Biotechnol. Prog. 1001, 7, 99-1 10
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Immobilization of Escherichia coli JM103[pUC8] in K-Carrageenan Coupled with Recombinant Protein Release by in Situ Cell Membrane Permeabilization Wen Ryan and Satish J. Parulekar* Department of Chemical Engineering, Illinois Institute of Technology, Chicago, Illinois 60616
Immobilization of Escherichia coli JM103[pUC8] was carried out with K-carrageenan as the support matrix. Substantial natural excretion of P-lactamase, attributable to the less intact membrane of plasmid-harboring cells, was observed in immobilized cell cultures. Nevertheless, a significant portion of the P-lactamase produced was retained in the cells. As compared to suspension cultures, much higher 0-lactamase activities, especially in the extracellular liquid, and much longer retention of plasmid-bearing cells (improved plasmid stability) were observed in immobilized cell cultures. Further enhancement in excretion of the recombinant protein (P-lactamase) was achieved by permeabilization of cell membrane by periodic exposure of the immobilized cell cultures to ethylenediaminetetraacetic acid (EDTA). While the presence of EDTA led to some suppression of cell growth in suspension cultures, cell growth in gel beads was not affected by EDTA to the same extent, possibly due to lesser exposure of immobilized cells to EDTA. Exposure of immobilized cell cultures to EDTA presumably inhibited plasmid replication and led in turn to diversion of cellular resources for the support of expression of plasmid genes. Indeed, treatment of the immobilized cell cultures with EDTA resulted in increased production of P-lactamase when compared to the enzyme production in EDTA-free cultures. More frequent addition of EDTA increased the period of retention of plasmid-bearing cells in these cultures but did not have any noticeable adverse effect on synthesis of P-lactamase. Improvement in plasmid stability in EDTA-treated immobilized cell cultures was ascribed to the reduction in the growth rate differential between plasmid-free and plasmid-bearing cells, since plasmid-free cells were subject to more reduction in specific growth rate than were plasmid-bearing cells.
Introduction Recombinant DNA technology provides tremendous possibilities for enhanced production of many valuable biologicals (Imanaka, 1986; Georgiou, 1988; Wang, 1988). The use of plasmids as extrachromosomal DNA elements for transfer of foreign genes into suitable hosts is made difficult by unstable plasmid maintenance (plasmid instability) and increased behavioral complexities of recombinant systems, when compared to nonrecombinant systems, due to host-vector interactions (Imanaka and Aiba, 1981; Imanaka, 1986;Peretti and Bailey, 1987;Georgiou, 1988; Ryan et al., 1989; Ryan and Parulekar, 1990). Escherichia coli has been by far the most popular host species for application of recombinant DNA technology owing to the wealth of genetic and physiological information about it. Various approaches, including alterations in genetic characteristics of plasmids [such as plasmid copy number (Seoand Bailey, 1985)and plasmid size (Zund and Lebek, 1980; Summers and Sherratt, 1984; Bron and Luxen, 1985; Cheah et al., 1987; Ryan et al., 198911, manipulation of cultivation conditions (Ryan et al., 1989; Ryan and Parulekar, 1990), and use of operational strategies such as two-stage operation (Siege1 and Ryu, 1985;Lee et al., 1988), cyclic operation (Stephens and Lyberatos, 1988;Impoolsup et al., 1989;Weber and San, 1989),
* To whom correspondence should be addressed. 8756-7938/91/3007-0099$02.50/0
and whole-cellimmobilization (Dhulster et al., 1984;Georgiou et al., 1985; de Taxis du Poet et al., 1986; Bailey et al., 1987;Berry et al., 1988;Marin-Iniesta et al., 1988;Barbotin et al., 1989; Marin-Iniesta, 1989; Sayadi et al., 1989) have been investigated for improving plasmid stability and for enhancing expression of plasmid genes in recombinant E. coli. Whole-cell immobilization facilitates the separation of cells from extracellular products and permits the use of continuous reactors while avoidingcell washout even under conditions of negligible cell growth (Karel et al., 1985). Higher volumetric reaction rates, higher overall productivity compared to suspension cultures, flexibility in reactor choice, and improved thermal and operational stability are additional advantages of whole-cell immobilization (Karel et al., 1985). Development of techniques for genetic manipulation of industrially important microorganisms and animal and plant cells have provided additional incentives for further development of immobilization techniques. Whole-cell immobilization enables bioreactor operation at dilution rates greater than those up to which steady-state operation of continuous suspension cultures is possible (increasing, thereby, up to a certain extent, volumetric productivity) and in the case of recombinant systems may result in redirection of cellular resources to promote plasmid-related activities. All of these attributes of course have a positive impact on the
0 199 1 American Chemical Society and American Institute of Chemical Engineers
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overall productivity of recombinant systems. For recombinant microbial systems subject to segregational plasmid instability, immobilization of recombinant cells has been reported to result in improved plasmid stability due to increased plasmid copy number and/or prolonged retention of plasmid-bearing cells in the support matrix (Bailey et al., 1987; Berry et al., 1988;Marin-Iniesta et al., 1988; Sayadi et al., 1989). The characteristics of various cellular processes such as growth and survival of cells, plasmid replication, and synthesis of plasmid-encoded products may be altered upon immobilization of recombinant cells owing to significant resistances in the support matrices to the transport of nutrients, including oxygen. Limitations on transport of one or more nutrients can significantly influence effectiveness and productivity of immobilized cell cultures (Karel et al., 1985). Excretion of the target product is essential in order for whole-cell immobilization to be an effective technique for continuous recovery of the product in the bulk liquid phase. Most of the proteins produced by E. coli are largely retained in the cells. Such retention further complicates downstream processing and may have detrimental effects on cellular physiology, especially when the target protein is overproduced. Furthermore, excessive accumulation of the target protein may result in a loss of its activity due to proteolytic degradation (Georgiou, 1988; Wang, 1988). Such accumulation of foreign proteins may also lead to formation of inclusion bodies. Recent studies have demonstrated that inclusion bodies may be formed in both cytoplasm and periplasm, and recovery of proteins from such protein aggregates is usually difficult (Georgiou et al., 1986;Georgiou and Shuler, 1988). Since most proteins are synthesized in the cytoplasm, excretion of a target protein begins with its secretion across the cytoplasmic membrane into the periplasm. Excretion of periplasmic proteins, including some enzymes such as 8-lactamase produced by E. coli, is regulated by the outer membrane. Such excretion results from the disorganization ofthe outer membrane, causing leakage of periplasmic proteins from the cell. It has been reported (Oliver, 1985) that it is due to the lack of a general support machinery for protein export in E. coli that anumber of proteins that are excreted naturally by the donor species (from which genes encoding the proteins are derived) are not excreted or excreted to only a small degree by recombinant E. coli. Although some sugars are useful for protein stabilization (Arakawa and Timasheff, 1982; Georgiou et al., 1986; Bowden and Georgiou, 1988),the requirement of the presence of these sugars at high concentrations makes their application in situ impractical. The development of alternative methodologies for in situ protein excretion in cultures of recombinant E. coli is therefore of critical importance. The efficacy of protein release in batch suspension cultures of E. coli JM103[pUC8] by in situ cell membrane permeabilization with ethylenediaminetetraacetic acid (EDTA) and phenethyl alcohol (PEA) was examined in one of our previous studies (Ryan and Parulekar, 1991b). While application of PEA in growing cultures was difficult due to the undesirable interference of PEA with processing of the precursor @-lactamase,EDTA was found to be appropriate for enhancing the protein excretion in this recombinant system without sacrificing the overall &lactamase production. The deteriorated cell envelope of recombinant cells, resulting from harboring of the recombinant plasmid a t high DNA content, was observed in this previous study (Ryan and Parulekar, 1991b) to be partially responsible for the profound growth rate differential between plasmid-free and plasmid-bearing cells.
