Effect of cell growth rate on the performance of a two-stage continuous culture system in a recombinant Escherichia coli fermentation

As part of the process optimization of a two-stage continuous culture system, the effect of growth rate mu(2) (app) on the performance of the second stage (production stage) was studied in a recombinant Escherichia coli K12 (DeltaH1Deltatrp/pPLc23trpA1). Important parameters considered were specific gene expression rate, plasmid content, and plasmid stability, all of which were closely related to the cell growth rate and the production rate of the cloned gene product (trpalpha). When operating conditions were maintained constant (T(1) = 35 degrees C, D(1) = 0.9 h(-1), T(2) = 40 degrees C, and D(2) = 0.7 h(-1)) and mu(2) (app) was varied, plasmid content in the second stage showed its maximum at mu(2) (app) = 0.4 h(-1) and decreased thereafter. Specific gene expression rate linearly increased with increasing mu(2) (app), while plasmid stability decreased. Optimum cell growth rate giving the maximum value in overall productivity was observed at around mu(2) (app) = 0.4 h(-1). The contribution or role of the three parameters, specific gene expression rate, plasmid content, and plasmid stability in exhibiting the maximum productivity at the optimal mu(2) (app) is discussed.     

Effect of environmental growth conditions on plasmid stability, plasmid copy number, and catechol 2,3-dioxygenase activity in free and immobilized Escherichia coli cells

In order to better understand the high plasmid stability in immobilized recombinant E. coli cells, the effects of dilution rate on the pTG201 plasmid stability, the copy number, and the catechol 2,3-dioxygenase (encoded by XyIE gene) production were, at first, studied in free E. coli W3101 continuous cultures in minimal media. It was found that decreasing specific growth rate increased the plasmid copy number and the catechol 2,3-dioxygenase activity but the stability decreased. In continuous culture with immobilized cells, an increase was shown in plasmid copy number and catechol 2,3-dioxygenase activity probably due to the distribution of growth in the gel beads. Besides mechanical properties of gel beads which may allow limited cell divisions, the increase in plasmid copy number is involved in enhanced plasmid stability in immobilized cells. In the same way, an experiment conducted in LB medium dealing with competition between pTG201-free and pTG201-containing E. coli B cells was described. It was shown that the competition was not more pronounced in gel bead compared to a free system. The effects of nutritional limitations on pTG201 plasmid stability and catechol 2,3-dioxygenase activity during chemostat cultivations in free and immobilized E. coli B cells were also investigated. It was found that immobilization of cells increased the stability of pTG201 even under glucose, nitrogen, or phosphate limited cultures. However in the case of magnesium depleted culture, pTG201 was shown to be relatively instable and a decrease in viable cell number during the immobilized continuous culture was observed. By contrast to the free system, the catechol 2,3-dioxygenase activity increased in immobilized cells under all culture conditions used.     

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.     

Construction of new vectors for cloning of promoters

Vectors for cloning promoter-DNA fragments were derived from plasmid pBR313 (Bolivar et al., 1977). These have several unique restriction sites and carry the trpA gene from Escherichia coli as a selective marker. The selection is based on an enhancement of the growth rate of those bacteria in which the expression of trpA is directed by the cloned promoter. The expression of trpA can be determined quantitatively, independently of the copy number of the vector, and should reflect the apparent strength of the promoter, since the DNA segment located before trpA contains translational stop signals in all three reading frames.     

Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda pL promoter.

Two multiple-copy, ColE1-type, plasmid cloning vehicles, pHUB2 and pHUB4, have been constructed that carry four different single restriction sites down-stream from the phage lambda promoter pL. The promoting activity of pL is switched off at low temperature in the presence of a cIts gene that specifies a temperature-sensitive repressor but could be activated by heat induction. cIts was located either on the host chromosome, or on a second plasmid pRK248 that is compatible with the cloning vehicle, or on the vehicle itself. Three different restriction fragments, each carrying the gene trpA of Salmonella typhimurium or Shigella dysenteriae, have been inserted into the EcoRI, BamHI and SalI sites, respectively, of these plasmids and pL dependent expression of the inserted gene in Escherichia coli was determined by measuring the enzymatic activity of the trpA gene product. Heat induction resulted in a level of expression of trpA corresponding to 1 to 6.6% of the total soluble cell protein as trpA protein. The level of trpA protein production depended on the particular insert and the plasmid used.     

