Recombinant protein synthesis and plasmid instability in continuous cultures of Escherichia coli JM103 harboring a high copy number plasmid

Escherichia coli JM103[pUC8] was employed as a model to investigate the behavior of a recombinant microbial system harboring a plasmid at high copy numbers. Experiments with batch and continuous cultures of recombinant and plasmid-free cells were conducted in a well-controlled bio-reactor. In batch experiments, plasmid copy number varied typically from an average of 500 during the exponential growth phase to as high as 1250 during the stationary phase. While the segregational plasmid instability was negligible in batch experiments, severe segregational instability occurred in continuous experiments conducted over a range of dilution rates, resulting in complete loss of plasmid-bearing cells from the continuous cultures within few residence times after transition to continuous operation. The profound differences in the specific growth rates and mass yields of the plasmid-free and plasmid-bearing cells resulting from the extra metabolic burden on the plasmid-bearing cells mainly due to excessive plasmid DNA content was the major cause for the plasmid instability. Plasmid multirnerization was detected in batch and continuous cultures and was found to have significant influence on the effective copy number and was partially responsible for the severe segregational instability in continuous cultures. A quasi-steady state representative of plasmid-bearing cells was established in the initial portion of each continuous culture experiment. Due to the profound growth rate differential between the two types of cells, transients of considerable duration were observed in each continuous culture experiment (initiated with a pure culture of plasmid bearing cells) following the slow accumulation of plasmid-free cells near the end of the quasi-steady state. Significant variations in various culture parameters (including a rapid decline in the plasmid-bearing fraction of the total cell population) occurred during this period, leading ultimately to a steady state for a culture dominated entirely by plasmid-free cells. In continuous cultures, plasmid copy number during the quasi-steady states increased with decreasing dilution rate from 50 (at 0.409 h(-1)) to 941 (at 0.233 h(-1)). Production of the plasmid-encoded protein (beta-lactamase) in these experiments was maximized at an intermediate dilution rate, corresponding to an optimum copy number of about 450. A similar optimum copy number was observed in batch cultures. Significant excretion of beta-lactamase was observed at both low and high dilution rates.     

Plasmid retention and gene expression in suspended and biofilm cultures of recombinant Escherichia coli DH5alpha(pMJR1750)

Differences in plasmid retention and expression are studied in both suspended and biofilm cultures of Escherichia coli DH5alpha(PMJR1750). An alternative mathematical model is proposed which allows the determination of plasmid loss probability in both suspended batch and continuously fed biofilm cultures. In our experiments, the average probability of plasmid loss of E. coli DH5alpha(pMJR1750) is 0.0022 in batch culture in the absence of antibiotic selection pressure and inducer. Under the induction of 0.17 MM IPTG, the maximum growth rate of plasmid-bearing cells in suspended batch culture dropped from 0.45 h(-1) to 0.35 h(-1) and the beta-galactosidase concentration reached an experimental maximum of 0.32. pg/cell 4 hours after the initiation of induction. At both 0.34 and 0.51 mM IPTG, growth rates in batch cultures decreased to 0.16 h(-1), about 36% of that without IPTG, and the beta-galactosidase concentration reached an experimental maximum of 0.47 pg/cell 3 hours after induction.In biofilm cultures, both plasmid-bearing and plasmid-free cells in increase with time reaching a plateau after 96 hours n the absence of both the inducer and any antibiotic selection pressure. Average probability of plasmid loss for biofilm-bound E. coli DH5beta(pMJR1750) population was 0.017 without antibiotic selection. Once the inducer IPTG was added, the concentration of plasmid-bearing cells in biofilm dropped dramatically while plasmid-free cell numbers maintained unaffected. The beta-galactosidase concentration reached a maximum in all biofilm experiments 24 hours after induction; they were 0.08, 0.1, and 0.12 pg/cel under 0.17, 0.34, and 0.51 mM IPTG, respectively. (c) 1993 John Wiley & Sons, Inc.     

Immobilization of Escherichia coli JM103[pUC8] in kappa-carrageenan coupled with recombinant protein release by in situ cell membrane permeabilization.