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The presence of a cell membrane permeabilizing agent decreased substantially the growth rate differential between E. coli JM103 and E. coli JM103[pUC8]. These results suggest, therefore, that the segregational plasmid instability observed in continuous cultures may be alleviated to a certain extent by periodic exposure of recombinant cell cultures to EDTA. In this paper, we report on improvement in plasmid stability and increase in synthesis and excretion of the plasmid-encodedprotein 6-lactamase for this recombinant system via whole-cell immobilization. EDTA was employed in some experiments on a periodic basis for permeabilization of the cell membrane so as to enhance p-lactamase release and reduce the growth rate differential between plasmid-free cells (formed in the absence of antibiotic selection pressure) and plasmid-bearing cells. The support material used for whole-cell immobilization was K-carrageenan. The motivation for its selection was the ease of formation and disintegration of gel beads, the latter being important for convenient measurement of activities or concentrations of various species in the beads.
Materials and Methods Bacterial Host and Plasmid. The host used in this study was Escherichia coli JM103 (Vieira and Messing, 1982). The plasmid employed, pUC8 (a relaxed plasmid) (Messing, 1983), is harbored in JM103 a t high copy numbers and encodes the protein p-lactamase, conferring the host resistance to ampicillin (Ryan and Parulekar, 1990, 1991a,b). The calcium treatment method of Maniatis et al. (1982)was used for transformation of the host. Stock cultures of plasmid-bearing cells were maintained on LB-agar (10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast extract, and 15 g/L agar) (Miller, 1972) plates at 4 "C (for routine usage) and in LB-glycerol medium at -80 "C (for long-term culture maintenance) in the presence of 0.1 g/L ampicillin (sodium salt, Sigma Chemical Co.). Cultivation. A phosphate-enriched minimal medium, M9P (with 2 g/L glucose as the limiting carbon source), was used for cultivation of recombinant cells (Miller, 1972; Ryan et al., 1989; Ryan and Parulekar, 1990, 1991a,b). Besides glucose, this medium contained (per liter) 12.0 g of NazHP04, 6.0 g of KH~POI,1.0 g of NH4C1, 0.5 g of NaC1, 0.24 g of MgS04, 0.015 g of CaC12.2H20, 0.06 g of L-proline, and 0.001 g of thiamine hydrochloride. For cultivation of immobilized cells, the medium was supplemented with 0.1 M KCl to preserve the integrity and material strength of gel beads. The inocula for preparation of cell-laden gel beads were obtained by serial subculturings in LB and M9P, ampicillin being present in each subculture a t 0.1 g/L. Cells from the exponential growth phase of the M9P subculture were used to inoculate 40 mL of M9P medium containing 0.1 g/L ampicillin, the inoculum size being 1.0 mL of the subculture. The resulting culture, after the recombinant cells were allowed to grow into early exponential growth phase, was warmed up to 42 "C and used for preparation of the gel-cell mixture as described next. Whole-Cell Immobilization and Bioreactor Operation. A 3% (w/v) gel solution was prepared by dissolving2.4g of K-carrageenan (Type 111,Sigma Chemical Co.) in 80 mL of a saline solution (containing 8.5g/L NaC1) with mild heating. After being autoclaved for 20 min, the gel solution was cooled to 42 "C and mixed with a 40-mL culture of plasmid-bearing cells a t this temperature. Gel beads were generated by pumping the gel-cell mixture (maintained at 42 "C) dropwise through a 0.1-mL glass pipet (Fisher Scientific) with a peristaltic pump into a
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bioreactor (1-L Bellco spinner flask, Model 1968-SOO26) containing 400 mL of an ice-cold 0.3 M KC1 solution to harden the gel beads. Gel beads with an average diameter of 4 mm were obtained with a pumping rate of 3 mL/min. About 3600 beads were generated for each experiment. After preparation of gel beads, the solution in the bioreactor was replaced with a fresh 0.3 M KC1solution and the bioreactor was placed in an ice bath with mild aeration for at least 16 h for further curing of the gel beads. The cured gel beads were washed twice at room temperature by pumping a fresh KC1 solution into the bioreactor and pumping out the waste fluid. After removing the second batch of waste fluid, the bioreactor was filled with sterile KC1-supplementedM9P medium to a total working volume of 400 mL. The feed and effluent volumetric flow rates were fixed at the desired values. Vigorous circulation of the gel beads and the gel bead free suspension was achieved in bioreactor operation without mechanical agitation by employinga high aeration rate (7.29 L/min). During the first 90 min subsequent to initiation of liquid feed, the liquid medium was allowed to saturate with oxygen at room temperature. The dissolved oxygen (DO) level was adjusted to 100% saturation upon switching the set point for bioreactor temperature to 37 "C (time zero). During the transition period (first 90 min) following this switch, DO level usually increased beyond 100% saturation due to the influence of temperature on mass transfer of oxygen across the gasliquid interface. Frequent calibration around 100% saturation was carried out until the DO level stopped increasing. The culture pH, measured by a pH probe (Ingold Electrode), was controlled at 7.4 with a pH controller (Horizon Ecology Co., Chicago, IL) by addition of 0.5 N NaOH as appropriate. Where applicable, in situ forced release of P-lactamase was accomplished by periodic addition of 8 mL of a 200 mM EDTA solution. Assays. Each culture sample contained 10-15 gel beads. After these were separated from gel bead free suspension and washed with 25 mL of a 8.5 g/L NaCl solution, 10 of these were completely disintegrated, by warming to sufficiently high temperature, in 5 mL of a 10 g/L sodium citrate solution (Fisher Scientific). The resulting solution was analyzed to measure concentrations of viable plasmidbearing and plasmid-free cells in gel beads and activities of P-lactamase in the abiotic and biotic phases of the gel beads. The bulk suspension remaining after removal of gel beads was analyzed for glucose, viable plasmid-bearing and plasmid-free cells, and intracellular and extracellular p-lactamase to measure activities/concentrations of these in the gel bead free suspension. Cell growth in gel beads and in gel bead free suspension was followed by measurement of viable cell counts of suitably diluted portions of appropriate suspensions on LB plates. Plasmid stability was routinely monitored by comparing colony numbers on LB plates without ampicillin [colony counts of total (plasmid-bearingand plasmidfree) cells] and with 0.1 g/L ampicillin (colony counts of plasmid-bearing cells). Glucose concentration was measured with a YSI glucose analyzer (Model 27, Yellow Springs Instruments, Inc.). The 0-lactamase in the abiotic phase was recovered from each sample (the gel bead free suspension or the cell-liquid suspension obtained upon complete disintegration of gel beads, as appropriate) after removal of cells from the sample by centrifugation for 5 min with a microcentrifuge(Model 235C, Fisher Scientific). The 0-lactamase retained in the cells was released by the osmotic shock method described elsewhere(Neu and Chou, 1967; Ryan et al., 1989) and the resulting cell debris was
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removed by centrifugation before the activity of the released P-lactamase was measured. The activity of 0-lactamase was measured spectrophotometrically by using cephalothin (sodium salt, Sigma Chemical Co.) as the substrate at 255 nm with a Bausch & Lomb Spectronic 21 spectrophotometer (O'Callaghan et al., 1968; Ryan et al., 1989). One unit of p-lactamase is defined as the amount of p-lactamase that can hydrolyze 1pmol of cephalothin in 1 min at pH 7.0 and 25 "C.
