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.     

Stability of plasmid-bearing microorganisms in batch and continuous cultures

The stability of five microbial strains bearing a domestic and/or exotic plasmid was investigated in continuous culture to obtain basic information on the fate of genetically engineered microorganisms released in the natural environment.The three strains with an exotic plasmid were constructed by the conjugal or mobilized transfer of conjugative plasmid R100-1 and non-conjugative plasmid RSF2124. Plasmid loss occurred only at the declining growth phase of batch culture of the transconjugants; the ratio of plasmid-free cells was 40–50% at the end of the culture, independent of the strains, whereas the plasmid in the native host cells was maintained at almost 100% of stability.In continuous culture of the transconjugant cells, the population ratio of plasmid-free cells at the pseudo-steady state was between 5–80% depending on the strain. The plasmid-bearing cells were not washed out of the continuous fermentor for 43 generations but maintained their quasi-stable concentration with some degree of oscillation. Simultaneous loss and retransfer of the plasmid from and to its host cells is suggested for the explanation.     

Plasmid stability of recombinant Pseudomonas sp. B13 FR1 pFRC20P in continuous culture

Plasmid stability of recombinant Pseudomonas sp. B13 FR1 pFRC20P, a strain capable of mineralizing 3- and 4-chlorobenzoate and 4-methylbenzoate, was investigated in continuous culture. The hybrid cosmid pFRC20P enables the strain to mineralize 4-methylbenzoate. Rapid plasmid loss was observed under nonselective conditions using 3-chlorobenzoate as the substrate. Plasmid stability decreased with increasing dilution rate. Despite the growth advantage of the generated plasmid free cells a total depletion of plasmid bearing cells was not observed. After approximately 50 generations the fraction of plasmid bearing cells reached a constant level of 10%, which was stably maintained during the next 25 generations. Cells from this stage were used to inoculate a new culture that resulted in a stable level of 50% plasmid bearing cells. By a temporary substrate change to selective conditions (4-methylbenzoate), this level could be further increased to 70%. Literature models on plasmid stability could not be applied to describe the experimental data. Therefore, a new but unstructured model was developed to describe the experimental results. The model is based on the existence of three subpopulations: a plasmid free one, an original plasmid bearing one with a growth disadvantage compared to plasmid free cells, and a second plasmid bearing subpopulation with increased stability that is generated from the original one and has a growth rate comparable to the plasmid free cells.     

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.     

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.     

Testing plasmid stability of Escherichia coli using the Continuously Operated Shaken BIOreactor System

Plasmids are common vectors to genetically manipulate Escherichia coli or other microorganisms. They are easy to use and considerable experience has accumulated on their application in heterologous protein production. However, plasmids can be lost during cell growth, if no selection pressure like, e.g., antibiotics is used, hampering the production of the desired protein and endangering the economic success of a biotechnological production process. Thus, in this study the Continuously Operated Shaken BIOreactor System (COSBIOS) is applied as a tool for fast parallel testing of strain stability and operation conditions and to evaluate measures to counter such plasmid loss. In specific, by applying various ampicillin concentrations, the lowest effective ampicillin dosage is investigated to secure plasmid stability while lowering adverse ecological effects. A significant difference was found in the growth rates of plasmid‐bearing and plasmid‐free cells. The undesired plasmid‐free cells grew 30% faster than the desired plasmid‐bearing cells. During the testing of plasmid stability without antibiotics, the population fraction of plasmid‐bearing cells rapidly decreased in continuous culture to zero within the first 48 h. An initial single dosage of ampicillin did not prevent plasmid loss. By contrast, a continuous application of a low dosage of 10 µg/mL ampicillin in the feed medium maintained plasmid stability in the culture. Consequently, the COSBIOS is an apt reactor system for measuring plasmid stability and evaluating methods to enhance this stability. Hence, decreased production of heterologous protein can be prevented. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1418–1425, 2016     

Influence of plasmid origin and promoter strength in fermentations of recombinant yeast

