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.     

Analysis of unstable recombinant Saccharomyces cerevisiae population growth in selective medium

Widely applied selection strategies for plasmid-containing cells in unstable recombinant populations are based upon synthesis in those cells of an essential, selection gene product. Regular partitioning of this gene product combined with asymmetric plasmid segregation produces plasmid-free cells which retain for some time the ability to grow in selective medium. This theory is elaborated here in terms of a segregated model for an unstable recombinant population which predicts population growth characteristics and composition based upon experimental data for stable strain growth kinetics, plasmid content, and selection gene product stability. Analytical solutions from this model are compared with an unsegregated phenomenological model to evaluate the effective specific growth rate of plasmid-free cells in selective medium. Model predictions have been validated using experimental growth kinetics and flow cytometry data for Saccharomyces cerevisiae D603 populations containing one of the plasmids YCpG1ARS1, YCpG1DeltaR8, YCpG1DeltaR88, YCpG1DeltaH103, YCpG1DeltaH200, pLGARS1, and pLGSD5. The recombinant strains investigated encompass a broad range of plasmid content (from one to 18 plasmids per cell) and probability alpha of plasmid loss at division (0.05 相似文献   

A novel feeding strategy for enhanced plasmid stability and protein production in recombinant yeast fedbatch fermentation

A novel feeding strategy in fedbatch recombinant yeast fermentation was developed to achieve high plasmid stability and protein productivity for fermentation using low-cost rich (non-selective) media. In batch fermentations with a recombinant yeast, Saccharomyces cerevisiae, which carried the plasmid pSXR125 for the production of beta-galactosidase, it was found that the fraction of plasmid-carrying cells decreased during the exponential growth phase but increased during the stationary phase. This fraction increase in the stationary phase was attributed to the death rate difference between the plasmid-free and plasmid-carrying cells caused by glucose starvation in the stationary phase. Plasmid-free cells grew faster than plasmid-carrying cells when there were plenty of growth substrate, but they also lysed or died faster upon the depletion of the growth substrate. Thus, pulse additions of the growth substrate (glucose) at appropriate time intervals allowing for significant starvation period between two consecutive feedings during fedbatch fermentation should have positive effects on stabilizing plasmid and enhancing protein production. A selective medium was used to grow cells in the initial batch fermentation, which was then followed with pulse feeding of concentrated non-selective media in fedbatch fermentation. Both experimental data and model simulation show that the periodic glucose starvation feeding strategy can maintain a stable plasmid-carrying cell fraction and a stable specific productivity of the recombinant protein, even with a non-selective medium feed for a long operation period. On the contrary, without glucose starvation, the fraction of plasmid-carrying cells and the specific productivity continue to drop during the fedbatch fermentation, which would greatly reduce the product yield and limit the duration that the fermentation can be effectively operated. The new feeding strategy would allow the economic use of a rich, non-selective medium in high cell density recombinant fedbatch fermentation. This new feeding strategy can be easily implemented with a simple IBM-PC based control system, which monitors either glucose or cell concentration in the fermentation broth.     

Phytase production by high cell density culture of recombinant Bacillus subtilis

Bacillus subtilis BD170, harboring a plasmid pGT44[phyC] carrying the phytase gene (phyC) and a phosphate-depletion inducible pst-promoter, was grown in a 2 l bioreactor. Using a controlled feeding of glucose, high cell densities of 32 and 56 g dry cell weight l^–1 were achieved with peptone and yeast extract, respectively, as the complex nitrogen sources in a semi-defined growth medium. The fed-batch protocol was applied to production of recombinant phytase and a high extracellular phytase activity (48 U ml^–1) was reached with peptone. Although the yeast extract feeding resulted in a higher cell density, it was unsuitable as a medium component for phytase expression due to its relatively high phosphate content.     

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.     