Immobilization of Escherichia coli JM103[pUC8] was carried out with kappa-carrageenan as the support matrix. Substantial natural excretion of beta-lactamase, attributable to the less intact membrane of plasmid-harboring cells, was observed in immobilized cell cultures. Nevertheless, a significant portion of the beta-lactamase produced was retained in the cells. As compared to suspension cultures, much higher beta-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 (beta-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 beta-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 beta-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.     

Recombinant protein excretion in Escherichia coli JM103[pUC8]: Effects of plasmid content, ethylenediaminetetraacetate, and phenethyl alcohol on cell membrane permeability

The presence of a high copy number plasmid (pUC8) was found to affect integrity of the cell envelope of Escherichia coli JM103, causing in turn significant release of the plasmid-encoded protein (beta-lactamase). The alterations in cell membrane permeability were evident from the increased susceptibility of recombinant cells to deoxycholic acid and methylene blue, which did not have appreciable effect on plasmid-free cells. The deteriorated cell membrane structure also resulted in a substantial reduction in specific growth rate and mass yield of plasmid-bearing cells. Further enhancement in beta-lactamase excretion was achieved by permeabilizing cell membrane with ethylenediaminetetraacetate (EDTA) and phenethyl alcohol (PEA). Unlike other commonly used physical and chemical methods for releasing the enzymes accumulated in the cells, application of EDTA and PEA at appropriate concentrations neither led to cell death nor interrupted synthesis of the plasmid-encoded protein. While in situ application of PEA was complicated due to interference with beta-lactamase activity, in situ application of EDTA was found to be an efficient way of releasing the recombinant protein without sacrificing its productivity. The experimental results demonstrate that the presence of EDTA and PEA can substantially reduce the growth rate differential between plasmid-free and plasmid-bearing cells, suggesting possible improvement of plasmid stability by application of these cell membrence-permeabilizing agents on a periodic basis.     

Plasmid stability in immobilized and free recombinant Escherichia coli JM105(pKK223-200): importance of oxygen diffusion, growth rate, and plasmid copy number.

Stability of the plasmid pKK223-200 in Escherichia coli JM105 was studied for both free and immobilized cells during continuous culture. The relationship between plasmid copy number, xylanase activity, which was coded for by the plasmid, and growth rate and culture conditions involved complex interactions which determined the plasmid stability. Generally, the plasmid stability was enhanced in cultured immobilized cells compared with free-cell cultures. This stability was associated with modified plasmid copy number, depending on the media used. Hypotheses are presented concerning the different plasmid instability kinetics observed in free-cell cultures which involve the antagonistic effects of plasmid copy number and plasmid presence on the plasmid-bearing/plasmid-free cell growth rate ratio. Both diffusional limitation in carrageenan gel beads, which is described in Theoretical Analysis of Immobilized-Cell Growth, and compartmentalized growth of immobilized cells are proposed to explain plasmid stability in immobilized cells.     

Plasmid stability in immobilized and free recombinant Escherichia coli JM105(pKK223-200): importance of oxygen diffusion, growth rate, and plasmid copy number

Stability of the plasmid pKK223-200 in Escherichia coli JM105 was studied for both free and immobilized cells during continuous culture. The relationship between plasmid copy number, xylanase activity, which was coded for by the plasmid, and growth rate and culture conditions involved complex interactions which determined the plasmid stability. Generally, the plasmid stability was enhanced in cultured immobilized cells compared with free-cell cultures. This stability was associated with modified plasmid copy number, depending on the media used. Hypotheses are presented concerning the different plasmid instability kinetics observed in free-cell cultures which involve the antagonistic effects of plasmid copy number and plasmid presence on the plasmid-bearing/plasmid-free cell growth rate ratio. Both diffusional limitation in carrageenan gel beads, which is described in Theoretical Analysis of Immobilized-Cell Growth, and compartmentalized growth of immobilized cells are proposed to explain plasmid stability in immobilized cells.     