Results and Discussion The recombinant E. coli strain employed in the present study has been found to excrete significant fractions of p-lactamase synthesized into extracellular liquid in both batch and continuous suspension cultures under a variety of environmental and operating conditions (Ryan and Parulekar, 1990,1991a). The significant natural excretion of the plasmid-encoded protein has been linked to the deterioration of cell envelope resulting primarily due to high plasmid DNA content of plasmid-bearing cells (Ryan and Parulekar, 1991b). While excretion is highly desirable as far as recovery of the plasmid-encoded protein is concerned, the defective cell membrane hampers growth of recombinant cells. The growth rate differential between plasmid-free and plasmid-harboring cells is increased as aresult. Aside from the metabolic burden due to plasmidmediated activities, including plasmid replication and expression of plasmid genes, the deteriorated cell membrane (which contributes in part to the significant growth rate differential) may in fact be the more immediate cause for the severe segregational plasmid instability observed in continuous cultures in the absence of a selection pressure (Ryan and Parulekar, 1990, 1991a,b). Although the manner in which plasmid DNA interferes with cell membrane structure remains largely unclear, it is obvious that there are both advantages and disadvantages associated with the defective cell envelope. Thorough consideration of these is essential while developing rational strategies for improving the performance of recombinant microbial systems (increased production of the target plasmid-encoded product(s) and improved plasmid stability). In view of the above, it appears that plasmid stability may be improved by subjecting recombinant cell cultures to environments such that both plasmid-freeand plasmidbearing cells have defective cell envelopes, thereby reducing the growth advantage of the former cell type over the latter. After all, release of the target product from cells is highly desirable and fixing the envelope of plasmidbearing cells may not be easy, especially when the benefits associated with high plasmid copy numbers are to be preserved. In a previous study (Ryan and Parulekar, 1991b), the presence of a cell membrane permeabilizing agent (EDTA) in batch suspension cultures of E. coli JM103[pUC8] led to a reduction in the growth rate differential between plasmid-bearing and plasmid-free cells and substantial enhancement in release of the plasmid-encoded protein without affecting its overall production. Furthermore, p-lactamase was efficiently preserved when excreted into the abiotic phase as opposed to the severe in vivo degradation the target protein was subject to when retained in the cells. Since the specific growth rates of both plasmid-free and plasmid-bearing cells are significantly reduced in the presence of EDTA, it may not be practical to employ this chemical in continuous suspension cultures owing to the possibility of accelerated washout of recombinant cells. In this regard, whole-cell immobilization appears to be an attractive alternative in cultivations involving EDTA
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o ICR (immobilized) ICR (released) a
FCR
0.8.
u
0.6.
\ A-
N, Figure 1. Profiles of plasmid-bearing fractions of the cell populations (i) in gel beads (0) and in the gel bead free suspension (0) in an immobilized cell reactor (ICR) experiment (dilution rate = 0.395 h-1) and (ii) in a continuous suspension culture [a, free cell reactor (FCR)]. The data for the continuous suspension culture (dilution rate = 0.398 h-l) are from Ryan and Parulekar (1991a). C+ and C represent concentrations of viable plasmidbearing cells and viable total (plasmid-bearing plus plasmidfree) cells, defined as number of cells per unit volume (volume of mobile cell-liquid suspension or volume of gel beads as appropriate). In this figure and where applicable in the figures that follow, N , represents the number of residence times after initiation of continuous operation (Le., product of real time after initiation of continuous operation and dilution rate).
treatment. With this in mind, E. coli JM103[pUC8] was immobilized in a K-carrageenan gel to investigate the efficacy of cell entrapment for improving plasmid stability and synthesis and excretion of the plasmid-encoded product (P-lactamase). The effects of dilution rate and EDTA treatment on productivity of immobilized recombinant cells were examined. The measurement of plasmid DNA content by the method described previously (Ryan and Parulekar, l990,1991a,b) was unsuccessful due to unexpected interference a t the stage of restriction enzyme cleavage. Arguments concerning the response of plasmid DNA content of recombinant cells to whole-cell immobilization and EDTA treatment are therefore based on our previous studies with suspension cultures of this recombinant system (Ryan and Parulekar, l990,1991a,b) and results of other investigators (Dhulster et al., 1984;de Taxis du Poet et al., 1986; Berry et al., 1988; Marin-Iniesta et al., 1988; Sayadi et al., 1989). In the discussion that follows, the term total enzyme activity refers to the sum of extracellular and intracellular P-lactamase activities. Comparison of Immobilized Cell Cultures and Suspension Cultures. The profiles of plasmid-bearing fractions of the cell populations (i) in gel beads and in the mobile (gel bead free) suspension in an experiment involving immobilized recombinant cells and (ii) in a continuous suspension culture (Ryan and Parulekar, 1991a) are shown in Figure 1. With the exception of dilution rate, all operating parameters in these experiments were identical, while the dilution rates (based on the total bioreactor volume) were nearly identical. The dilution rate for experiments with immobilized cells is defined as the ratio of volumetric feed rate to volume of suspension of gel beads (total bioreactor volume). The cell concentrations in this paper represent numbers of viable cells per unit volume (volume of gel beads or gel bead free suspension, as appropriate). The number of residence
times after initiation of continuous operation (Nr,product of real time after initiation of continuous operation and dilution rate) is an indicator of the real time. Due to the significant transients observed in each continuous operation, where the specific growth rate of neither plasmidbearing nor plasmid-free cells is time-invariant, it is not prudent to consider the number of generations of either cell type as an accurate indicator of real time. It is interesting to observe the similarity in patterns of decline in the plasmid-bearing fractions ( C + / C ) of the cell populations (a) entrapped in the gel beads and (b) in the continuous suspension culture. The improvement in plasmid stability upon immobilization of recombinant cells is evident from the profiles in Figure 1. Some insight into the improvement in plasmid stability can be obtained by comparing concentrations of plasmidbearing and total cells in the two experiments. In the immobilized cell reactor experiment, the gradual decline in the plasmid-bearing fraction of immobilized cell population from 1.0 to 0.05 was accompanied by a decline in total cell concentration in gel beads from nearly 7.5 x 108 to nearly 5 X lo8 cells/mL-I (see Figures 1 and 3). The notation mL-F and mL-I denotes mL of gel bead free suspension culture and mL of immobilized phase (gel), respectively. During this period, concentration of plasmidbearing cells in gel beads thus decreased from nearly 7.5 X lo8to nearly 2.5 X lo7cells/mL-I. For identical decline (from 1.0 to 0.05) in the plasmid-bearing fraction of the total cell population in the continuous suspension culture experiment (Ryan and Parulekar, 1991a), it is estimated that concentration of plasmid-bearing cells decreased from nearly 3.95 X lo8to nearly 9.9 X lo7cells/mL-F, while the total cell concentration increased from nearly 3.95 X 108 to nearly 2 x lo9 cells/mL-F. The estimations for the continuous suspension culture experiment are based on experimental data on total cell mass concentration and plasmid-bearing fraction of the total cell population and correlations between number densities and mass concentrations of plasmid-bearing and plasmid-free cells reported in Ryan and Parulekar (1991a). I t follows, therefore, that although the concentration of plasmid-bearing cells (prior to significant accumulation of piasmid-free cells) was greater in gel beads than in the continuous suspension culture, the concentration of plasmid-free cells (when the cell population was composed primarily of these cells) in gel beads (nearly 4.75 X lo8cells/mL-I) was substantially lower than that in the continuous suspension culture (nearly 1.9 X lo9cells/mL-F). It is therefore evident that the improvement in plasmid stability upon cell entrapment is attributable to a higher concentration of recombinant cells and restricted and slower accumulation of plasmidfree cells in gel beads. It is worth noticing that the profile of plasmid-bearing fraction of the cell population in the gel bead free suspension in the experiment employing immobilized cells lies between the profiles of the same in the continuous suspension culture and in the gel beads in the former experiment. This indicates that a significant fraction of the plasmid-bearing cell population in the bulk suspension (bioreactor volume not occupied by the gel beads) is derived from leakage of plasmid-bearing cells from gel beads and that such leakage is continuous. Each gel bead can therefore be regarded as a "mini" continuous culture in which cells grow and some of these escape from the bead. If the supply of essential nutrients to the gel beads is adequate, the rates of cell growth in and cell leakage from the gel beads may be nearly equal and the cell concentrations may remain nearly unchanged over a certain time
103 (a)
80 I
0
ICR FCR
"-
a
\
N, Figure 2. Profiles of extracellular enzyme (P-lactamase) activity (EE)and total enzyme activity (TE) in an immobilized cell reactor experiment (0, dilution rate = 0.395 h-l) and in a continuous suspension culture (0, dilution rate = 0.398 h-'). The data for the continuous suspension culture are from Ryan and Parulekar (1991a). The enzyme activities are expressed as 0-lactamase units per milliliter of mobile cell-liquid suspension.