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 segregated model for plasmid content and product synthesis in unstable binary fission recombinant organisms

Plasmid propagation in populations of unstable, binary fission recombinant organisms has been studied using a segregated, population balance mathematical model. Segregated models have the advantage of direct incorporation of basic information on mechanisms and kinetics of plasmid replication and segregation at the single-cell level. The distribution of cellular plasmid content and specific rates of plasmid gene expression have been obtained for several single-cell models of plasmid replication, partition, and gene expression. Plasmid replication kinetics during cell growth significantly influence the plasmid content distribution. In the case of transient growth of plasmid-containing and plasmid-free cells in partially selective medium, the degree of selection required for stable maintenance of plasmid-containing cells has been determined. Guidelines are presented for applicability of simpler, nonsegregated models and for evaluation of the parameters in these models based on single-cell mechanisms and associated parameters.     

Large-scale preparation of ribulosebisphosphate carboxylase from a recombinant system in Escherichia coli characterized by extreme plasmid instability

An ampicillin-resistant, RecA- strain of Escherichia coli (HB101) harboring the multicopy pBR322 plasmid containing the structural gene for ribulosebisphosphate carboxylase from Rhodospirillum rubrum was used to prepare large quantities of the carboxylase protein. This recombinant system was characterized by extreme plasmid instability, which resulted in part from the 1.7-fold faster growth rate of plasmid-free cells and in part from very rapid rates of plasmid segregation. The plasmid-containing organisms produced and excreted a large amount of beta-lactamase activity, with the result that ampicillin selection could only be maintained for a very short period of time, after which the plasmid-containing (carboxylase-producing) cells were overgrown by plasmid-free cells. The instability was so severe that even isolated colonies prepared on ampicillin-containing plates were impure and contained plasmid-free cells. Nevertheless, large quantities of carboxylase protein could be obtained from this system by using a highly dilute inoculum which allows selection of ampicillin-resistant (carboxylase-producing) organisms for a sufficient period of time so that the period of growth under nonselective conditions was minimized, and cells harvested at high cell densities contained large amounts of the carboxylase protein. In the present instance, 300-liter fermentations were initiated with a 0.3-microliter inoculum of freshly grown cells. After 20 h of growth in rich medium containing ampicillin, the harvested cells contained 74 g of ribulosebisphosphate carboxylase protein (average of two separate cultures). These results are discussed in terms of the general nature of plasmid instability and protocols available to minimize the effects of such instability.     

Cultivation of the hyperthermophilic archaeonSulfolobus solfataricus in low-salt media

Two low-salt complex media, bactopeptone and desalted yeast extract, were used for high density cultivation of the hyperthermophilic archaeonSulfolobus solfataricus (DSM 1617). Bactopeptone, which has low mineral ion content among various complex media, was good for cell growth in batch cultures; the maximal cell density in bactopeptone was comparable to that in yeast extract. Howver, cell growth was rather poor when bactopeptone was added by the fed-batch procedure. Since several vitamins are deficient in bactopeptone, the effect of vitamins on cell growth was examined. Among the vitamins tested, pyridoxine was found to improve the growth rate ofS. solfataricus. To reduce the growth inhibition caused by mineral ions, yeast extract was dialyzed against distilled water and then fed-batch cultures were carried out using a feed medium containing desalted yeast extract. Although the concentrations of mineral ions in yeast extract were significantly lowered by the dialysis procedure, fed-batch cultivation with desalted yeast extract was unsatisfactory. To examine whether low molecular weight solutes in yeast extract are crucial for cell growth, we investigated the effect of trehalose, a most abundant compatible solute in yeast extract, on the growth pattern. Cell densities were increased and the length of the lag phase was markedly shortened by the presence of trehalose, indicating that trehalose plays an important role in the growth ofS. solfataricus.     

