has been the ability to create genetically tailored strains of bacteria which could be dictated to express target genes at high levels.1-3 Such overexpressing recombinant bacteria have been proven a benefit from both commercial and academic standpoints since they enabled production of desired product in large quantities. Though a host-vector relationship and promoter strength are important factors in expression of a foreign gene in the host, the importance of nutritional and metabolic parameters cannot be undermined. Foreign protein synthesis in the host is found to enhance maintenance coefficient4 and is reported to impose metabolic burden.5 It has thus been considered important to investigate the effect of a factor such as oxygen which plays a crucial role in aerobic metabolism,6*7 on the formation of foreign gene product in E. co/i. The target gene in the present case is MspI DNA methyltransferase (M.

from the restriction-modification system of Mostrain of E. coli harborin the target gene has been constructed as reported earlier.‘, ? ’ In the present communication, we report overexpression of the target gene in this strain of recombinant E. co/i as affected by variations in dissolved oxygen concentration and oxygen mass transfer.

MspI)

raxeEla sp.* The recombinant

Materials and methods Chemicals and reagents Restriction endonuclease MspI and lambda DNA were obtained from New England Biolabs, Beverly, MA, USA. Lysozyme, ampicillin, isopropyl-P--o-thiogalactopyranoside (IPTG), protamine sulfate, sodium dodecyl sulfate (SDS), S-adenosyl-L-methionine (AdoMet) and reagents used for polyacrylamide gel electrophoresis were procurred from Sigma, St. Louis, MO. The protein assay kit from Bio-Rad (Richmond, CA) was used as recommended.

Bacterial strain and plasmid Address reprint request to Dr. Ashok K. Dubey, Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology-Delhi, Hauz Khas, New Delhi, 110016, India Received 9 August 1995; accepted 13 June 1996

Enzyme and Microbial Technology 20:355-360, 1997 0 1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

Escherichia coli K-12 strain ER1727 [A(mcrBC-) hsdRMS mrr) 2::TnlO, mcrA1272::TnlO, F’lacproABlacIqA(lacZ)-Ml_51 was kindly provided by Dr. E. Raleigh from New England Biolabs. This strain of E. coli was used for overexpression of the target gene

0141-0229/97/$17.00 PII SO141-0229(96)00151-2

Papers which was placed downstream of the T7 promoter regulated by the luc operator in the expression vector pMSP.‘O The E. coli strain

ER1727 harboring recombinant plasmid pMSP was cultivated in 2 1 of Luria Broth” containing 150 pg ml-’ ampicillin at 37’C. The cultivation vessel was a Bioengineering AG Fermenter of 4-l capacity equipped with provisions for control and measurement of pH, temperature, dissolved oxygen concentration, agitation, and aeration.

Preparation of cell-free extract and expression of the target gene The steps involved in preparation of cell-free extract as crude M. MspI were similar to those described earlier.12 In brief, the harvested cells (0.5 g) were suspended in 2 ml of buffer containing 10 mM potassium phosphate (pH 7.4), 1 mM N%EDTA, 14 rn~ B-mercaptoethanol, 0.3 M NaCl, and 10% glycerol. The cell suspension was sonicated for 3 min with burst and gap periods of 15 s at 14 pm amplitude in a Soniprep 150 (New Brunswick, Edison, NJ). The temperature during sonication was maintained at 5 f 2’C. The sonicated cell suspension was centrifuged at 31,000 g for 30 min. Recovered clear supematant was treated as crude preparation containing the foreign gene product (@I methylase). This crude preparation was used to quantitate M. MspI protein to measure its expression in the host cells. Au adequately diluted aliquot of this cell-free crude extract was incubated with 1 pg lambda DNA at 37°C for 1 h in 20 pl mixture containing 50 mu Tris-HCI (pH 7.6) 50 mu NaCl, 10 rn~ EDTA, 5 mM B-mercaptoethanol, and 80 PM AdoMet. Following the methylation reaction, the lambda DNA in the reaction mixture was subjected to R. MspI restriction to check the level of protection achieved. One unit of M. MspI was defined as the minimum volume of crude methylase preparation required for complete protection of 1 pg of lambda DNA from restriction by MspI endonuclease. Protein quantitation was based on the method of Bradford13 and was performed using a protein assay kit from Bio-Rad by following the protocol provided by the supplier. Specific methylase activity (units mg-’ protein) was based on the above measurements. The rate of target gene expression was expressed in terms of specific activity per plasmid per hour (U mg-’ p-l h-l). Specific activity per plasmid (U mg-’ p-i) was used to measure the level of expression of the target gene.