period. A quasi-steady state for recombinant cells in the gel beads may result in such situations as typified by the results discussed later (see Figure 3). Production of @-lactamasein the continuous-flow immobilized cell reactor (ICR) and the continuous suspension culture (free cell reactor-FCR) are compared in Figure 2. The total 0-lactamase activity (TE) represents the sum of intracellular and extracellular (EE) P-lactamase activities. For the experiments involving immobilized recombinant cells, p-lactamase activities are expressed on the basis of gel bead free suspension (units/mL-F) as if all the P-lactamase synthesized is released into the abiotic phase of the gel bead free suspension. Such expression of the enzyme activities is very convenient for assessing the volumetric productivity of the enzyme for the recombinant system. The significantly higher total 8-lactamase activities in the experiment employing immobilized cells when compared to those in the suspension culture experiment can be attributed mainly to maintenance of higher concentrations of the recombinant cells at lower specific growth rates and longer retention of these cells in the immobilized state. It is anticipated that, under comparable conditions in the bulk liquid phase, immobilized recombinant cells will have a lower specific growth rate than their freely suspended counterparts due to substantial diffusional resistances for transport of nutri-
ents essential for growth, especially oxygen, in the former state. One would therefore expect agreater extent of plasmid-related activities (including plasmid replication and synthesis of plasmid-encoded products) in immobilized recombinant cells in this situation. The activity of @-lactamasein all experiments was estimated by measuring the rate of disappearance of cephalothin (substrate for enzymatic hydrolysis). This rate would represent the rate of hydrolysis of cephalothin by p-lactamase provided that there is no other source of disappearance of cephalothin such as its possible nonspecific binding to carrageenan. Great care was taken while separating gel beads from gel bead free suspension in each sample withdrawn from the reactor. The suspension of the separated gel beads in the sodium citrate solution was warmed to sufficiently high temperature to ensure complete disintegration ofgel beads. The following control experiments were performed to estimate the extent of binding, if any, of cephalothin to carrageenan present (in dissolved state) in the cell-liquid suspension resulting from complete disintegration of gel beads. Cell-free gel beads were prepared following the procedure described under Materials and Methods, cured for an extended period, and then disintegrated as described under Materials and Methods. The resulting solution (0.1-0.5 mL) was mixed with 0.1 mL of 1.0 mM cephalothin solution and an appropriate amount of 0.1 M NaZHP04 solution (pH 7.0) to a final volume of 1.0 mL (Ryan et al., 1989). The absorbance of the mixture (which is proportional to cephalothin concentration) was recorded every 15 s for 2 min. No significant alteration in cephalothin concentration was observed in any of the control experiments, which indicates that the binding of the substrate to carrageenan in the solution was, if any, negligible. In our previous studies with batch and continuous suspension cultures of the recombinant cells (Ryan and Parulekar, 1990, 1991a), the plasmid DNA content was observed to increase with decreasing specific growth rate of the cells to copy number levels close to, or in some cases in excess of, 1000 plasmid monomer equiv per cell. Consequently, plasmid DNA content of the immobilized recombinant cells is expected to be greater than that of freely suspended recombinant cells due to slower growth of the former. Sayadi et al. (1989) have indeed observed plasmid copy numbers for immobilized recombinant cells (E.coli W3101[pTG201])to be greater than those for freely suspended recombinant cells. In view of this and our previous observations (Ryan and Parulekar, 1991a,b) regarding connection between plasmid-related activities and integrity of the envelope of plasmid-bearing cells, one would expect that the envelopes of recombinant cells will be less intact when the cells are immobilized than when they are freely suspended owing to further promotion of plasmid-related activities in the former state. This would translate into higher extent of excretion of p-lactamase by recombinant cells in an immobilized state than in a freely suspended state. In this regard, it is interesting to observe that significantly greater portions of the total p-lactamase activities (TE) were detected in the abiotic phase [i.e., extracellular @-lactamase activities (EE) were greater] in the immobilized cell reactor experiment than in the continuous suspension culture experiment (see Figure 2). As the results to be presented later indicate, the intracellular 0-lactamase activities for cells in the gel bead free suspension were a small fraction of the intracellular 0-lactamase activities for the gel-entrapped cells, indicating that a very large fraction of the total P-lactamase produced was synthesized
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by immobilized recombinant cells. The increased percentage of the total @-lactamaseactivity appearing in the abiotic phase in the immobilized cell reactor is therefore attributable mainly to immobilization of recombinant cells. The results in Figures 1 and 2 for immobilized cells were obtained from an experiment conducted a t a dilution rate of 0.395 h-l based on a total culture volume (including volume of gel beads) of 400 mL. Not all of the total culture volume is accessible to the cell-liquid suspension and therefore the reciprocal of the dilution rate defined on such a basis exceeds the average residence time of the mobile cell-liquid suspension in the bioreactor. The dilution rate based on the gel bead free volume of the culture (approximately 290 mL) would be 0.545 h-l. The volume of the bioreactor that is occupied by the cell-liquid suspension is actually greater than the gel bead free volume of the bioreactor, since the porous space in the gel beads is also accessibleto the cell-liquid suspension. The "actual dilution rate" therefore was between 0.395 and 0.545 h-l. The porosity of gel beads prepared from a 3% (w/v) K-carrageenan solution has been reported to be 0.835 (Moon and Parulekar, 1991). The dilution rate basedon the bioreactor volume accessible to cell-liquid suspension therefore may have been 0.42 h-l or in excess of it. Under the environmental and operating conditions employed for this experiment, the results of our previous study (Ryan and Parulekar, 1991a) with continuous suspension cultures indicate that retention of recombinant cells in a suspension culture is not possible a t or beyond the dilution rate of 0.416 h-I due to their physical washout. Considering this, retention of recombinant cells in the gel bead free suspension in the immobilized cell reactor experiment over a period much longer than that for which such retention was possible in the continuous suspension culture (at a dilution rate of 0.398 h-l) indicates that significant leakage of recombinant cells from the gel beads was largely responsible for the presence of these cells in the bulk suspension. Such cell leakage has also been observed in other bacterial systems immobilized by using gel beads and hollow fibers (Berry et al., 1988; Marin-Iniesta et al., 1988; Sayadi et al., 1989; Karel and Robertson, 1989). Effect of Dilution Rate on Cell Growth. One of the merits of whole-cellimmobilization is that it permits bioreactor operation a t dilution rates in excess of the maximum specific cell growth rate. Accumulation of cells in the bulk liquid phase can be minimized in these situations. Profiles of the total (plasmid-free plus plasmid-bearing) cell concentrations in the gel beads (CI) and in the gel bead for two dilution rates are presented free suspension (CF) in Figure 3. Profiles of the plasmid-bearing fraction of the immobilized cell population for these dilution rates are presented in Figure 4. At the dilution rate of 0.395 h-l, concentration of cells in the mobile (gel bead free) suspension is substantial and a two-stage phenomenon, associated with the severe segregational plasmid instability, is observed for the cell population in the gel bead free suspension. This two-stage phenomenon was also observed in continuous suspension cultures of this recombinant system (Ryan and Parulekar, 1990, 1991a). During the quasi-stationary state, the culture in the mobile suspension is composed mainly of plasmid-bearing cells, while it is increasingly dominated by plasmid-free cells as the steady state is approached. The increase in the total cell concentration in the gel bead free suspension during the period between the quasi-steady and steady states is due to the substantially higher yield of plasmid-free cells compared to that of plasmid-bearing cells. Significant transients in most of the uncontrolled culture parameters,
(a) 0
D = 0.T4S hr-I CI (calb/mGI)
C F (celb/mL-F)
40
.CI
20
eo
40
j $ 4
BO
100
0
30
0
40
60
Nr Figure 3. Profiles of the total viable cell concentrations (i) in gel beads [o,CI: cells per milliliter of immobilized phase (gel)] and (ii) in gel bead free suspension ( 0 ,CF: cells per milliliter of gel bead free suspension) for immobilized cell reactor experiments conducted at dilution rates (D)of 0.395 h-* and 0.745 h-l. Y
0.6.