Effects of medium composition and nutrient limitation on loss of the recombinant plasmid pLG669-z and β -galactosidase expression by Saccharomyces cerevisiae

The effects of medium composition, nutrient limitation and dilution rate on the loss of the recombinant plasmid pLG669-z and plasmid-borne β -galactosidase expression were studied in batch and chemostat cultures of Saccharomyces cerevisiae strain CGpLG. The difference in growth rates between plasmid-free and plasmid-containing cells (Δμ) and the rate of segregation (R) were determined and some common factors resulting from the effect of medium composition on plasmid loss were identified. Glucose-limited chemostat cultures of CGpLG grown on defined medium were more stable at higher dilution rates and exhibited Δμ -dominated plasmid loss kinetics. Similar cultures grown on complex medium were more stable at lower dilution rates and exhibited R-dominated plasmid loss kinetics. Overall plasmid stability was greatest in phosphate-limited chemostat cultures grown on defined medium and was least stable in magnesium-limited cultures grown on defined medium. Δμ decreased and R increased with increased dilution rate, irrespective of medium composition. Increased plasmid loss rates at high or low dilution rates would appear to be characteristic of loss kinetics dominated by R or Δμ, respectively. Growth of glucose-limited chemostat cultures on complex medium decreased Δμ values but increased R values, in comparison to those cultures grown on defined medium. Any increased stability that a complex medium-induced reduction of Δμ may have conferred was counteracted by an increased R value. Increased β-galactosidase productivity was correlated with increased plasmid stability only in glucose-limited chemostat cultures grown on defined medium and not in those grown on complex medium. Previous studies have yielded contrasting responses with regard to the effect of dilution rate on recombinant plasmid loss from S. cerevisiae. Our findings can account for these differences and may be generally valid for the stability of similar yeast plasmid constructs. This information would facilitate the design of bioprocesses, where recombinant plasmid instability results in reduced culture productivity. Received 08 July 1996/ Accepted in revised form 14 January 1997     

Enhancement of cloned gene product synthesis via autoselection in recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae autoselection strains with mutations in the ura3, fur1, and urid-k genes have been obtained through a sequential isolation procedure. This autoselection system is an extension of one described by Loison et al. The mutations effectively block both the pyrimidine biosynthetic and salvage pathways and in combination are lethal to the host. Therefore, a plasmidencoded URA3 gene is essential for cell viability regardless of the growth conditions, and complex (traditionally nonselective) media can be employed without the risk of plasmid loss. The effects of medium enrichment on growth and cloned gene product synthesis were examined in batch culture for two autoselection strains. The plasmid gene product beta-galactosidase was under the control of the yeast GAL1 promoter, and two methods of induction were employed; one strain was induced via temperature shift while the other was induced by galactose addition. Three nutrient media were investigated: a lean selective medium (SD), a richer semidefined medium (SDC), and a rich complex medium (YPD). The results demonstrated the improvements in cloned gene productivity possible when the growth medium is enriched, with up to 10-fold increases in beta-galactosidase productivity observed. Plasmid instability and mutation reversion were not problems for the autoselection strains, even in uracil-containing medium. Short-term plasmid stabilities were approximately 90% in all three media tested. During continuous culture of the autoselection temperature-sensitive strain, long-term plasmid stability was excellent and beta-galactosidase expression remained high after more than 25 residence times under inducing conditions. In contrast, both beta-galactosidase specific activity and plasmid stability decreased linearly with time for an analogous nonautoselection strain. The introduced fur1 and uridk mutations were very stable; after more than 50 generations of growth in complex medium, stability values of 99-100% were measured. (c) 1993 Wiley & Sons, Inc.     

Large-scale preparation of ribulosebisphosphate carboxylase from a recombinant system in Escherichia coli characterized by extreme plasmid instability.

An ampicillin-resistant, RecA- strain of Escherichia coli (HB101) harboring the multicopy pBR322 plasmid containing the structural gene for ribulosebisphosphate carboxylase from Rhodospirillum rubrum was used to prepare large quantities of the carboxylase protein. This recombinant system was characterized by extreme plasmid instability, which resulted in part from the 1.7-fold faster growth rate of plasmid-free cells and in part from very rapid rates of plasmid segregation. The plasmid-containing organisms produced and excreted a large amount of beta-lactamase activity, with the result that ampicillin selection could only be maintained for a very short period of time, after which the plasmid-containing (carboxylase-producing) cells were overgrown by plasmid-free cells. The instability was so severe that even isolated colonies prepared on ampicillin-containing plates were impure and contained plasmid-free cells. Nevertheless, large quantities of carboxylase protein could be obtained from this system by using a highly dilute inoculum which allows selection of ampicillin-resistant (carboxylase-producing) organisms for a sufficient period of time so that the period of growth under nonselective conditions was minimized, and cells harvested at high cell densities contained large amounts of the carboxylase protein. In the present instance, 300-liter fermentations were initiated with a 0.3-microliter inoculum of freshly grown cells. After 20 h of growth in rich medium containing ampicillin, the harvested cells contained 74 g of ribulosebisphosphate carboxylase protein (average of two separate cultures). These results are discussed in terms of the general nature of plasmid instability and protocols available to minimize the effects of such instability.     