Effects of temperature and dilution rate on the copy number of recombinant plasmid in continuous culture of Bacillus stearothermophilus (pLP11)

The penicillinase yield Y(P/X) (U/g cell) of a transformant Bacillus stearothermophilus CU21 (pLP11) in continuous culture of LGP broth at 44, 47.5, and 50 degrees C, respectively, depended not only on temperature, theta ( degrees C), but also on dilution rate, D (h(-1)), in a peculiar fashion that could not have been realized if the product were from the gene on the chromosome rather than the plasmid. The gene dosage effect could account for the unusual dependence of Y(P/X) on theta and D, because the mode of Y(P/X) as a function of D at a given temperature resembled that of plasmid content C(p) (mg plasmid/g cell) vs. D at the same temperature. In other words, C(p), corresponding to the copy number of plasmid per grams of cell, could be controlled by either theta, D, or both in this instance. However, when the gene expression efficiency, epsilon (U/ng plasmid), was plotted versus C(p), the effects of theta and D on the expression became indistinct.     

High-level expression of human BSF-2/IL-6 cDNA in Escherichia coli using a new type of expression-preparation system

BSF-2 (B cell stimulatory factor-2/IL-6) is a member of the lymphokine family and responsible for B cell differentiation. Expression plasmids of human BSF-2 cDNA were constructed using a trp promotor/operator and a trpA terminator. In an extract of Escherichia coli HB101 holding "direct" expression plasmid pBSF-2D, activity of BSF-2 was detected, but overproduction was not observed. A "fused" expression system was therefore developed to prepare the recombinant protein. In this system, cDNA was expressed as a fused protein with human IL-2 N-terminal peptide. In the case of the fused BSF-2 expression plasmid, pBSF-2F, inclusion bodies were observed and overproduction of the protein occurred. As this fused protein had a Phe-Arg-Ala sequence at the junction of hIL-2 and BSF-2, it was possible to process mature BSF-2 from the fused BSF-2 by treatment with kallikrein and aminopeptidase P. From 1 liter of E. coli culture, 45 mg of mature BSF-2 was purified; it had a relative biological activity equal to that of natural BSF-2 purified from T cells.     

Effect of specific production rate of recombinant protein on multimerization of plasmid vector and gene expression level

In fed-batch cultures of recombinant Escherichia coli BL21(DE3)[pT7-G3IL2] at high cell concentration, the post-induction specific growth rate was carefully regulated by controlled medium feed to maximize the synthesis level of recombinant fusion interleukin-2, G3.IL-2. A maximum concentration of G3.IL-2 (11.25 g l(-1)) was achieved in the induced recombinant culture growing at the rate of 0.056 h(-1). A steep decrease in the expression level of G3.IL-2 was observed at the post-induction specific growth rates higher than its optimal value (0.056 h(-1)). In the induced recombinant cultures, plasmid multimerization was observed and highly dependent on specific growth and production rate: a higher post-induction specific growth rate and an increased specific production rate tended to significantly promote it much further. Moreover, plasmid stability was found to decrease rapidly in a faster growing culture.     

Plasmid stability and kinetics of continuous production of glucoamylase by recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in an airlift bioreactor

Production of glucoamylase by recombinant Saccharomyces cerevisiae C468/pGAC9 (ATCC 20690) in a continuous stirred tank bioreactor was studied at different dilution rates. Plasmid stability was found to be growth (dilution rate) dependent; it increased with the dilution rate. Bioreactor productivity and specific productivity also increased with the dilution rate. A kinetic equation was used to model the plasmid stability kinetics. The growth rate ratio between plasmid-carrying and plasmid-free cells decreased from 1.397 to 1.215, and segregational instability or probability of plasmid loss from each cell division decreased from 0.059 to 0.020 as the dilution rate increased from 0.10 to 0.37 1/h. The specific growth rates increased with dilution rate, while the growth rate difference between plasmid-carrying and plasmid-free cell populations was negligible. This was attributed to the low copy number of the hybrid plasmid pGAC9. Thus, the growth rate had no significant effect on plasmid instability. The proposed kinetics was consistent with experimental results, and the model simulated the experimental data well.     