Growth profile, induction with IPTG, and plasmid copy number The growth curve for the present recombinant strain of E. coli was established by plotting optical density (600 mu) versus time with a sampling interval of 15 min. The physiological state of the culture suitable for induction to get maximum expression of M. MspI was determined by inducing the culture with 1 mu IPTG at different time points during exponential and stationary phases of growth followed by estimation of the rate of target gene expression. To determine the postinduction period of cultivation to achieve maximum intracellular concentration of the induced protein, samples of the induced culture were withdrawn at 30-min intervals to quantitate the M. MspI synthesized. The plasmid copy number per genome was determined using the method of Projan et ~2.‘~ Briefly, the cell lysate was prepared by employing a freezethaw technique. The cycles were carried out at -7O’C and 40°C five times. The viscous mass was microfuged at 12,000 g for 20 min at 4’C. The supematant was analyzed by gel electrophoresis on 0.8% (w/v) agarose. The fraction of DNA present as plasmid was determined from a densitometric scan of chromosomal and plasmid DNA fractions on the agarose gel. Plasmid DNA content

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in copies per genome was computed using data as obtained above. Calculations were based on the method as referred above.

Volumetric oxygen transfer coeflcient (K,a), oxygen uptake rate (OUR), and dissolved oxygen (DO) The dynamic method of Taguchi and Humphery” was employed to measure K,a. The volumetric oxygen transfer coefficient was

varied by changing the agitation rate in the range of 250-1,200 rpm (Table I). Any change in agitation rate outside this range did not produce any significant variation in K,_a. Air flow rate has been known to have relatively small effect on K,_a values;i6 therefore, it was not used as a variable to change K,a in the bioreactor and was kept constant at 2.5 vvm. The higher rate of aeration (2.5 vvm) was chosen to ensure a sufficient supply of oxygen. The OUR values for induced and uninduced cultures were determined by the procedure described by Pirt.” Measurements of cell productivity (g 1-l h-l) and expression of foreign protein (U mg-’ p-l) were performed at periodic intervals close to K,a estimation time points. The effects of DO on cell productivity and target gene expression was observed by performing measurements at various DO levels ranging from 0.055X1.22 mmoles 1-l. The DO levels were maintained constant by adjusting air flow manually during the period of cell cultivation while keeping the agitation rate fixed at 500 rpm. In another set of experiments DO values were not maintained constant and a profile depicting change in DO levels during the course of cultivation was obtained.

Results Induction with IPTG IFTG at a 1 mM concentration was used to switch on the promoter controlling M. MspI gene; however, it is important to induce the cells only during a particular phase of growth which is physiologically favorable for the host cells to undertake the imposed synthesis of a foreign protein. An experiment was designed, therefore, to determine the phase of growth suitable for induction to get maximum target gene expression. The cells were induced at different periods during growth to obtain the corresponding initial rate of expression of the target gene (U mg-’ p-l h-l). The results as contained in Figure la indicated that there was an increase in the rate of target gene expression until the culture entered mid-exponential phase where the maximum value of 2.7 U mg-’ p-l h-’ was maintained during the entire mid-log phase (for about 1.25 h) but declined sharply subsequently in the late log and deceleration phases; however, a certain

Table 1 Agitation rates and corresponding ric oxygen transfer coefficients (K,a) Agitation (rpm) 250 500 650 750 1,000 1,200

rate

K,a (h-‘) (Uninduced) 42 56 59 61 84 102

values of volumet-

K,a (h-l) (Induced) 48 65 71 84 104 128

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Time (h) Figure 1 Induction of recombinant E. co/i with IPTG. Determination of growth phase suitable for induction (a). Rate of expression of target gene (0); and growth curve (0). Determination of period required to attain maximum expression of target gene following induction with IPTG (b). Growth curve of uninduced (0) and induced cells (0). Data points which are common for induced and uninduced cells (0) and expression of target gene (A)

level of expression (1.45 U mg-’ p-l h-l) was maintained during the stationary phase of growth. The growth curve of E. co/i strain ER1727 harboring recombinant plasmid pMSP had 4 h of exponential phase, 4 h of deceleration phase, and subsequently a long stationery phase of over 12 h. In view of the above observation, a period of induction was chosen to correspond to a mid-log phase in the experiments that followed. Addition of inducer (IPTG) to culture in the mid-log phase forced it to enter the lag phase (Figure lb) which continued for 3 h. This period corresponded to the rest of the exponential phase and early deceleration phase if the culture remained uninduced. The induced culture picked up exponential growth following the lag. This phase of exponential growth stretched over a long period of 11 h. As evident from the results presented in Figure 1b, most of the foreign protein was synthesized during the

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Time Ih) Figure 2 Dissolved oxygen concentration and target gene expression. A plot indicating maximum values for target gene expression and cell productivity that were obtained at respective DO concentrations in the bioreactor (a). Profiles were obtained for target gene expression and cell produotivity under oxygen deficient conditions: DO concentration = 0,055 mmoles I-’ (b) and oxygen-sufficient conditions: DO concentration = 0.22 mmoles I-’ (c). Parameters under investigation were evaluated periodically during 20 h of cell cultivation in a bioreactor. Cell productivity (0); expression of target gene (0); and DO level (x)



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Papers postinduction lag. The foreign protein expression remained constant after reaching its peak value of 6.7 x lo2 U mg-’ p-l during the first hour of postinduction log. A characteristic difference in the log phase of induced and uninduced cultures was observed with re ard to the specific growth rate which was higher (0.4 h- P) for uninduced cells compared to induced ones (0.12 h-l).