Plasmid instability in an industrial strain ofBacillus subtilis grown in chemostat culture

Summary A pUB110-derived plasmid/Bacillus subtilis host combination was segregationally unstable when grown in chemostat culture with complex or minimal medium and under starch, glucose or magnesium limitation. The kinetics of plasmid loss were described in terms of the difference in growth rates between plasmid-containing and plasmid-free cells (d) and the rate at which plasmid-free cells were generated from plasmid-containing cells (R). Loss of plasmid-containing cells from the population was d dominated. Changes in medium composition and the nature of growth limitation caused variations in both d and R. The plasmid was most stable in glucose-limited chemostat cultures with minimal medium and least stable under starch limitation with complex complex medium. R and d were smaller for cultures in complex media than those in minimal media. Limitation by starch induced expression of the plasmid-encoded HT amylase gene and was associated with increased values of R and d. Magnesium limitation in minimal medium caused a significant increase in d and a decrease in R.Abbreviations Cm chloramphenicol - Kan kanamycin - Cm^r cells resistant to chloramphenicol (5 mg L^–1) - Kan^r cells resistant to kanamycin (5 mg L^–1) - Cm^sKan^s cells sensitive to chloramphenicol and kanamycin     

Effect of medium composition on the maintenance of a recombinant plasmid in Bacillus subtilis.

Recombinant plasmid pCED3 [confers beta-galactosidase production (LacZ+) and kanamycin resistance (Kmr)] in Bacillus subtilis was found to be both segregationally and structurally unstable. Since many solutions to segregational instability are already available, the problem of structural instability was specifically addressed by inclusion of kanamycin in the growth media. Culture instability was found to be highest in complex and defined media supporting high growth rates. Stabilization over the duration of the experiment (40 generations) was achieved by use of a recently developed chemically defined medium supporting a lower growth rate. Slowing down growth by decreasing temperature was much less effective. A major effect of the growth medium appears to be that of decreasing the growth rate advantage held by cells with plasmid deletions over parental cells containing the intact plasmid.     

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.     

Effects of medium composition on hirudin production in recombinant Saccharomyces cerevisiae

Summary The enriched medium based on yeast nitrogen basc(YNB)increased hirudin synthesis and secretion in rccombinant Saccharomyces cerevisiae in batch and fed-batch cultures. Fed-batch fermentation with the defined medium yielded 342mg hirudin/l but supplementation of yeast extract increased the final hirudin concentration to 461mg hirudin/l. The defined medium, however, produced the product protein with higher purity of 21% and hence will allow easy separation of secreted hirudin from other contaminated polypeptides present in the growth medium. In a continuous culuture, the defined medium yielded higher concentrations of cell mass and hirudin than the complex medium.     

Studies on maintenance of vector pBK2 in Corynebacterium acetoacidophilum by co-culture experiments in a continuous bioreactor

A plasmid vector pBK2 was tested for maintenance in Corynebacterium acetoacidophilum and found to be 100% stable for 90 generations. In co-culture experiments in absence of any selection pressure it was able to cause washout of plasmid free cells. Deletion mutagenesis of the plasmid was done to identify the regions of DNA responsible for its stable maintenance. This led to the identification of a region of DNA in pBB1, an endogenous plasmid used in the construction of pBK2, which is responsible for providing a growth rate advantage to the plasmid containing cell in co-culture with plasmid-free cells. To explain this observed stability we postulate the production of a “growth-inhibiting factor” by the plasmid-containing cells. Inclusion of a death rate term for the plasmid free cells in the computer simulation of the growth kinetics in a chemostat successfully predicts, depending upon the magnitude of the kinetic parameters, a stable coexistence of the two or washout of the plasmid-free cells, an observation that matches the experimental data. Because of its stable maintenance and its ability to select against plasmid-free cells this host-vector system is a natural candidate for cloning and use in large-scale fermentations.     