Effect of air supplement on the performance of continuous ethanol fermentation system

For the purpose of improving ethanol productivity, the effect of air supplement on the performance of continuous ethanol fermentation system was studied. The effect of oxygen supplement on yeast concentration, cell yield, cell viability, extracellular ethanol concentration, ethanol yield, maintenance coefficient, specific rates of glucose assimilation, ethanol production, and ethanol productivity have been evaluated, using a high alcohol tolerant Saccharomyces cerevisiae STV89 strain and employing a continuous fermentor equipped with an accurate air metering system in the flow rate range 0-11 mL air/L/h. It was found that, when a small amount of oxygen up to about 80mu mol oxygen/L/h was supplied, the ethanol productivity was significantly enhanced as compared to the productivity of the culture without any air supplement. It was also found that the oxygen supplement improved cell viability considerably as well as the ethanol tolerance level of yeast. As the air supply rate was increased, from 0 to 11 mL air/L/h while maintaining a constant dilution rate at about 0.06 h(-1), the cell concentration increased from 2.3 to 8.2 g/L and the ethanol productivity increased from 1.7 to 4.1 g ethanol/L/h, although the specific ethanol production rate decreased slightly from 0.75 to 0.5 g ethanol/g cell/h. The ethanol yield was slightly improved also with an increase in air supply rate, from about 0.37 to 0.45 ethanol/g glucose. The maintenance coefficient increased by only a small amount with the air supplement. This kind of air supplement technique may very well prove to be of practical importance to a development of a highly productive ethanol fermentation process system especially as a combined system with a high density cell culture technique.     

Sustained and constitutive high levels of protein production in continuous cultures of bacillus subtilis

The feasibility of continuous production of proteins in chemostat cultures of Bacillus subtilis was investigated. An expression system consisting of the bacterium B. subtilis BR151 carrying plasmid p602/19 was used. The plasmid contains the cat (chioramphenicol acetyltrans-ferase) gene downstream of a strong vegetative T5 promoter. It was found that, at a dilution rate of 0.2 h(-1) production of relatively high levels of CAT protein (about 4% ofcellular protein) can be sustained. But, experiments at a higher dilution rate of 0.4 h(-1) were unproductive because of high acidformation and washout. Combination of low cell yield, which results from excessive acid formation, and low dilution rate led to a low volumetric CAT productivity. Our recent work with the nonrecombinant cells, has demonstrated that uptake of small amounts of citrate significantly reduces or entirelyeliminates the acid formation. This superior performance in the presence ofcitrate was hypothesized, based on strong experimental evidence, to be the result of a reduction in glycolysis flux through a sequence of events leading to a reduction in pyruvate kinase and phosphof- ructokinase activities, the regulatory enzymes of glycol-ysis. In this study, it is demonstrated that cofeeding of glucose and citrate substantially reduces theorganic acid formation and significantly increases the recombinant culture productivity. The combination of high specific CAT activity and cell density resulted in a total of six- to tenfold higher culture productivitywhen citrate and glucose were cometabolized than when glucose was the only carbon source. (c) 1995 John Wiley & Sons Inc.     

Production of cloned human leukocyte interferon by Bacillus subtilis: optimal production is connected with restrained growth.

Bacillus subtilis, transformed with a plasmid containing the human alpha-2 (leukocyte) interferon gene, was cultivated in batch and continuous culture in a complex medium. In continuous culture with dissolved oxygen of less than 10% of air saturation, the extracellular interferon titer decreased sharply when the growth rate was lower or higher than the optimal one (mu = 0.14 h-1). Thus, a relatively low growth rate was best for extracellular interferon production, and oxygen limitation enhanced interferon production. The mean output rate in batch culture after successful harvest was 20 X 10(6) IU/liter per h and the maximal output rate in continuous culture was 14 X 10(6) IU/liter per h.     

