Plasmid maintenance and protein overproduction in selective recycle bioreactors

A new plasmid construct has been used in conjunction with selective recycle to successfully maintain otherwise unstable plasmid-bearing E. coli cells in a continuous bioreactor and to produce significant amounts of the plasmid-encoded protein beta-lactamase. The plasmid is constructed so that pilin expression, which leads to bacterial flocculation, is under control of the tac operon. The plasmid-bearing cells are induced to flocculate in the separator, whereas cell growth and product synthesis occur in the main fermentation vessel without the inhibiting effects of pilin production. Selective recycle allows for the maintenance of the plasmid-bearing cells by separating flocculent, plasmid-bearing cells from nonflocculent, segregant cells in an inclined settler, and recycling only the plasmid-bearing cells to the reactor. As a result, product expression levels are maintained that are more than ten times the level achieved without selective recycle. All experimental data agree well with theoretical predictions.     

Continuous recombinant bacterial fermentations utilizing selective flocculation and recycle.

Selective recycle has successfully been used to maintain an unstable plasmid-bearing bacterial strain as dominant in a continuous reactor, whereas the culture reverts to 100% segregant cells when selective recycle is not used. The plasmid-bearing strain is slower growing and flocculent; however, when the cells lose their plasmid, the resulting segregant cells are nonflocculent and grow at a faster rate due to their decreased metabolic burden. Both types of cells exit a chemostat and enter an inclined settler where the flocculent plasmid-bearing cells are separated from the nonflocculent segregant cells by differential sedimentation. The underflow from the cell separator, which is enriched with plasmid-bearing cells, is recycled back to the chemostat, while the segregant cells are withdrawn off the top of the settler and discarded. The experimental results agree well with selective recycle reactor theory. On the basis of the theory, a criterion is presented that has been shown to successfully predict whether or not a selective recycle reactor can maintain a plasmid-bearing strain.     

A new oxygen-regulated promoter for the expression of proteins in Escherichia coli

Several properties of a new oxygen-regulated promoter, OXYPRO, were tested in small-scale Escherichia coli cultures. Using OXYPRO, maximal activity of a reporter gene encoding chloramphenicol acetyltransferase (CAT) occurred in cultures that were tightly capped immediately after inoculation. This is probably a result of the reduced oxygen concentration attained in capped cultures, a condition known to be required for OXYPRO induction. CAT levels were significantly higher when the cells were grown in a glycerol-based medium. Similar levels of CAT expression were obtained when OXYPRO was compared to the trp-lac (tac) promoter. In addition, regulated expression of CAT occurred in a wild type strain of E. coli, suggesting that OXYPRO will be useful in most E. coli strains. Thus, OXYPRO provides a simple, inexpensive, and unobtrusive method to achieve high levels of cloned protein expression in most strains of E. coli. OXYPRO is available in a high copy plasmid with a convenient multiple cloning site for the insertion of genes for direct expression in E. coli.     

A novel structured kinetic modeling approach for the analysis of plasmid instability in recombinant bacterial cultures

The instantaneous specific growth rate of a recombinant bacterial culture is directly calculated using a simple structured kinetic modeling approach. Foreign plasmid replication and foreign protein expression represent metabolic burdens to the host cell. The individual effects of these plasmid-mediated activities on the growth rate of plasmid-bearing cells are estimated separately. The dynamic and steady state simulations of the model equations show remarkable agreement with widely observed experimental trends in plasmid copy number and foreign protein content. The model provides an important tool for understanding and controlling plasmid instability in recombinant bacterial fermentations. The modeling framework employed here is suitable for studying the metabolism and growth of a variety of microbial cultures.     

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.     

Overexpression of cloned genes using recombinant Escherichia coli regulated by a T7 promoter: II. Two-stage continuous cultures and model simulations

A two-stage culture strategy was studied for continuous high-level production of a foreign protein in the chemically inducible T7 expression system. The first stage is dedicated to the maintenance of plasmid-bearing cells and the second stage to the target protein synthesis by induction of cells coming from the first stage. On entering the second stage, recombinant cells undergo a gradual induction of the target gene expression. These plasmid-bearing cells experience dynamic changes in intracellular compositions and specific growth rates with their individual residence times. Therefore, the overall cultural characteristics in the production stage are really averages of the contributions from the various cells with different residence times. The behavior of the two-stage culture is described by a model, which accounts for dynamic variations of cell growth and protein synthesis rates with cell residence times. Model simulations were compared with experimental results at a variety of operating conditions such as inducer concentration and dilution rate. This model is useful for understanding the behavior of two-stage continuous cultures. (c) 1993 John Wiley & Sons, Inc.     

Dual control of C-reactive protein gene expression by interleukin-1 and interleukin-6.

