Escherichia coli growth and plasmid copy numbers in continuous cultivations

Summary AnEscherichia coli K-12 strain harbouring either the plasmid pBR322, or the recombinant plasmid pKTH1220, a 14 kb derivative of pBR322, or no plasmid was grown in a chemostat. The cultivations were continued for 300–400 bacterial generations.E. coli hosts harbouring pBR322 or no plasmid grew in a similar way, but the growth of the host containing the big recombinant plasmid was slower. The plasmid copy numbers increased up to 2–3 fold as the dilution rate was increased from 0 to ca. 1 h^–1. After this point the increase in dilution rate seemed to induce a rapid decrease in the plasmid copy numbers. High copy numbers could be maintained using dilution rates resulting in good productivity of the cell mass.     

An unusual mutation in RepA increases the copy number of a stringently controlled plasmid (Rtsl derivative) by over one hundred fold

A copy number mutant of the Rtsl replicon (copy number 1–2 copies/cell) was obtained. A one-base substitution in the repA region results in a single amino acid change from histidine to asparagine at position 159. This mutation increased the plasmid copy number by up to 120-fold depending upon the growth conditions. At 42.5° C the plasmid with the wild type replicon was unstable while the mutated replicon was relatively stable.     

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.     

Protein production using recombinant yeast in an immobilized-cell-film airlift bioreactor

A recombinant Saccharomyces cerevisiae C468/pGAC9 (ATCC 20690), which expresses Aspergillus awamori glucoamylase gene under the control of the yeast enolase I (ENO1) promoter and secretes glucoamylase into the extracellular medium, was used as a model system to investigate the effect of cell immobilization on bioreactor culture performance. Free suspension cultures in stirred-tank and airlift bioreactors confirmed inherent genetic instability of the recombinant yeast. An immobilized-cell-film airlift bioreactor was developed by employing cotton cloth sheets to immobilize the yeast cells by attachment. Enhanced enzyme productivity and production stability in the immobilized-cell system were observed. Experimental data indicated that the immobilized cells maintained a higher proportion of plasmid-bearing cells for longer periods under continuous operation. The higher plasmid maintenance with immobilized cells is possibly due to reduced specific growth rate and increased plasmid copy number. Double-selection pressure was used to select and maintain the recombinant yeast. The selected strain showed better production performance than the original strain. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 241-251, 1997.     

High-Level Production of Plasmid DNA by Escherichia coli DH5α ΩsacB by Introducing inc Mutations

For small-copy-number pUC-type plasmids, the inc1 and inc2 mutations, which deregulate replication, were previously found to increase the plasmid copy number 6- to 7-fold. Because plasmids can exert a growth burden, it was not clear if further amplification of copy number would occur due to inc mutations when the starting point for plasmid copy number was orders of magnitude higher. To investigate further the effects of the inc mutations and the possible limits of plasmid synthesis, the parent plasmid pNTC8485 was used as a starting point. It lacks an antibiotic resistance gene and has a copy number of ∼1,200 per chromosome. During early stationary-phase growth in LB broth at 37°C, inc2 mutants of pNTC8485 exhibited a copy number of ∼7,000 per chromosome. In minimal medium at late log growth, the copy number was found to be significantly increased, to approximately 15,000. In an attempt to further increase the plasmid titer (plasmid mass/culture volume), enzymatic hydrolysis of the selection agent, sucrose, at late log growth extended growth and tripled the total plasmid amount such that an approximately 80-fold gain in total plasmid was obtained compared to the value for typical pUC-type vectors. Finally, when grown in minimal medium, no detectable impact on the exponential growth rate or the fidelity of genomic or plasmid DNA replication was found in cells with deregulated plasmid replication. The use of inc mutations and the sucrose degradation method presents a simplified way for attaining high titers of plasmid DNA for various applications.     

A new extant respirometric assay to estimate intrinsic growth parameters applied to study plasmid metabolic burden

Start‐up phenomena in microbial biokinetic assays are not captured by the most commonly used growth‐related equations. In this study we propose a new respirometric experimental design to estimate intrinsic growth parameters that allow us to avoid these limitations without data omission, separate mathematical treatment, or wake‐up pulses prior to the analysis. Identifiability and sensitivity analysis were performed to confirm the robustness of the new approach for obtaining unique and accurate estimates of growth kinetic parameters. The new experimental design was applied to establish the metabolic burden caused by the carriage of a pWW0 TOL plasmid in the model organism Pseudomonas putida KT2440. The metabolic burden associated was manifested as a reduction in the yield and the specific growth rate of the host, with both plasmid maintenance and the over‐expression of recombinant proteins from the plasmid contributing equally to the overall effect. Biotechnol. Bioeng. 2010;105: 141–149. © 2009 Wiley Periodicals, Inc.     