Mechanisms of plasmid stable maintenance with special focus on plasmid addiction systems

The stable inheritance of bacterial plasmids is achieved by a number of different mechanisms. Among them are resolution of plasmid oligomers into monomers, active plasmid partitioning into dividing cells and selective killing of plasmid-free segregants. A special focus is given to the last mechanism. It involves a stable toxin and an unstable antidote. The antidotes neutralize their cognate toxins or prevent their synthesis. The different decay rates of the toxins and the antidotes underlie molecular mechanisms of toxin activation in plasmid-free cells. By eliminating of plasmid-free cells from the population of plasmid-bearing ones the toxin-antidote couples therefore act as plasmid addiction systems.     

A single-cell assay of beta-galactosidase in recombinant Escherichia coli using flow cytometry

A flow cytometric method was developed for the assay of beta-galactosidase in single Escherichia coli cells. A new fluorogenic substrate for beta-galactosidase, C(12)FDG, contains a lipophilic group that allows the substrate to penetrate through cell membranes under normal conditions. When the substrate is hydrolyzed by intracellular beta-galactosidase, a green fluorescent product is formed and retained inside the cell. Consequently, the stained beta-galactosidase-positive cells exhibit fluorescence, which is detected by flow cytometry. This new assay was used to analyze the segregational instability caused by a reduction in specific growth rate of the plasmid-bearing cells in the T7 expression system. Induction results in a substantial accumulation of intracellular beta-galactosidase along with a rapid increase in the fraction of plasmid-free cells. Once the cells lose the plasmid, they no longer produce beta-galactosidase, which is reduced by at least half every generation; thus, after staining, the fluorescent, plasmid-bearing cells can be distinguished from the nonfluorescent, plasmid-free cells using flow cytometry. This article describes the feasibility of the flow cytometric assay for single E. coli cells and reports the optimal assay conditions. A direct relationship between beta-galactosidase activity and green fluorescence intensity was found, and the fractions of recombinant cells in batch cultures were analyzed after various levels of induction.     

Optimal fed-batch operation of recombinant cells subject to plasmid instability and death

Optimal feed rate strategy is studied for fed-batch culture of recombinant cells with plasmid instability and with different death rates for the plasmid-free cells (PFC) and plasmid-bearing cells (PBC). Most of the fed-batch fermentation is known to have first-order singularity and therefore a single singular arc. However, this study shows that a singular arc with second-order singularity and therefore two distinct singular arcs are possible for a recombinant cell process if PFC and PBC are subjected to death, and their specific growth rates are proportional to each other. Two types of singular arcs are elucidated and analyzed. The optimal policies over the singular arcs are theoretically explored as these findings reveal qualitative information on the singular arc, which is critically important in providing the optimal initial conditions in numerical computation of optimal feed rate profile.     

Stability of a recombinant plasmid carrying a-amylase gene in Bacillus subtilis

Plasmid pSB6 is a streptococcal recombinant plasmid carrying the a-amylase gene of Bacillus amyloliquefaciens and the chloramphenicol resistance gene. The segregational and structural instabilities of this plasmid were examined under non-selective conditions in Bacillus subtilis. These instabilities were modelled according to a kinetic expression derived from the difference in the growth between plasmid-bearing and plasmid-free cells. This plasmid showed slight segregational instability and much higher levels of structural instability under the conditions examined.     

Stability of a recombinant plasmid carrying a-amylase gene in Bacillus subtilis

Plasmid pSB6 is a streptococcal recombinant plasmid carrying the a-amylase gene of Bacillus amyloliquefaciens and the chloramphenicol resistance gene. The segregational and structural instabilities of this plasmid were examined under non-selective conditions in Bacillus subtilis. These instabilities were modelled according to a kinetic expression derived from the difference in the growth between plasmid-bearing and plasmid-free cells. This plasmid showed slight segregational instability and much higher levels of structural instability under the conditions examined.     

Data analysis of plasmid stability in continuous culture of recombinantSaccharomyces cerevisiae

Summary A simple method using non-linear regression is developed to analyse experimental data from plasmid stability studies of recombinantSaccharomyces cerevisiae grown in continuous cultures with non-selective and selective media. This method simultaneously provides quantitative information on the probability of plasmid loss due to segregation during cell division and the specific growth rates of plasmid-containing and plasmid-free cells at particular dilution rates. The method is applied to a set of experimental data. The three-parameter model, together with the estimated parameter values, provides a good fit to the experimental data.     