Human C-reactive protein (CRP) is the major acute phase reactant during acute inflammation. The human CRP promoter is expressed in an inducible and cell-specific manner when linked to the bacterial CAT gene and transfected into human hepatoma cell cultures. In this paper we analyze the effect of several recombinant cytokines or CRP promoter inducibility in human Hep3B cells. When cytokines are tested singly the major inducer of CRP-CAT fusions is interleukin-6 (IL-6). Maximal CAT gene expression, however, is only achieved when both interleukin-1 beta (IL-1 beta) and IL-6 are present. The response to the two cytokines is cooperative. Cooperativity is maintained when the CRP promoter is linked to a different coding region, that of the bacterial neomycin phosphotransferase II gene. With a series of 5' and 3' deletions we show the existence of two distinct and independent regions responsive to IL-6 and located upstream to the TATA box. The IL-1 effect is exerted at the level of downstream sequences that are probably important for optimal mRNA translatability or nuclear-cytoplasmic transport. Inducibility is not influenced by the activation of protein kinases C or A and does not require new protein synthesis.     

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.     

A novel BK virus-based episomal vector for expression of foreign genes in mammalian cells.

A composite mammalian cell-E. coli shuttle vector was developed based on the human papova virus BK and pSV-neo. The vector contains a dioxin-responsive enhancer (DRE) controlling a mouse mammary tumor virus (MMTV) promoter for the inducible expression of inserted genes. In human cells the vector replicates episomally, presumably utilizing the BKV rather than the SV40 origin, and expresses the BK T/t antigens. A deletion in the late BK region precludes the expression of the core/capsid proteins VP1, VP2, and VP3, thereby preventing the infectious lytic cycle. HeLa cells which were transfected with this vector and selected for resistance to the antibiotic G418 maintained the construct primarily in episomal form during more than one year of continuous culture, with little or no integration into the host genome. Transformed cells cultured in higher concentrations of G418 contained higher copy numbers of the vector. This permits one to vary the dosage of an inserted gene easily and reversibly without the need of conventional amplification techniques and clonal analysis. Using a chloramphenicol acetyl transferase (CAT) reporter gene inserted downstream of the MMTV promoter, we found that CAT expression was greater in clones with higher vector copy number. CAT expression was inducible with 2,3,7,8-tetrachlorodibenzo-p-dioxin, but inducibility was found to be inversely proportional to the copy number. Transformation of bacteria with plasmid molecules retrieved from the mammalian host was efficient, making this vector well adapted for the screening of cDNA libraries for the ability to express a phenotype in mammalian cells. Moreover, DNA sequences were stable during long-term passage in mammalian cells; vector passaged continuously for more than one year retained fully functional bacterial genes for resistance to chloramphenicol and ampicillin.     

Population dynamics of a continuous fermentation of recombinant Saccharomyces cerevisiae using flow cytometry

The plasmid instability of genetically modified microorganisms during prolonged bioreactor operations is one of the major problems to be overcome in the production of recombinant proteins. The use of flow cytometry to monitor a fermentation process with recombinant cells in a CSTR is reported here. This technique has been applied to determine the fraction of plasmid-bearing cells (P+) of a recombinant Saccharomyces cerevisiae strain harboring the EXG1 gene in a continuous stirred tank bioreactor with a working volume of 2 L. The different levels in the expression of the EXG1 gene, which encodes the enzyme exo-beta-glucanase, were used to determine the P+ fraction. Other parameters such as viability, cellular protein, cell size and structure were also monitored using flow cytometry. This technique has two main advantages over the conventional method of determining the P+ fraction (plating in selective and non-selective solid media): (a) it takes a very short period of time to obtain a measurement that provides multiple parametric information; and (b) it is more representative of the bioreactor cell population since it can analyze thousands of cells in the same sample. A continuous operation (432 h) with the recombinant strain in a CSTR was carried out to test the application of this technique. Measurements of cellular exo-beta-glucanase activity and cellular protein content closely correlates to the measured fraction of plasmid-containing cells in the population. Moreover, the standard deviation of the fraction of P+ cells determined using this technique was very low (about 2%). Recombinant protein production also increased the size of the yeast cells, whereas the recombinant cells also had a more complex internal structure than the non-recombinant host strain.     

Influence of immobilization on the stability of pTG201 recombinant plasmid in some strains of Escherichia coli

The stability of pTG201 plasmid was examined by continuous culture in three genetically different Escherichia coli hosts. Two types of experiment were carried out, one with free cells and one with immobilized cells. When cells were cultivated in free continuous culture in the absence of antibiotic selection, the plasmid was maintained with various degrees of stability in the three host organisms. By contrast, in continuous culture with immobilized cells, plasmid pTG201 was stably maintained in the three strains. We showed that the increase in pTG201 stability in immobilized cells is due neither to plasmid transfer between immobilized cells nor to an increase of the plasmid copy number of immobilized cells. We also showed that plasmid-free cells, when coimmobilized and grown in competition with plasmid-containing cells, cannot overrun the culture.     