The effects of plasmid promoter strength and origin of replication on cloned gene expression in recombinant Saccharomyces cerevisiae have been studied in batch and continuous culture. The plasmids employed contain the Escherichia coli lacZ gene under the control of yeast promoters regulated by the galactose regulatory circuit. The synthesis of beta-galactosidase was therefore induced by the addition of galactose. The initial induction transients in batch culture were compared for strains containing plasmids with 2mu and ARS1 origins. As expected, cloned gene product synthesis was much lower with the ARS1 plasmid: average beta-galactosidase specific activity was an order of magnitude below that with the 2mu-based plasmid. This was primarily due to the low plasmid stability of 7.5% when the plasmid origin of replication was the ARS1 element. The influence of plasmid promoter strength was studied using the yeast GAL1, GAL10, and hybrid GAL10-CYC1 promoters. The rate of increase in beta-galactosidase specific activity after induction in batch culture was 3-5 times higher with the GAL1 promoter. Growth rate under induced conditions, however, was 15% lower than in the absence of lacZ expression for this promoter system. The influence of plasmid promoter strength on induction behavior and cloned gene expression was also studied in continuous fermentations. Higher beta-galactosidase production and lower biomass concentration and plasmid stability were observed for the strain bearing the plasmid with the stronger GAL1 promoter. Despite the decrease in biomass concentration and plasmid stability, overall productivity in continuous culture using the GAL1 promoter was three times that obtained with the GAL10-CYC1 promoter.     

Control of the cell cycle ofCandida utilis by external conditions

A mathematical model of the cell cycle ofCandida utilis in a continuous culture was formulated with respect to dilution rate. It makes it possible to express the duration of morphological stages in minutes, separately for mother cells and daughter cells. These values were compared with equivalent parameters in batch cultures. Duration of the morphological stage with buds was much longer in batch cultures as compared with the same value determined for a continuous culture according to the mathematical model. When using cultivation apparatus with a higher aeration capacity the (S + G₂) phase, i.e. the stage bearing the bud, was reduced also in the batch cultures and approached the values determined for the continuous culture by means of the mathematical model.     

A model for growth of Saccharomyces cerevisiae containing a recombinant plasmid in selective media

A major problem in the use of plasmids as recombinant vectors is the problem of plasmid-free cell generation from plasmid shedding and subsequent growth. A common technique for controlling the population of plasmidfree cells is the use of selective media against these cells using an auxotrophic host and a plasmid that has the ability to produced the essential metabolite. A distributed model describing the growth of Saccharomyces cerevisiae containing a recombinant plasmid in selective media was developed. The model allows for growth and production of a metabolite by the plasmid-carrying strain and growth of the plasmid-free cells on resulting metabolite concentrations. Through a determination of system constants and numerical solution to the equations, experimental batch and continuous culture results for cell concentration transients could be simulated by the model. The results indicated that despite selective pressure, plasmid-free cell growth was significant.     

Activity and stability of a recombinant plasmid-borne TCE degradative pathway in biofilm cultures

The activity and stability of the TCE degradative plasmid TOM31c in the transconjugant host Burkholderia cepacia 17616 was studied in selective and non-selective biofilm cultures. The activity of plasmid TOM31c in biofilm cultures was measured by both TCE degradative studies and the expression of the Tom pathway. Plasmid loss was measured using continuous flow, rotating annular biofilm reactors, and various analytical and microbiological techniques. The probability of plasmid loss in the biofilm cultures was determined using a non-steady-state biofilm plasmid loss model that was derived from a simple mass balance, incorporating results from biofilm growth and plasmid loss studies. The plasmid loss model also utilized Andrew's inhibition growth kinetics and a biofilm detachment term. Results from these biofilm studies were compared to similar studies performed on suspended cultures of Burkholderia cepacia 17616-TOM31c to determine if biofilm growth has a significant effect on either plasmid retention or Tom pathway expression (i.e., TCE degradation rates). Results show that the activity and expression of the Tom pathway measured in biofilm cultures was significantly less than that found in suspended cultures at comparable growth rates. The data obtained from these studies fit the plasmid loss model well, providing plasmid loss probability factors for biofilm cultures that were equivalent to those previously found for suspended cultures. The probability of plasmid loss in the B. cepacia 17616-TOM31c biofilm cultures was equivalent to those found in the suspended cultures. The results indicate that biofilm growth neither helps nor hinders plasmid stability. In both the suspended and the biofilm cultures, plasmid retention and expression could be maintained using selective growth substrates and/or an appropriate plasmid-selective antibiotic. Copyright 1998 John Wiley & Sons, Inc.     