A mathematical method for analysing plasmid stability in micro-organisms

A mathematical model describing the instability of plasmids in micro-organisms has been developed. The model is based on the assumption that the overall causes of plasmid instability are described by the segregational instability of the plasmid, R (i.e. the rate at which plasmid-free cells are generated from plasmid-bearing cells), and the growth rate difference, d mu (i.e. the difference in growth rate between plasmid-free and plasmid-bearing cells). A method for determining the values of R and d mu (accompanied by 95% confidence limits) for any plasmid-bearing micro-organism is described. This method is based on the observation that, depending on the plasmid, various exponential patterns of plasmid instability are observed. The stability of Escherichia coli 1B373(pMG169), where d mu much greater than R, and E. coli RV308(pHSG415), where R much greater than d mu, are analysed in order to demonstrate the method.     

Stability in continuous cultures of recombinant bacteria: A metabolic approach

Continuous-culture population dynamics of recombinant bacteria are predicted with a structured kinetic model. The instantaneous specific growth rates of the plasmid-bearing and plasmidfree cells are explicitly calculated from their metabolic activities. The resultant growth-rate differential (between plasmid-bearing and plasmid-free cells) is dynamic and changes over the course of a fermentation. Further, the growth-rate differential is a function of dilution rate. We present the experimental determination of model constants governing plasmid replication and foreign protein expression for a host/vector systemE. coli RR1 [pBR329]. For a different experimental system, we estimate the increased polypeptide expression from a DNA insert solely from the instability population dynamics. Stability predictions agree quite well with experimental observations from the literature and our lab.     

Using a kinetic model that considers cell segregation to optimize hEGF expression in fed-batch cultures of recombinant Escherichia coli

Growth inhibition of recombinant Escherichia coli during the expression of human epidermal growth factor was observed. The recombinant cells could be segregated into three populations based on their cell division and plasmid maintenance abilities: dividing and plasmid-bearing cells, dividing and plasmid-free cells, and viable-but-non-culturable (VBNC) cells. Fed-batch fermentations were performed to investigate the effect of cell segregation on the kinetics of growth and foreign protein production. The results showed that a low concentration of inducer caused weak induction, whereas high levels cause strong induction, resulting in cells segregating into VBNC bacteria and producing a low foreign protein yield. A kinetic model for cell segregation was proposed and its predictions correlated well with experimental data for cell growth and protein expression. The optimal induction strategy could then be predicted by the model, and this prediction was then verified by experimentally deriving the conditions necessary for maximum expression of recombinant protein.     

Analysis of continuous cultures of recombinant methylotrophs

Static and dynamic characteristics of continuous cultures of recombinant methylotrophs, which are designed to improve the selectivity of plasmid-bearing cells and the plasmid stability, are investigated in detail. Operational regions in which coexistence (survival of plasmid-bearing and plasmid-free cells) operation is feasible have been identified in the entire space of kinetic parameters and operating variables. The stability characteristics of each steady state are examined. The existence of oscillatory states around the coexistence steady state is investigated using the dynamic (Hopf) bifurcation analysis. For proper startup of the continuous culture operation, it is critical to identify the sets of initial conditions, if any, which lead to transients that ultimately result in washout of plasmid-bearing cells and avoid such conditions. For the numerical illustrations presented, the coexistence steady state happens to be locally stable over much of its region of existence, particular for the operating conditions corresponding to maximum productivity.     