Control of Plasmid R1 Replication: Kinetics of Replication in Shifts Between Different Copy Number Levels

Plasmid R1 replication was studied in shifts between two steady states of copy number. The copy number was varied in two ways. First, we utilized the fact that it decreases with increasing growth rate. To minimize the metabolic effects of changes in the growth rate, the downshifts were obtained by adding α-methylglucoside to cultures growing in glucose-minimal medium, and the upshifts were obtained by adding glucose to cultures growing in the presence of glucose plus α-methylglucoside. Second, we used a temperature-dependent copy mutant of plasmid R1 (pKN301). Plasmid pPK301 shows a threefold higher copy number at 40 than at 30°C. In both types of shift, plasmid replication immediately adjusted to the postshift differential rate. The copy number asymptotically adjusted to the new steady state. Hence, the system that controls plasmid R1 replication sets the frequency of replication without measuring the actual copy number. It has been suggested that plasmid R1 replication is under negative control by an R1-mediated repressor protein. Among the replication control models that involve negative control, the Pritchard inhibitor dilution model, the Sompayrac-Maaløe autorepressor model, and the plasmid λdv system all predict gene dose-independent copy number control.     

Effect of various irradiation treatments of plant protoplasts on the transformation rates after direct gene transfer

Summary In P. hybrida and B. nigra an enhancement of transformation rates (direct gene transfer) of about six to seven-fold was obtained after irradiation of protoplasts with 12.5 Gy (X-ray). The effect of protoplast irradiation was similar in experiments where protoplasts were irradiated 1h before transformation (X-ray/DNA) or 1h after completion of the transformation procedure (DNA/X-ray). Increased X-ray doses up to 62.5 Gy resulted in further enhancement of percentages of transformed colonies, indicating a correlation between relative transformation frequencies (RTF) and the doses applied. Estimation of degradation rates of plasmid sequences in plant protoplasts yielded a reduction of plasmid concentration to 50% 8–12 h after transformation. In 1-day-old protoplasts, the level of plasmid fragments dropped to 0%–10% compared to 1h after transformation. The results demonstrate that the integration rates of plasmid sequences into the plant genome may in part be governed by DNA repair mechanisms. This could be an explanation for the observed genotypic dependence of transformation rates in different plant species and plant genotypes. Gene copy number reconstructions revealed enhanced integration rates of plasmid sequences in transformed colonies derived from irradiated protoplasts.     

Characterization of a Large, Stable, High-Copy-Number Streptomyces Plasmid That Requires Stability and Transfer Functions for Heterologous Polyketide Overproduction

A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene, spd, and a partition gene, parAB, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (actII-ORF4) and biosynthetic genes (eryA) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance.     

Influence of temperature rise on kinetic parameters during batch propagation of Kluyveromyces fragilis in cheese whey under ambient conditions

Three 5 l working volume fermenters were used to investigate the growth of the yeast Kluyveromyces fragilis in acid cheese whey under ambient temperature in order to assess the specific growth rate and yield, the lactose and oxygen uptake rates during the various phases of batch culture, the effect of increasing temperature on the various kinetic parameters, and the need for a cooling unit for single cell production batch systems. The initial dissolved oxygen in the medium was 5.5 mg l^–1 and the pH was maintained at 4.5. The observed lag phase, specific growth rate and maximum cell number were 4 h, 0.2 h^–1 and 8.4 × 10⁸ cells ml^–1, respectively. About 99% of the lactose in cheese whey was utilized within 20 h, 85% during the exponential growth phase. The specific lactose utilization rates by K. fragilis were 0.20 × 10^–12, 1.457 × 10^–12, 0.286 × 10^–12 and 0.00 g lactose cell^–1 h^–1, for the lag, exponential, stationary and death phases, respectively. The dissolved oxygen concentration in the medium decreased as the cell number increased. The lowest oxygen concentration of 1.2 mg l^–1 was observed during the stationary phase. The volumetric oxygen transfer coefficient was 0.41 h^–1 and the specific oxygen uptake rates were 0.32 × 10^–12, 2.14 × 10^–12, 0.51 × 10^–12 and 0.003 × 10^–12 mg O₂ cell^–1 h^–1, for the lag, exponential, stationary and death phases, respectively. The maximum temperature recorded for the medium was 33 °C, indicating that a cooling unit for batch production of single cell protein at ambient temperature is not needed for this type of bioreactor. The increase in medium temperature affected the cell growth and the lactose and oxygen uptake rates.     