Enhanced production of alpha-amylase in fed-batch cultures of Bacillus subtilis TN106[pAT5

Growth of Bacillus subtilis TN106[pAT5] and synthesis of plasmid-encoded protein (alpha-amylase) are investigated in batch, continuous, and fed-batch cultures using a defined medium containing glucose and/or starch as the carbohydrate source. The batch culture studies reveal that reduced availability of arginine hampers growth of recombinant cells (which lack an arginine synthesis gene) but promotes production of alpha-amylase and substitution of glucose by starch as the carbohydrate source leads to slower growth of recombinant cells and increased production of alpha-amylase per unit cell mass. Retention of recombinant cells over prolonged periods in continuous cultures is not possible without continuous application of antibiotic selection pressure owing to segregational plasmid instability. Fed-batch experiments with constant volumetric feed rate demonstrate that alpha-amylase production is enhanced at lower feed concentration of starch (sole carbohydrate source) and lower volumetric feed rate. Such slow addition of starch is however not conducive for growth of recombinant cells. The expression of the thermostable alpha-amylase gene carried on the recombinant plasmid pAT5 (derived from a plasmid isolated from a thermophilic bacterium) is promoted at higher temperatures, while growth of recombinant cells is depressed. In all batch and fed-batch experiments, production of alpha-amylase is observed to be inversely related to growth of recombinant cells. The efficacy of two-stage bioreactor operations, with growth of recombinant cells being promoted in the first stage and alpha-amylase production in the second stage, in attaining increased bulk alpha-amylase activity is demonstrated. (c) 1993 John Wiley & Sons, Inc.     

Kinetic study of instability of recombinant plasmid pPLc23trpAl in E. coli using two-stage continuous culture system

Derepression of the phage lambda p(L) promoter on recombinant plasmid pPLc 23-trpAl caused a rapid increase of plasmid free segregants in the population. In continuous culture, increased production of trpA protein follwing derepression was accompanied by a continuous deceleration of specific growth rate. In the repressed condition, plasmid loss per generation in continuous culture decreased as dilution rate increased from 0.06 to 1.08 h(-1). Over this range, the concentration of plasmid DNA within the cell decreased eightfold corresponding to a decrease in plasmid number from 74 to 32 molecules/cell. The use of a two-stage continuous culture system coupled with a temperature sensitive expression system allows a high trpA productivity from the derepressed plasmid for more than 48 h and also offers a possibility of minimizing the instability problem of high expression recombinants. Such a system also permits the critical study of the effects of fermentation and other regulatory parameters on expression under better controlled conditions than is possible in a batch culture or single-stage continous culture.     

Continuous isolation of plasmid DNA by annular chromatography.

Continuous chromatographic separations, especially of multicomponent mixtures, constitute interesting options for biotechnological downstream processing. Taking the separation of plasmid DNA from clearified lysates on hydroxyapatite as a pertinent example, we discuss the potential of continuous annular chromatography (CAC) in comparison with conventional (preparative) batch chromatography. In CAC the column is realized in the form of a thin (5 mm, height 210 mm) slowly rotating annulus. The performance of such a CAC column is compared to that of an ("analytical") batch column of similar thickness (diameter) and length (4 x 250 mm) and that of a ("preparative") batch column of similar cross-sectional surface area and height (50 x 210 mm). The quality of the obtained plasmid as defined by the appearance of the corresponding agarose gels (native and linearized plasmid), the 260/280 ratio and the biological activity (transient transfection of HEK 293 cells) was found to be identical in all three cases. The yields are also shown to be equivalent. The loading factor is found to be the most decisive parameter for the transfer of a given separation method between the continuous and the batch columns. Under nonoptimized conditions, plate numbers tended to be lower in the continuous compared to the batch columns. This is shown to be largely due to an artifact created by the CAC design (collection of averaged fractions at the outlets) and can be overcome by optimizing the rotation speed. Surprisingly the large batch column consistently gave better plate numbers than either the small batch or the CAC column. Compared to the preparative batch column, wall effects are more pronounced in the CAC (respectively the small diameter batch column), which may translate into better bed stability but conceivably also contributes to an increase in plate height, due to the reduction in bed density usually observed in the proximity of the wall. The CAC is shown to be a powerful approach to continuous chromatography, which allows a direct and straightforward upscale of chromatographic bioseparation methods.     

A generalized model of plasmid replication

A simple model is developed that permits the determination of cellular extrachromosomal DNA content for a large number of host-plasmid systems. The model incorporates host, vector, and environmental influences on plasmid replication through the use of empirical expressions. The model successfully predicts the plasmid content of many host-vector systems in a balanced growth situation, both in continuous and batch cultures. Techniques for determining model parameters are also presented. The model parameters, which characterize the effect of plasmid on its own synthesis, are given physical interpretation through the development of a structured model for a particular class of plasmids and subsequent comparison of the predictions of the two models. The simplicity of the model expressions should, in the absence of: (1) discriminatory criteria such as plasmid concentration under transient growth conditions or (2) detailed knowledge on molecular mechanisms of plasmid replication, prove useful in the study of host-vector systems for genetic engineering applications.     

Effects of segregation and selection on instability of plasmid pACYC184 in Escherichia coli B.