Influence of immobilization on the stability of pTG201 recombinant plasmid in some strains of Escherichia coli.

The stability of pTG201 plasmid was examined by continuous culture in three genetically different Escherichia coli hosts. Two types of experiment were carried out, one with free cells and one with immobilized cells. When cells were cultivated in free continuous culture in the absence of antibiotic selection, the plasmid was maintained with various degrees of stability in the three host organisms. By contrast, in continuous culture with immobilized cells, plasmid pTG201 was stably maintained in the three strains. We showed that the increase in pTG201 stability in immobilized cells is due neither to plasmid transfer between immobilized cells nor to an increase of the plasmid copy number of immobilized cells. We also showed that plasmid-free cells, when coimmobilized and grown in competition with plasmid-containing cells, cannot overrun the culture.     

Purification of recombinant antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein antigen using the expression system pH6EX3 followed by metal chelating affinity chromatography.

A novel plasmid expression vector (pH6EX3) that directs the synthesis of a fusion protein with a histidine hexapeptide at its N-terminus and a foreign protein at its C-terminus was constructed. The fusion gene is controlled by a strong tac promoter, leading to high-level expression of recombinant protein in several bacterial strains; the protein is deposited mainly as an insoluble mass in inclusion bodies. The fusion protein can be purified from the insoluble cell fraction by one-step affinity chromatography based on the selective interaction between the histidine hexapeptide and a metal chelating matrix charged with Ni2+ ions. The principle of this new system was tested by expressing and purifying antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein autoantigen. With the use of column chromatography and pH gradient elution, about 25 micrograms recombinant protein/ml of bacterial culture was obtained.     

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.     

Use of synthetic ribosome binding site for overproduction of the 5B protein of insertion sequence IS5.

Insertion sequence IS5 is a bacterial transposable element which contains three open reading frames designated 5A, 5B and 5C. Although there was no detectable expression from the 5B open reading frame when it was preceded by the native promoter and ribosome binding site or by a tac promoter and the native ribosome binding site, we have overproduced a 5B protein both in vitro and in Escherichia coli cells by using a tac promoter and a specially-designed synthetic ribosome binding site. beta-galactosidase fusion studies suggested that the synthetic binding site is at least 150-fold more efficient than the native binding site. The 5B protein amounted to 80-85% of the total protein made in vitro and 20-25% of the total protein pulse-labelled in whole cells. It is stable in vitro but rapidly degraded in vivo. Thus expression of the 5B gene appears to be limited by both poor translation initiation and protein degradation.     

Stability fluctuations of plasmid-bearing cells: immobilization effects

The maintenance of the plasmid vectors pTG201 and pTG206 (which both carry the Pseudomonas putida xylE gene) and pB lambda H3 in Escherichia coli hosts was studied in free and immobilized continuous cultures. pTG201, containing the strong lambda PR promoter, was more quickly lost than plasmid pTG206, containing the tetracycline resistance gene promoter. The instability of pTG201 seems to be related to high expression of the cloned xylE genet. Fluctuations in the proportion of pTG201-containing cells were observed in the free system, suggesting the appearance of adaptive descendants (with and without plasmid) from the initial strains. The loss of plasmid vectors from E. coli cells and the fluctuations in the proportion of plasmid-containing cells could be prevented by immobilizing plasmid-containing bacteria in carrageenan gel beads.     

Sequence and expression characteristics of a shuttle chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli

An efficiently transforming chloramphenicol-resistance (CmR) shuttle marker for Saccharomyces cerevisiae and Escherichia coli has been characterized in terms of its primary structure and expression characteristics. The complete nucleotide (nt) sequence of the CmR marker is given, with details on restriction sites, apparent expression signals for both organisms, and translation of the Cm acetyltransferase (CAT)-coding sequence. SDS-polyacrylamide gel electrophoresis and Western blotting have confirmed that the marker produced an identical CAT protein in yeast and E. coli. Each copy of the marker, whether present in multiple copies or as a single copy, gave rise to approx. 0.1% of the total soluble protein as CAT in haploid yeast cells. When compared with homologous expression of alcohol dehydrogenase (ADH-I) by the same ADC1 promoter, this represents a 27-fold reduction for CAT expression, which is typical of heterologous gene expression in yeast. When the marker was on a multicopy plasmid in yeast, up to 2.1% of the total soluble cell protein was produced as CAT, but this did not adversely affect the growth of host cells. Increase of the Cm concentration in the medium did not result in an increase in the number of plasmids nor the amount of CAT protein produced, showing that plasmid copy number and marker expression are regulated independently of the selection pressure. In E. coli, the ADC1 yeast-promoter DNA was found to contain both forwards and backwards promoter activity. The level of expression provided by these promoters was equivalent to that of an average E. coli gene.