DO and target gene expression The supply of oxygen to the culture is a function

of the dissolved oxygen concentration in the medium; therefore, experiments have been conducted at various DO levels ranging from 0.055-0.22 mmoles 1-l to evaluate their effect on recombinant cell productivity and expression of foreign protein (Figure 2~2). It is evident from the data that both cell productivity and target gene expression increased with increasing DO level. The maximum value was attained at 0.22 mmoles 1-l. Detailed data obtained during a cultivation period of 20 h are, however, given only for DO values representing oxygen deficient (0.055 mmoles 1-l; Figure 2b) and oxygen sufficient (0.22 mmoles 1-l; Figure 2c) conditions. Under oxygen-deficient conditions the cell productivity and target gene expression followed a similar profile except for the later period (between 15-20 h) where expression went down by more than 50% while cell productivity decreased only marginally (Figure 2b). Under oxygen-sufficient conditions, target gene expression attained its maximum value of 6.6 x 10 U mg ’ p by T = 6 h (Figure 2c) while under deficient conditions, a significantly lower peak value of 3.1 x lo2 U mg-’ p-l was reached after a much longer duration (T = 10 h). Contrary to the observation made under oxygen-deficient conditions, neither cell productivity nor the target gene expression showed any decline after attaining their respective peaks during the 20-h study period under the oxygen-sufficient conditions. In another set of experiments, studies were aimed at observing changes in the DO level of the medium during cell cultivation in the bioreactor to determine the behavior of culture with respect to oxygen consumption. The results pertaining to these experiments are presented in Figure 3. The induced and uninduced cultures demonstrated a marked difference in their oxygen consumption. The DO level went down only marginally (6-10%) during O-5 h (which corresponded to an active phase of growth) if the culture remained uninduced. For the rest of the period, the DO level was maintained around a constant value except in case of oxygen-deficient conditions (0.055 mmoles 1-l) where it was observed to fall continuously (Figure 3~). Upon induction with IPTG, there was a sudden decrease in the DO level at all oxygen concentrations studied (0.055-0.22 mmoles 1-l). During the first three hours after induction, the DO level of 0.22 mmole 1-l decreased by more than 15% while that of 0.055 mmoles 1-l decreased below the detection level. Subsequently, DO levels were maintained approximately constant up to 15 h and then displayed an increasing tendency (Figure 3b).

Oxygen mass transfer and target gene expression As mentioned above, induction of the culture to turn on synthesis of the M. MspI protein led to a significant drop in 358



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Figure 3 Profile of dissolved oxygen concentration during cultivation of uninduced (a) and induced (b) ceils. The DO profiles were obtained by conducting the cell cultivation at initial DO values ranging from 0.055-0.22 mmoles I-’ in a bioreactor for 20 h under conditions mentioned in MATERIALSAND METHODS

the DO level implying that oxygen demand and consumption was raised, and thus, the need to investigate the effect of oxygen mass transfer on expression of the target gene. The related parameters such as oxygen uptake rate and cell productivity were also evaluated at different K,_a values in the range of 40-l 20 h-i. The cell productivity of uninduced culture increased from 0.3-0.98 g 1-l h-’ when K,a was raised from 43-61 h-l, but its further increase beyond 61 h-’ had no effect. A lower peak value of cell productivity (0.88 g 1-l h-l) was, however, obtained for induced culture. The OUR of uninduced culture also increased with increasing K,a and reached its maximum value of 0.095 mM 1-l h-’ at 84 h-’ (Figure 4~). Though the patterns of increase in OUR with respect to K,_u were comparable for induced and uninduced cultures, the values were higher in the former

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Figure 4 Expression of target gene, cell productivity, and oxygen uptake rate as a function of oxygen mass transfer. The different &_a values were obtained by changing agitation rates in the range of 250-1.200 rpm at a constant aeration of 2.5 wm for uninduced (a) and, induced (b) cultures. Expression of target gene (0); cell productivity (0); and oxygen uptake rate (A)

compared to the latter; for example, the maximum OUR was observed to be 0.135 and 0.095 mM 1-l h-’ for induced and uninduced cultures, respectively. The OUR of induced culture reached its peak value as stated above at K,_a of 104 h-l. A K,a of 84 h-’ obtained at an agitation speed of 750 rpm was found to be optimum for both target gene expression and cell productivity since their res ective peak values of 6.7 x lo2 U mg-’ p-l and 0.88 g I- P h-’ were attained there (Figure 4b).