We use a mathematical model to analyze the dynamics of loss of nonconjugative pACYC184 from populations of Escherichia coli B in glucose-limited continuous culture. This model incorporates both plasmid segregation and selection against plasmid carriage. It is concluded that there is intense selection against plasmid carriage (s = 0.3 per culture generation), which amplifies the frequency of segregants arising de novo.     

Effects of temperature on plasmid stability and penicillinase productivity in a transformant of Bacillus stearothermophilus

After transforming host cells of Bacillus stearothermophilus CU21 with a recombinant plasmid pLP11 that harbored constitutive penicillinase genes of B. licheniformis CO1, both the stability of the plasmid and specific rate of penicillinase production were studied. The temperature at which the plasmid could be kept in a stable fashion in the transformant of B. stearothermophilus CU21 (pLP11) ranged nearly from 44 to 50 degrees C, irrespective of batch and continuous cultures. Continuous and steady-state cultures of the transformant could only be realized within this narrower temperature range. Indeed, the approximate temperature ranges of growth for the host and transformant were from 40 to 70 degrees C and from 40 to 63 degrees C, respectively. Clearly, the upper limit for the growth temperature of host cells decreased when they were transformed. Kinetic patterns of penicillinase production in continuous culture of the transformant (with plasmid) from 44 to 50 degrees C differed remarkably from that of B. licheniformis CO1 (without plasmid) at 37 degrees C.     

Stability of a lac operon in Zymomonas mobilis in batch and continuous culture

ZM6100(RP1::Tn951), a strain of Zymomonas mobilis containing the lactose transposon Tn951 on the broad host range plasmid RP1, progressively lost all plasmid markers in batch culture under non-selective conditions. After 120 generations less than 0.1% of the population retained the plasmid markers. ZM6306, derived from ZM6100(RP1::Tn951) by prolonged tetracycline selection, showed 100% stability for all plasmid markers when grown without selection pressure in both batch and continuous culture. In continuous culture, the synthesis of β-galactosidase was induced by the addition of lactose, and low levels of galactose were detected together with a small increase in ethanol concentration.     

Flow Cytometry and Real-Time Quantitative PCR as Tools for Assessing Plasmid Persistence

The maintenance of a plasmid in the absence of selection for plasmid-borne genes is not guaranteed. However, plasmid persistence can evolve under selective conditions. Studying the molecular mechanisms behind the evolution of plasmid persistence is key to understanding how plasmids are maintained under nonselective conditions. Given the current crisis of rapid antibiotic resistance spread by multidrug resistance plasmids, this insight is of high medical relevance. The conventional method for monitoring plasmid persistence (i.e., the fraction of plasmid-containing cells in a population over time) is based on cultivation and involves differentiating colonies of plasmid-containing and plasmid-free cells on agar plates. However, this technique is time-consuming and does not easily lend itself to high-throughput applications. Here, we present flow cytometry (FCM) and real-time quantitative PCR (qPCR) as alternative tools for monitoring plasmid persistence. For this, we measured the persistence of a model plasmid, pB10::gfp, in three Pseudomonas hosts and in known mixtures of plasmid-containing and -free cells. We also compared three performance criteria: dynamic range, resolution, and variance. Although not without exceptions, both techniques generated estimates of overall plasmid loss rates that were rather similar to those generated by the conventional plate count (PC) method. They also were able to resolve differences in loss rates between artificial plasmid persistence assays. Finally, we briefly discuss the advantages and disadvantages for each technique and conclude that, overall, both FCM and real-time qPCR are suitable alternatives to cultivation-based methods for routine measurement of plasmid persistence, thereby opening avenues for high-throughput analyses.     

Investigation of subpopulation heterogeneity and plasmid stability in recombinant escherichia coli via a simple segregated model

Many microbial and cell cultures exhibit phenomena that can best be described using a segregated modeling approach. Heterogeneties are more marked in recombinant cell cultures because subpopulations, which often exhibit different growth and productivity characteristics, are more easily identified by selective markers. A simple segregated mathematical model that simulates the growth of recombinant Escherichia coli cells is developed. Subpopulations of different growth rate, plasmid replication rate, and plasmid segregation probability are explicitly considered. Results indicate that a third mechanism of plasmid instability, referred to here as a "downward selective pressure," is significant when describing plasmid loss in batch and chemostat cultures. Also, the model agrees well with experimental data from cultures under antibiotic selective pressure. Finally, model simulations of chemostat cultures reveal the importance of initial conditions on culture stability and the possible presence of nonrandom partitioning functions. (c) 1993 John Wiley & Sons, Inc.