Production of cloned human leukocyte interferon by Bacillus subtilis: optimal production is connected with restrained growth

Bacillus subtilis, transformed with a plasmid containing the human alpha-2 (leukocyte) interferon gene, was cultivated in batch and continuous culture in a complex medium. In continuous culture with dissolved oxygen of less than 10% of air saturation, the extracellular interferon titer decreased sharply when the growth rate was lower or higher than the optimal one (mu = 0.14 h-1). Thus, a relatively low growth rate was best for extracellular interferon production, and oxygen limitation enhanced interferon production. The mean output rate in batch culture after successful harvest was 20 X 10(6) IU/liter per h and the maximal output rate in continuous culture was 14 X 10(6) IU/liter per h.     

Determination of kinetic parameters related to the piasmid instability: for the recombinant fermentation under repressed condition

The plasmid stability under the repressed state of cloned gene was studied theoretically as well as experimentally using recombinant E. coli K12DeltaH1Deltatrp/pPLc23trpA1 as a "host-vector" model system. The important kinetic parameters studied were the plasmid loss rate (theta) describing the rate at which the plasrnid-harboring cells lose plas-mids and the plasmid-free cells are generated per unit time and the difference in growth rates (Delta) between the two genotypes. These parameters were carefully defined, studied, and compared with other key kinetic parameters involved in the recombinant fermentation to further our understanding of metabolism of recombinants. The ratio of the concentration of plasmid-free cells to plasmid-harboring cells (Omega) was introduced, and the mathematical model was derived and used for the determination of the kinetic parameters associated with plasmid instability. These methods developed based on the theoretical considerations were tested experimentally. The results of these methods were compared, and the best method was selected and recommended. The effect of temperature and dilution rate on kinetic parameters theta and Delta were also studied in continuous culture, in order to provide some practical information related to the operation and control of recombinant fermentation processes.     

High production of acetone-butanol-ethanol with high cell density culture by cell-recycling and bleeding

A continuous acetone-butanol-ethanol (ABE) production system with high cell density obtained by cell-recycling of Clostridium saccharoperbutylacetonicum N1-4 has been studied. In conventional continuous culture of ABE without cell-recycling, the cell concentration was below 5.2 g l(-1) and the maximum ABE productivity was only 1.85 g l(-1)h(-1) at a dilution rate of 0.20 h(-1). To obtain a high cell density at a faster rate, we concentrated the solventogenic cells of the broth 10 times by membrane filtration and were able to obtain approximately 20 g l(-1) of active cells after only 12h of cultivation. Continuous culture with cell-recycling was then started, and the cell concentration increased gradually through cultivation to a value greater than 100 g l(-1). The maximum ABE productivity of 11.0 gl(-1)h(-1) was obtained at a dilution rate of 0.85 h(-1). However, a cell concentration greater than 100 gl(-1) resulted in heavy bubbling and broth outflow, which made it impossible to carry out continuous culture. Therefore, to maintain a stable cell concentration, cell-bleeding was performed together with cell-recycling. At dilution rates of 0.11h(-1) and above for cell-bleeding, continuous culture with cell-recycling could be operated for more than 200 h without strain degeneration and the overall volumetric ABE productivity of 7.55 gl(-1)h(-1) was achieved at an ABE concentration of 8.58 gl(-1).     

Stabilization of anE. coli plasmid by a mini-F fragment of DNA

Summary In anEscherichia coli K-12 strain (trpA trpE tnaA) cultured in LB broth without selective pressure, a pBR322 derivative containing the gene for tryptophan synthase (pBR322-trpBA) was found to be unstable. After 70 cell-number doublings, only 50% of the host cells retained the gene for ampicillin resistance (Ap^r). Insertion of the mini-F fragment of F factor DNA into this plasmid could effectively reduce the plasmid loss. Partial derepression of the tryptophan promotor-operator by 3-indopleacrylic acid further decreased the stability of the pBR322-trpBA but not that of the mini-F inserted plasmid (pBR322F-trpBA) The vector pBR322F-trpBA could be maintained at high copy number in the culture after 100 generations of growth; the culture was able to overproduce tryptophan synthase in the presence of 3-indoleacrylic acid.l-Tryptophan was produced from indole andl-serine using andE. coli host transformed with.pBR322F-trpBA DNA. After 8 h of incubation, the expression level was approximately 180 g/l.