Discussion It is important to note that the recombinant cells could express target gene optimally only when induced within a specified period which stretched little over 1 h during the later half of the exponential phase. Induction of cells during periods other than that as specified above resulted in the expression of the target gene at suboptimal levels; thus, the metabolic state of the host cells is an important parameter to be determined while overexpressing a foreign gene. Addition of IPTG to the medium immediately arrested the growth of exponentially growing cells which lead to a lag period of 3 h characterized by target gene expression. Enforced diversion of protein synthesizing machinery of the

mass transfer

on: S. Bhattacharya

and A. Dubey

host E. co/i to undertake large-scale synthesis of foreign protein at the cost of its own cellular proteins required for growth and metabolism appeared to be the cause for this lag. Also evident from the experimental data is the fact that most of the foreign protein was synthesized by the induced cells during the above lag period. When induced culture entered the exponential phase, the cells divided more slowly compared to uninduced culture and consequently had a longer exponential phase. The specific growth rate of uninduced culture (0.4 h-‘) was in good agreement with other strains of E. coli.” Upon induction, the specific growth rate decreased by more than three times (0.12 h-‘) which explained nearly three times expansion of log phase of induced culture over that of uninduced culture, Only scant information is available in the literature regarding physiological imbalance created in recombinant cell-harboring cloned genes placed under IPTG inducible promoter. George et ~1.‘~ reported that E. coli strain JM109 carrying a fusion gene under the tat promoter accumulated pyruvate in addition to acetate when induced with IPTG. The uninduced cells accumulated acetate and no pyruvate; thus, IPTG induction had clearly caused changes in the physiology and metabolism of the cells. The onset of the lag phase and considerable reduction in the specific growth rate due to IPTG induction as observed in the present work further confirms this conclusion. Dissolved oxygen is reported to have variable effects on heterologous gene expression in recombinant bacteria. An increase in the DO level caused an increase in P-lactamase expression in Streptomyces lividans2’ contrary to a previous report by Ryan et a1.21 where p--lactamase expression in recombinant E. coli strains was found to decrease with an increase in the DO level. Decrease in production of a cloned antigen in E. coli with increasing DO level was also observed in another study.22 In the present istudy, a significant increase (>50%) in M. MspI synthesis occurred when DO was raised from 0.055 to 0.12 mmoles 1-l. The cause of variable oxygen effects as reported in various studies may be attributed to different host-vector systems as well as different expression systems used. Our results indicated that under oxygen-deficient conditions, the level of target protein came down drastically when cultivated beyond 10 h. This implies that while synthesized methylase degraded, no new synthesis occurred; thus, there was no or minimal expression during prolonged cultivation under oxygendeficient conditions. Under conditions of surplus oxygen, constancy of the synthesis peak for foreign protein during prolonged cultivation might be a consequence of equal rates of synthesis and degradation. A continuous fall in the DO level for three hours upon IPTG induction suggested that with increasing synthesis of foreign protein, oxygen consumption increased. Since protein synthesis is an energydependent process, accelerated synthesis of a target protein might impose a burden on cellular energy.3 To overcome such an energy burden, oxygen consumption of the culture should increase because oxygen is crucial for energy generation in aerobic microorganisms.’ Increase in OUR by 50% upon induction (Figure 4) would enable the cells to enhance their oxygen consumption. A greater part of the consumed oxygen under conditions of enhanced OUR following induction was likely to be utilized toward the synEnzyme

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thesis of foreign protein in large quantities. In view of cessation of cell growth during this period, only a small part of the consumed oxygen would, however, be expected to be utilized for maintaining cell viability. A rising tendency in DO level during the later period of cell cultivation might be due to a declining tendency in expression level during that period as was observed (compare Figure 2c and Figure 3b, DO = 0.22 mmoles 1-l). It has been retorted that OUR is affected by agitation rate and hence K,a 3; therefore, K,a in the bioreactor should be maintained so as to meet the demand of enhanced oxygen uptake u on induction. In the present system, a K,a value of 84 h- P was best for optimal expression of the target gene. Low cell productivity of induced culture might be due to greater allocation of cellular resources in overexpression of the target gene which causes reduction in growth. As emerged from the present studies, target gene overexpression and cell productivity were not colinear. Foreign protein synthesis at high levels invariably caused a reduction in cell productivity; furthermore, operating conditions for achieving optimal overexpression of the target gene in question were a DO of 0.16 mmoles 1-l and KLa of 84 h-‘; however, to arrive at a generalized conclusion regarding viable correlation between cell production, overexpression, and the parameters examined, studies involving different overexpression systems would be necessary.

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