The yeast 2 micron plasmid: strategies for the survival of a selfish DNA

Summary The designation of the yeast 2 circle as a selfish DNA molecule has been confirmed by demonstrating that the plasmid is lost with exponential kinetics from haploid yeast populations grown in continuous culture. We show that plasmid-free yeast cells have a growth rate advantage of some 1.5%–3% over their plasmid-containing counterparts. This finding makes the ubiquity of this selfish DNA in yeast strains puzzling. Two other factors probably account for its survival. First, the rate of plasmid loss was reduced by allowing haploid populations to enter stationary phase periodically. Second, it was not possible to isolate a plasmid-free segregant from a diploid yeast strain. Competition experiments demonstrated that stability in a diploid is conferred at the level of segregation and that plasmid-free diploid cells are at a selective advantage compared with their plasmid-containing counterparts. Yeast cells in nature are usually homothallic and must frequently pass through both diploid and stationary phases. The 2 plasmid appears to have evolved a survival strategy which exploits these two features of its host's life cycle.     

Production of recombinant human granulocyte-colony-stimulating factor in high cell density yeast cultures

The recombinant human granulocyte-colony-stimulating factor (rhG-CSF) was synthesized in a fusion protein using a GAL1-10 UAS in recombinant Saccharomyces cerevisiae and the intracellular KEX2 cleavage led excretion of mature rhG-CSF into the extracellular culture broth. The recombinant yeast growth in fed-batch cultures was controlled by precise computer-aided medium feed. The optimal C/N ratio in preinduction (glucose/Casamino acids) and post-induction (galactose/yeast extract) feed media was determined at 3 and 2, respectively. The final rhG-CSF and cell concentration was more than 60 mg/L and 70 g/L, respectively, with around 90% plasmid stability and negligible ethanol accumulation. Comparing the cell growth between the hG-CSF + and hG-CSF - recombinant strains shows that the cloned gene product does not hamper the host cell growth.     

Production of poly(3-hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia coli.

Recombinant Escherichia coli XL1-Blue harboring a high-copy-number plasmid containing the Alcaligenes eutrophus polyhydroxyalkanoate synthesis genes could efficiently synthesize poly(3-hydroxybutyrate) (PHB) in a complex medium containing yeast extract and tryptone but not in a defined medium. One of the reasons for the reduced PHB production in a defined medium was thought to be severe filamentation of cells in this medium. By overexpressing an essential cell division protein, FtsZ, in recombinant E. coli producing PHB, filamentation could be suppressed and PHB could be efficiently produced in a defined medium. A high PHB concentration of 149 g/liter, with high productivity of 3.4 g of PHB/liter/h, could be obtained by the pH-stat fed-batch culture of the filamentation-suppressed recombinant E. coli in a defined medium. It was also found that insufficient oxygen supply at a dissolved oxygen concentration (DOC) of 1 to 3% of air saturation during active PHB synthesis phase did not negatively affect PHB production. By growing cells to the concentration of 110 g/liter and then controlling the DOC in the range of 1 to 3% of air saturation, a PHB concentration of 157 g/liter and PHB productivity of 3.2 g of PHB/liter/h were obtained. For the scale-up studies, fed-batch culture was carried out in a 50-liter stirred tank fermentor, in which the DOC decreased to zero when cell concentration reached 50 g/liter. However, a relatively high PHB concentration of 101 g/liter and PHB productivity of 2.8 g of PHB/liter/h could still be obtained, which demonstrated the possibility of industrial production of PHB in a defined medium by employing the filamentation-suppressed recombinant E. coli.