0.4.
0.2 '
0.0 0
20
40
00
80
I
N, Figure 4. Profiles of the plasmid-bearing fraction of the immobilized cell population for immobilized cell reactor experiments conducted at dilution rates of 0.395 h-l (0) and 0.745 h-l (0). CI+ and CI denote concentrations of plasmid-bearing cells and total cells, respectively, in gel beads and are expressed as cells per milliliter gel bead.
including the plasmid-bearing fraction of the total cell population in the mobile suspension, were observed during this period. At this dilution rate, accumulation of cells in gel beads is restricted due to the rather limited availability of
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Table I. Average Glucose Concentration (S) and Average Dissolved Oxygen (DO) Level in Quasi-Stationary and Stationary States of Immobilized Cell Reactor (ICR) Experiments at Two Different Dilution Rates dilution rate 0.395 h-l 0.745 h-l quasi-stationary state
s, g / L
0.088 f 0.004 63.7 f 3.2
0.790 f 0.045 92.5 f 2.6
s, g / L
0.022 f 0.004 51.0 f 1.2
0.521 f 0.046 78.6 f 4.4
DO, 94 saturation stationary state DO, "; saturation
essential nutrients, presumably due to substantial consumption of the nutrients by the cells in the mobile suspension (see also Table I). As a result, the two-stage phenomenon observed for the culture in the gel bead free suspension is absent as far as the culture in gel beads is concerned. The difficulty for cell growth in gel beads should be evident from the observation that the total cell attains a maximum value concentration in gel beads (CI) and declines thereafter while the total cell concentration in the gel bead free suspension is low and increasing very slowly during the quasi-steady state of the gel bead free suspension culture. Plasmid-bearing cells are retained in the gel-entrapped cell population for a period in excess of 50 residence times after the initiation of continuous operation. At the higher dilution rate (0.745 h-l), the total cell concentration in the gel bead free suspension is substantially reduced owing to accelerated washout of both plasmid-bearing and plasmid-free cells at this dilution rate as compared to that at the lower dilution rate. It must be emphasized that, under the cultivation conditions employed here, prolonged retention of plasmid-bearing cells in the gel bead free suspension is impossible, while the dilution rate employed is not too far from the maximum specific growth rate of plasmid-free cells (Ryan and Parulekar, 1991a). A consequence of this is the increased transport of nutrients into porous region of gel beads, which results in substantial promotion of growth of immobilized cells. The total cell concentration in gel beads is therefore severalfold higher than that in the gel bead free suspension culture. Promotion of cell growth in gel beads owing to increased availability of nutrients gives rise to a two-stage phenomenon for the immobilized cell population, similar to the one observed for the cell population in the gel bead free suspension in the lower dilution rate (0.395 h-l) experiment. After an initial increase in the total cell concentration in gel beads during the first 20 residence times following initiation of continuous operation, a quasi-steady state was reached during which the total cell concentration in gel beads remained relatively unchanged. This suggests that the cell leakage from gel beads was significant, the leakage rate being close to the net cell growth rate. Plasmid-free cells accumulated gradually yet slowly in the gel beads during the quasi-steady state. A period of fast transients leading to what appeared to be a final steady state followed the quasi-steady state (see Figures 3 and 4). The plasmid-bearing fraction of the immobilized cell population declined rapidly during this period (see Figure 4), and commensurate with the growth advantage (higher cell yield) of plasmid-free cells over plasmid-bearing cells, the total cell concentration in the gel beads increased continuously during this period. It is anticipated that, as the stationary state was approached, the net cell growth rate and the rate of cell leakage from gel beads were nearly equal, the gel-entrapped cell population being composed almost entirely of plasmid-free cells.