Two-stage cultivation of recombinant Saccharomyces cerevisiae to enhance plasmid stability under non-selective conditions: experimental study and modeling

A leucine auxotroph strain of Saccharomyces cerevisiae was used to study plasmid stability and expression using a recombinant plasmid, which contained a foreign gene for firefly luciferase (luc). This recombinant yeast was tested in a series of continuous cultures in semi-defined media with varying concentrations of yeast extract in order to study its effect on stability. While the biomass concentration and luciferase activity increased with increasing concentrations of yeast extract, the plasmid stability declined. An analysis of the growth rates showed that the recombinants enjoyed a growth rate advantage over the plasmid-free cells at critically low yeast extract concentrations, possibly due to leucine starvation in the media. A two-stage cultivation strategy was designed in order to create a yeast extract limited environment so that plasmid-free cells could not grow and overtake the recombinant cells. The cells were cultivated in selective media in the first stage, and then transferred continuously to the second stage where the media was enriched by feeding yeast extract. The feed rate was kept low in order to ensure yeast extract and hence leucine starvation, thereby selecting against the plasmid-free cells. This strategy resulted in a stable existence of recombinant cells, which stabilized around 60% at steady state during the tested period of cultivation. The complex nitrogen feed helped in increasing the cell density and volumetric activity by approximately 9 and 18-fold respectively with respect to that achieved in minimal medium. The experimental data was used to formulate a mathematical model to predict cell growth and plasmid stability in two-stage cultivation, which correctly explained the experimental data.     

Kinetics of benzoate-induced loss of the TOL plasmid from Pseudomonas putida MT15 during growth in chemostat culture

Potassium-limited chemostat cultures of Pseudomonss putida MT15, grown on excess glucose, displayed approximately 100% plasmid loss after 60 generations of growth in the presence of 5 mM benzoate. The kinetics of plasmid loss indicated that plasmid-free cells displayed a growth rate advantage, which we attribute to selective inhibition of the growth of plasmid-containing cells by benzoate. However, stable, mixed populations of plasmid-free cells, deletants and plasmid-containing cells were selected during growth under glucose limitation in the presence of benzoate. This behaviour indicated that the plasmid-free cells in these cultures displayed a growth rate disadvantage and that their appearance was due entirely to benzoate-induced segregational instability of the plasmid.     

Investigation of the effect of growth environment on the stability of low-copy-number plasmids in Escherichia coli

The stability of a low-copy-number plasmid, pHSG415, in Escherichia coli, was investigated in batch and continuous culture. The plasmid was unstable in batch culture, but was significantly stabilized by growth in continuous culture with phosphate, nitrogen or potassium limitation. However, the plasmid was very unstable when grown in continuous culture with sulphate limitation. These results contrast with those obtained with multicopy plasmids such as pBR322, which is particularly unstable in carbon- or phosphate-limited continuous culture. The effect of growth rate on the stability of E. coli(pHSG415) grown in continuous culture with glucose limitation was also investigated. The plasmid was significantly more stable in cells grown at higher growth rates. The segregational instability (R) of the plasmid and the difference in growth rate between plasmid-free and plasmid-bearing cells (dmu) were calculated for each condition using the method of Cooper et al. (accompanying paper: Journal of General Microbiology 133, 1871-1880). It was found that the primary cause of the loss of pHSG415 from the cell population was the segregational instability of the plasmid.     

Amino acid supplementation decreases plasmid retention in Escherichia coli

The effect of amino acid supplementation on plasmid stability in Escherichia coli B/r was tested experimentally. Comparisons of experimental results to computer-predicted values were made using a detailed, structured single-cell model. The plasmid, pDW17 (a pBR322 derivative with a mutated tac promoter controlling the beta-lactamase gene), was used. In chemostat cultures, the amino acid supplemented cultures were always less stable than those grown in minimal medium. This effect was not a growth rate effect, as increasing growth rate imsproves stability for both cultures in minimal medium and in amino acid supplemented medium. The computer model also predicted a decrease in stability due to amino acid supplementation. The model also predicts that amino acid supplementation, combined with moderately strong plasmid-encoded protein expresion, results in a depletion of low-molecular-weight organics compared with plasmid-free cells. In minimal medium the same level of plasmid-encoded protein synthesis results in a strong reduction in amino acid pools compared with plasmid-free cells. With amino acid supplementation the growth differential between plasmid-bearing and plasmid-free cells may be due to an "energy limitation," while in minimal medium the size of the growth rate differential may be due to a "building block" limitation. (c) 1992 John Wiley & Sons, Inc.