The profiles in Figure 4 indicate that plasmid-bearing cells (in gel beads) are retained much longer with increasing dilution rate, a trend also observed in continuous suspension cultures of this recombinant strain (Ryan and Parulekar, 1991a). It must be realized that the presence of cells in the gel bead free suspension is inevitable even at high dilution rates, owing to considerable leakage of cells from gel beads. Nevertheless, from a comparison of cell concentrations in the gel bead free suspension and the porous space of gel beads (see Figures 3 and 4), it is obvious that cells are effectively retained in the reactor at high dilution rates. Table I provides a comparison of concentrations of two essential nutrients, glucose and oxygen, in the two immobilized cell reactor experiments. The dissolved oxygen remained at high levels in both experiments. While the fractional conversion of glucose fed was very high (>95 5% ) in the lower dilution rate (0.395 h-l) experiment, a significant fraction of the glucose fed remained unused in the higher dilution rate (0.745 h-l) experiment. In each experiment, glucose concentration in the final steady state (both immobilized and gel bead free suspension cultures being dominated by plasmid-free cells) was lower than glucose concentration in the quasi-stationary state (cultures dominated by plasmid-bearing cells). This trend, also observed with continuous suspension cultures of this recombinant strain (Ryan and Parulekar, 1991a),results from the tremendous growth advantage that plasmid-free cells have over plasmid-bearing cells. Synthesis and Distribution of 8-Lactamase. It is interesting to note that while the highest total P-lactamase activity was lower at the higher dilution rate (0.745 h-I), the difference in the highest total P-lactamase activities in these two experiments was not significant despite the substantial difference in dilution rates (see Figure 5). From the concentration profiles of total viable cells presented in Figure 3, one may deduce that, while at the lower dilution rate (0.395 h-l) plasmid-bearing cells in gel beads and in the gel bead free suspension produced the plasmid-encoded protein at comparable levels, production of this protein could mainly be attributed to plasmid-bearing cells in gel beads at the higher dilution rate (0.745 h-l) since the concentration of recombinant cells in the gel bead free suspension was very low at this dilution rate. The total 0-lactamase activity is the sum of (i) /?-lactamase activity in the abiotic phase, (ii) the enzyme activity retained in the immobilized cells, and (iii) the enzyme activity retained in the freely suspended cells, the three types of activities being measured (and expressed on the basis of per milliliter of gel bead free suspension) following the procedure described under Materials and Methods. From the results presented in Figure 5, it is obvious that a large fraction of the total p-lactamase activity appeared in the abiotic phase in both experiments. The high levels of release observed here, as far as P-lactamase is concerned, are attributable to deterioration in the integrity of the envelope of plasmid-bearing cells due to maintenance of the plasmid at high DNA content and conduct of plasmidrelated activities (Ryan and Parulekar, 1991b). I t is interesting that the extent of excretion of the plasmid-encoded protein in either experiment was higher than that observed in continuous suspension cultures maintained under comparable cultivation conditions [see Ryan and Parulekar (1991a)l. This demonstrates the effectiveness and significant potential of whole-cell immobilization for release of plasmid-encoded proteins normally retained in the host cells. While the 0-lactamase activity
Biotechnol. Prog., 1991, Vol. 7, No. 2
106
100 (a) D = 0.395 hr-’
o 0
rd
+,
(b) D = 0.745 hr-’
Figure 5. Cumulativedistributionsof total @-lactamaseactivity in immobilized cell reactor experiments conducted at dilution rates of 0.395 and 0.745 h-l. For each dilution rate, the bottom curve represents the profile of the 8-lactamaseactivity retained in the freely suspended cells, the top curve the profile of the total @-lactamase activity,and the middle curve the profile of the @-lactamase activity retained in immobilized and freely suspended cells. The contributions of (i) the enzyme activity in the abiotic phase, (ii) the enzyme activity retained in immobilized cells, and (iii) the enzyme activity retained in freely suspended cells to the profile of total /3-lactamaseactivity are therefore represented by (i) area indicated by wide bars, (ii) area indicated by narrow bars, and (iii) darkened area, respectively. retained by the immobilized plasmid-bearing cells was a significant fraction of the total P-lactamase activity, the P-lactamase activity retained by the freely suspended recombinant cells was negligible in comparison to the total P-lactamase activity. In both experiments, the P-lactamase activity retained in the plasmid-bearing cells in the mobile suspension was low primarily due to low cell concentrations (see Figure 3) and low P-lactamase synthesis rates [see Ryan and Parulekar (1991a)l. The profiles of p-lactamase activity in the abiotic phase (EE) in the two experiments are presented in Figure 6. The maximum extracellular p-lactamase activities in these experiments were not significantly different. While the productivity of P-lactamase is proportional to the product of dilution rate and the average 0-lactamase activity, the total amount of 0-lactamase produced is proportional to the product of the number of residence times for which plasmid-bearing cells are retained in the culture and the average P-lactamase activity during this period. Since the average total P-lactamase activities in the two experiments were com-
D = 0.995 hr-l D = 0.746 hr-I
Figure 6. Profiles of the extracellular8-lactamaseactivity (EE) for immobilized cell reactor experiments conducted at dilution and 0.745 h-’ (0). rates of 0.395 h-* (0) parable (see Figure 5) and so were the average P-lactamase activities in the abiotic phase (see Figure 6), the amounts and productivities of total as well as extracellular p-lactamase were much higher for the higher dilution rate experiment. As discussed next, a large fraction of the plasmid-encoded protein retained in the immobilized recombinant cells was recovered by further permeabilization of their envelopes by treatment of the cultures with ethylenediaminetetraacetic acid (EDTA). Cell Membrane Permeabilization via EDTA Treatment. The effect of EDTA on integrity of cell membrane is believed to be closely linked to the ability of EDTA to extract certain functional divalent cations, such as Ca2+and Mg2+,that are of vital importance to the structure of the cell envelope (Voll and Leive, 1970; Burman and Nordstrom, 1971;Yem and Wu, 1978). While EDTA is capable of partially breaking down the selective barrier of the cell membrane, cell death or lysis can be kept minimal and synthesis of major macromolecules can be kept uninterrupted when growing cells are treated with appropriate concentrations of EDTA. In our previous study (Ryan and Parulekar, 1991b), treatment of batch (pure) suspension cultures of E. coli JM103 and E. coli JM103[pUC8] with identical concentrations of EDTA (EDTA concentrations in the reactor up to 4.0 mM) resulted in greater percentage reductions in the specific growth rate of plasmid-free cells. Exposure of plasmid-bearing and plasmid-free cells to EDTA a t these levels did not result in stoppage of cell growth. The minimal inhibitory concentrations of EDTA for plasmidbearing and plasmid-free cells (concentrations at which cell growth is stopped) were estimated to be substantially greater than 4.0 mM (Ryan, 1990; Ryan and Parulekar, 1991b). A possible reason for greater influence of EDTA on plasmid-free cells is that since the envelope of plasmidbearing cells was already less intact due to the presence of a large number of copies of the plasmid (Ryan and Parulekar, 1991b), addition of EDTA did not have as significant impact on the integrity of the envelope of plasmid-bearing cells as it did on the integrity of the envelope of plasmid-free cells. It appears, therefore, that retention of plasmid-bearing cells in nonselective environments can be considerably prolonged by subjecting the cultures periodically to EDTA treatment. In this previous study (Ryan and Parulekar, 1991b), treatment with EDTA led to enhanced fi-lactamase excretion without significantly
Blotechnol. Prog., 1991, Vol. 7, No. 2
sacrificing the overall production of the plasmid-encoded protein. Sharp decreases in plasmid content, signifying substantial inhibition of plasmid replication, were observed after attainment of maximum copy numbers (closeto 1250) in the EDTA-treated cultures in contrast to the gradual changes in plasmid copy number (an increase in copy number up to 1250 followed by a decrease in the same) in the postexponential phases of cultures not exposed to EDTA (Ryan and Parulekar, 1991b). In the present study, EDTA was added periodically to a continuous-flow immobilized cell reactor. The period of injection of EDTA was varied from experiment to experiment. Each EDTA injection consisted of rapid pumping (in less than 30 s) of 8 mL of a 200 mM EDTA solution into the reactor, giving rise to an EDTA concentration of 5.5 mM (based on 290 mL of the gel bead free suspension) in the reactor. The results for two such experiments, conducted at a dilution rate of 0.745 h-l with EDTA being added every 12 and 24 h, are presented and discussed next. One would expect that, owing to additional resistances to transport of EDTA from the bulk abiotic phase into gel beads, the gel-entrapped cells would be subject to a more limited exposure to EDTA than are cells in the gel bead free suspension. Freely suspended plasmid-bearing and plasmid-free cells would therefore be subject to larger percentage reductions in specific growth rates than their respective immobilized counterparts. Indeed, commensurate with this, the total cell concentrations in the mobile suspension (CF) in cultures treated with EDTA were lower than those in cultures not exposed to EDTA (data not shown here). Suppression of growth of freely suspended cells increased the availability of nutrients for cells in gel beads, leading therefore to substantially higher concentrations of plasmid-bearing cells in gel beads in cultures treated with EDTA (see Figure 7). Upon exposure to EDTA, plasmid-free cells are subject to greater percentage reduction in specific growth rate than are plasmid-bearing cells (Ryan and Parulekar, 1991b). The preferential effect of EDTA on plasmid-free cells in gel beads should be apparent from the concentration profiles in Figure 7, top panel, which indicate a substantial reduction in concentration of plasmid-free cells in gel beads (CI-) in cultures subject to periodic EDTA treatment. Reduced generation of plasmid-free cells in gel beads and increased availability of nutrients in gel beads, due to suppression of cell growth in the gel bead free suspension upon exposure to EDTA, led to substantial increases in concentration of immobilizedplasmid-bearing cells (see Figure 7, bottom panel). Retention of plasmidbearing cells in gel beads was therefore considerably prolonged upon EDTA treatment. More frequent addition of EDTA led to more efficient suppression of growth of plasmid-free cells in gel beads (see Figure 7). An improvement in plasmid stability for immobilized cell cultures upon treatment with EDTA is evident from an alternate representation of the profiles in Figure 7, provided in Figure 8. For a fixed amount of EDTA injected, retention of plasmid-bearing cells in the reactor is prolonged with an increase in the frequency of EDTA injection. We reported previously (Ryan and Parulekar, 1991b) that resupplementation of shake-flask cultures of recombinant cells, operated in batch mode and treated with 3 mM EDTA for 4 h, with Ca2+and Mg2+ did not restore cell growth to the level existent prior to EDTA treatment. EDTA-treated cells in continuous immobilized cultures, on the other hand, were able to recover their growth in
107 60 0
0 0
I
EDTA ( 2 4 - h cycle) EDTA (12-hr cyclej EDTA-free
I
u" D
40
EDTA (12-hr cycle) EDTA-free
00
0
rl
W
+
6
Figure 7. Profiles of concentrations of plasmid-free and plasmidbearing cells in gel beads in immobilized cell reactor experiments conducted at a dilution rate of 0.745 h-l withlwithout periodic injection of EDTA. The symbols 0,0 and 0 represent data from experiments in which (i) EDTA was not added, (ii) EDTA was added every 24 h, and (iii) EDTA was added every 12 h, respectively. I
I
U
0.8.
u \ + u
r,
r,
0.6.
0.4
0.2.
D 0
0
0.0 I 0
EDTA-free EDTA (24-hr cycle) EDTA (12-hr cycle)
ao
60
40 N
80
100
T
Figure 8. Profiles of the plasmid-bearing fraction of the immobilized cell population in continuous reactor experiments conducted at a dilution rate of 0.745 h-l (i) without EDTA injection !O),(ii) with EDTA injection every 24 h ( O ) ,and (iii) with EDTA injection every 12 h ( 0 ) .
between consecutiveEDTA injections. It must be realized that when EDTA is injected periodically, the duration (in
Biotechnol. hog.., 1991, Vol. 7, No. 2
1601
*
I
EDTA (12-hr cycle) o EDTA-free
n
0
0
24-hr Cycle 12-hr Cycle
$
\
P
W
ww
I /.' OO
L..20
40
60
80
I
100
N, Figure 9. Profiles of extracellular 8-lactamaseactivity (EE) in immobilized cell reactor experiments conducted at a dilution rate of 0.745 h-1 (i) without EDTA injection (E),(ii) with EDTA and (iii) with EDTA injection every 12 injection every 24 h (O),
---
0
I
lo
Time (hour)
h (0).
Figure 10. Variations in extracellular0-lactamaseactivity (EE) in typical EDTA injection cycles for immobilized cell reactor experiments conducted at a dilution rate of 0.745 h-l with periods of EDTA injection being 24 h (0) and 12 h ( 0 ) . Time zero corresponds to EDTA injection.
an injection cycle) of the presence of EDTA in the continuous culture is much shorter (than that in a batch culture subject to single injection of EDTA) owing to continual loss of EDTA in the reactor effluent and continual dilution of EDTA remaining in the reactor by the EDTA-free feed. Further, in a continuous bioreactor operation, the culture is resupplemented continuouslywith Ca2+and Mg2+,divalent cations that are of vital importance to the structure of the cell envelope; and nutrients (including oxygen) necessary for continuation of energy metabolism (Leive, 1968),which is required for restoration ofthe damaged cell membranes, are supplied continuously. At this dilution rate (0.745 h-l), physical washout and dilution by incoming feed would cause the EDTA concentration in the immobilized cell reactor to decline exponentially with time from 5.5 to 4.0 mM (a concentration at which stoppage of growth of either cell type does not occur) and 0.055 mM (15% of the initial concentration) in nearly 25 min and 6.18 h, respectively. The actual rate of decline in EDTA concentration is expected to be much greater than this because of utilization of EDTA for chelation of the divalent cations in the reactor. It is therefore not surprising to see a quick recovery by EDTAtreated cells in the continuous bioreactor operation. Considering that (i) the minimum inhibitory concentrations of EDTA for both strains were substantially greater than 4.0 mM, (ii) there was a rapid decline in EDTA concentration (in each injection cycle) in the reactor, and (iii) the concentration of viable plasmid-bearing cells in gel beads increased upon exposure to EDTA, it is evident that EDTA treatment led to permeabilization of membranes of plasmid-bearing cells and did not cause significant lysis of these cells. Periodic injection of EDTA into the immobilized cell reactor enabled recovery of the p-lactamase activity retained in the immobilized cells by further permeabilization of membranes of the gel-entrapped plasmid-bearing cells. The profiles of extracellular 0-lactamase activity presented in Figure 9 provide an excellent demonstration of the marked effect of EDTA on release of 0-lactamase. It is important to note that more frequent addition of EDTA (every 1 2 h) to the culture did not adversely affect production of the plasmid-encoded protein since the 0-lactamase activity profiles for experiments with 12-h and 24-h EDTA cycles were nearly identical. In fact, with a
12-h cycle for EDTA injection, P-lactamase synthesis continued for a longer period due to prolonged retention of plasmid-bearing cells in the culture. It is important to point out that the maximum extracellular p-lactamase activity (EE) in the EDTA-treated cultures (see Figure 9) is substantially greater than the maximum total 0-lactamase activity (TE) in the EDTA-free culture (see Figure 5b). The EDTA-treated immobilized cell cultures are therefore superior producers of the plasmid-encoded protein. The enhancement in production of the protein is attributable primarily to much higher concentrations in EDTA-treated of plasmid-bearing cells in gel beads (CI+) cultures (see Figure 7), particularly during the first 50 residence times after initiation of continuous bioreactor operation. In view of the inhibitory effect of EDTA on plasmid DNA content discussed earlier and reported by Ryan and Parulekar (1991b),it is conceivable that exposure of cells to EDTA prevents excessive replication of the plasmid so that more cellular resources can be allocated for synthesis of p-lactamase. The significant fluctuations in the extracellular @-lactamase activity in the EDTA-treated cultures (see Figure 9) are not an aberration or an artifact of the experimental procedure followed. These fluctuations can be explained via a close-up of a portion of Figure 9 (presented in Figure 10) that portrays the response of extracellular @-lactamase activity to exposureof the cultures to EDTA in typical EDTA injection cycles; time zero in Figure 10corresponds to EDTA injection. The effectiveness of EDTA in releasing P-lactamasefrom the plasmid-bearing cells is evident from the substantial increase in the extracellular p-lactamase activity immediately after exposure of the cells to EDTA. It is interesting to note that the EDTA-treated cells were able to recover normal functions of their envelope within a short period, as indicated by the rapid decline in the extracellular 0-lactamase activity starting at about 1 h after addition of EDTA. Immediately upon its injection, the concentration of EDTA in the reactor was 5.5 mM, based on 290 mL of mobile suspension phase. The cultivation medium (M9P) contained 0.1 mM Ca2+and 2.0 mM Mg2+. A t these concentrations of EDTA and the divalent cations, it is therefore expected (Ryan and Parulekar, 1991b) that both divalent cations were completely chelated by EDTA and the cell membrane was
Biotechnol. FYog., 1991, Vol. 7, No. 2
significantly permeabilized, resulting in the immediate release of P-lactamase. As the EDTA concentration in the reactor decreased due to culture dilution and the culture was continuously resupplemented with calcium and magnesium ions through the feed, plasmid-bearing cells were able to fix their damaged membranes and/or synthesize intact membranes for their daughters. As a result, the release of p-lactamase decreased progressively, resulting in a corresponding decline in the extracellular /3-lactamase activity until 4-5 h after an EDTA injection.
Conclusions The production and excretion of a plasmid-encoded protein, p-lactamase, by E . coli JM103[pUC8] cells immobilized in K-carrageenan gel beads was investigated in this study. Substantial natural release of the plasmidencoded protein into the bulk abiotic phase was observed. This release was attributable to the less intact envelope of plasmid-bearing cells owing to the large plasmid DNA content of these cells. While the defective cell envelope is desirable in view of excretion of the plasmid-encoded protein, it contributes in part to the significant growth rate differentialbetween plasmid-freeand plasmid-bearing cells, which is the principal cause of severe segregational plasmid instability in continuous cultures. Immobilization of plasmid-bearingcells led to prolonged retention of these in continuous-flowbioreactors and hence to improved plasmid stability. As observed with continuous suspension cultures of this recombinant system (Ryan and Parulekar, 1991a), plasmid stability for immobilized cell cultures (retention of plasmid-bearing cells in the bioreactor) improved with increasing dilution rate. Excretion of 0-lactamase was enhanced further by periodic exposure of the immobilized cell cultures to appropriate amounts of EDTA. Such exposure led to suppression of cell growth in gel bead free suspension due to direct contact of freely suspended cells with the cell membrane permeabilizing agent EDTA. Exposure to EDTA led to a greater percentage reduction in the specific growth rate of plasmid-free cells since prior to the EDTA treatment the envelopes of these were intact, while the envelopes of plasmid-bearing cells were less intact due to the tremendous burden imposed by harboring of the plasmid and promotion of plasmid-related activities. The growth rate differential between the two cell types was therefore reduced upon exposure of the immobilized cell cultures to EDTA. This, in addition to the increased availability of nutrients, essential for cell growth, to immobilized cells owing to suppression of cell growth in the gel bead free suspension, led to promotion of growth of plasmid-bearing cells in gel beads. Plasmid-bearing cells were therefore retained longer in the immobilized cell cultures upon exposure of these to EDTA. More frequent addition of EDTA led to more efficient suppression of growth of plasmid-free cells in gel beads, further improving the plasmid stability. Efforts for measurement of plasmid DNA contents of immobilizedrecombinant cells were not successful because of some, as yet unknown, interference at the stage of restriction enzyme cleavage. On the basis of our previous observations on the effect of EDTA on the plasmid content of the recombinant cells in batch suspension cultures (Ryan and Parulekar, 1991b), it is likely that the presence of EDTA prevents excessive amplification of plasmid DNA. Beyond a certain level, amplification of plasmid DNA may not be beneficial to synthesis of the plasmid-encoded protein (Ryan and Parulekar, 1991a). The productivity of each recombinant cell, as far as synthesis of P-lacta-
109
mase is concerned, may therefore be increased owing to diversion of cellular resources from plasmid replication to expression of plasmid genes (including the 0-lactamase gene). It was as a result of these factors (viz., promotion of growth of immobilized plasmid-bearing cells, improved plasmid stability, and increased allocation of cellular resources for synthesis of p-lactamase vis-a-vis plasmid replication) that, in addition to increasing the extent of excretion of p-lactamase, exposure of immobilized cell cultures to EDTA led to increased production of the protein. It was also observed that more frequent addition of EDTA to immobilized cell cultures did not have any noticeable adverse effect on 8-lactamase synthesis. Production of the plasmid-encoded protein was in fact prolonged due to longer retention of plasmid-bearing cells in gel beads upon more frequent EDTA injection. The results reported in this article vividly illustrate the tremendous potential of cell membrane permeabilizing agents such as EDTA not only for forced release of recombinant proteins largely retained in the cells but also for increasing substantially the productivity and stability of recombinant systems.
Acknowledgment Financial support received from the Public Health Service (under Public Health ServiceBiomedicalResearch Support Grants 2-S07-RR07027-22,2-S07-RR07027-23, and 2-S07-RR07027-24), Amoco Foundation, and the Galvin Venture Fund of IIT is gratefully acknowledged.
Literature Cited Arakawa, T.; Timasheff, S. N. Stabilization of Protein Structure by Sugars. Biochemistry 1982, 21, 6536-6544. Bailey, K.; Vieth, W. R.; Chotani, G. K. Analysis of Bioreactors Containing Immobilized Recombinant Cells. Ann. N . Y.Acad. Sei. 1987,506, 196-207. Barbotin, J.-N.; Berry, F.; Briasco, C.; Huang, J.; Nasri, M.; Sayadi, s.;Thomas, D. Immobilization Effects on the Stability of Recombinant Microorganisms. Chim. Oggi. 1989, 7 ( l l ) , 4952. Berry, F.; Sayadi, S.;Nasri, M.; Barbotin, J.-N.; Thomas, D. Effect of Growing Conditions of Recombinant E. coli in Carrageenan Gel Beads upon Biomass Production and Plasmid Stability. Biotechnol. Lett. 1988, 10, 619-624. Bowden, G. A.; Georgiou, G. The Effect of Sugars on P-Lactamase Aggregation in Escherichia coli. Biotechnol. Prog. 1988, 4 (2), 97-99. Bron, S.;Luxen, E. Segregational Instability of pUB110-Derived Recombinant Plasmids in Bacillus subtilis. Plasmid 1985, 14, 235-244. Burman, L. G.; Nordstrom, K. Colicin Tolerance Induced by Ampicillin or Mutation to Ampicillin Resistance in a Strain of Escherichia coli K-12. J . Bacteriol. 1971, 106, 1-13. Cheah,U.E.; Weigand, W.A.;Stark,B. C.EffectsofRecombinant Plasmid Size on Cellular Processes in Escherichia coli. Plasmid 1987, 18, 127-134. De Taxis du Poet, P.; Dhulster, P.; Barbotin, J.-N.; Thomas, D. Plasmid Inheritability and Biomass Production: Comparison Between Free and Immobilized Cell Cultures of Escherichia coli BZ18[pTG201] Without Selection Pressure. J . Bacterial. 1986, 165, 871-877. Dhulster, P.; Barbotin, J.-N.; Thomas, D. Culture and Bioconversion Use of Plasmid-Harboring Strain of Immobilized E. coli. Appl. Microbiol. Biotechnol. 1984, 20, 87-93. Georgiou, G.; Chalmers, J. J.; Shuler, M. L.; Wilson, D. B. Continuous Immobilized Recombinant Protein Production from E . coli Capable of Selective Protein Excretion: A Feasibility Study. Biotechnol. Prog. 1985, 1 ( l ) , 75-79. Georgiou, G.; Telford, J. N.; Shuler, M. L.; Wilson, D. B. Localization of Inclusion Bodies in Escherichia coli Overproducing @-Lactamaseor Alkaline Phosphatase. Appl. Enuiron. Microbiol. 1986, 52, 1157-1161.
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