Stable continuous constitutive expression of a heterologous protein in Saccharomyces cerevisiae without selection pressure

The stability of heterologous protein expression in Saccharomyces cerevisiae during continuous culture without selection for plasmid-containing cells was investigated. The protein chosen was the leech thrombin inhibitor desulphato-hirudin, which is tolerated well by S. cerevisiae when over-expressed. Expression was from a 2- derived multicopy vector containing a synthetic hirudin gene under control of the constitutive glyceraldehyde-3-phosphate dehydrogenase derived GAPFL promoter. The behaviour of the system was studied at three dilution rates (D) corresponding to approximately 30% (0.06 h^–1), 60% (0.12 h^–1) and 90% (0.17 h^–1) of the estimated maximum D. The level of plasmid loss was low at all Ds, with only 5–10% plasmid-free cells observed at 75 generations. The plasmid was most stably maintained at the intermediate D of 0.12 h^–1, where the rate of loss was comparable to the loss of the native 2- plasmid. Hirudin expression was also highest at D=0.12 h^–1, possibly as a result of cell lysis at D=0.06 h^–1 and D=0.17 h^–1, leading to the release of vacuolar proteases and subsequent proteolysis of hirudin. Differences in expression levels were not a result of changes in plasmid copy number, which was in the range 40–60 throughout all three experiments. The high stability of this system at all Ds investigated shows that heterologous protein expression is not a burden to S. cerevisiae when the protein expressed is tolerated well. Correspondence to: M. Ibba     

Modeling of recombinant bacteria fermentation for enhanced productivity

Accurate kinetic models are necessary in order to scale biochemical processes which utilize recombinant organisms. In this work, the performance of a kinetic model proposed by Miao and Kompala (Biotechnol. Bioeng.,40, 787–796, 1992) has been tested against experimental data at different concentrations of substrates. The experimental results from the induction of the batch cultures with IPTG (isopropyl-β-D-thiogalactopyranoside) prove that the model equations are reliable in predicting the biomass and foreign protein concentrations as well as the optimum induction time.     

Effect of high density cultivation on plasmid copy number in recombinantEscherichia coli cells

Summary The copy number of plasmid harboringE. coli K 12 strains was examined in fed batch cultivations in semisynthetic and synthetic media. Under conditions of high cell density (45–50 g dry weight/I) the plasmid copy number reached a maximum level between 200 and 400 copies per cell. A decrease of phosphate concentration in the medium was obtained similar to the increase of copy number. A high segregational and structural stability of vectors used in this work was observed.     

Conjugative Transfer of the Large Plasmid p19 in Various <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Strains

Cryptic conjugative plasmid p19 from the environmental Bacillus subtilis strain 19 was labeled with the cat gene conferring resistance to chloramphenicol. The resulting plasmid, p19cat, was used to estimate the transfer frequency, to study the dynamics of plasmid transfer, and to detect some specific features of conjugation between various B. subtilis strains.__________Translated from Genetika, Vol. 41, No. 5, 2005, pp. 601–606.Original Russian Text Copyright © 2005 by Poluektova, Fedorina, Prozorov.     

Plasmid DNA fermentation strain and process‐specific effects on vector yield,quality, and transgene expression

Industrial plasmid DNA manufacturing processes are needed to meet the quality, economy, and scale requirements projected for future commercial products. We report development of a modified plasmid fermentation copy number induction profile that increases gene vaccination/therapy vector yields up to 2,600 mg/L. We determined that, in contrast to recombinant protein production, secretion of the metabolic byproduct acetate into the media had only a minor negative effect on plasmid replication. We also investigated the impact of differences in epigenetic dcm methylase‐directed cytosine methylation on plasmid production, transgene expression, and immunogenicity. While Escherichia coli plasmid production yield and quality are unaffected, dcm− versions of CMV and CMV‐HTLV‐I R promoter plasmids had increased transgene expression in human cells. Surprisingly, despite improved expression, dcm− plasmid is less immunogenic. Our results demonstrate that it is critical to lock the plasmid methylation pattern (i.e., production strain) early in product development and that dcm− strains may be superior for gene therapy applications wherein reduced immunogenicity is desirable and for in vitro transient transfection applications such as AAV production where improved expression is beneficial. Biotechnol. Bioeng. 2011;108: 354–363. © 2010 Wiley Periodicals, Inc.     

Age and growth of Pacific herring larvae based on length-frequency analysis and otolith ring number

Synopsis Age and growth in length and dry weight of cohorts of wild Pacific herring larvae, Clupea harengus pallasi, were measured using successive modes in the length-frequencies of the catches and the number of rings in the otoliths. Average linear rates of growth in length ranged from 0.36–0.41 mm d^–1, and average exponential rates of growth in dry weight ranged from 0.063–0.084 d^–1. Length-date and dry weight-age curves were best described using one-cycle and two-cycle Gompertz functions, respectively. Weight-length relationship were, therefore, curvilinear on double logarithmic plots and were best described by a non-linear allometric function. Average rates of otolith ring deposition were 0.90, 1.09 and 0.73–1.26d^–1. Rings were deposited daily from the day of complete yolk absorption in the first two cohorts, but interrupted ring deposition was observed over the first 27 d of the third cohort. Relatively low water temperatures, <9° C, coincided with the interruption and may have caused it.     

Light and temperature effects on the growth rate of three freshwater [2pt] algae isolated from a eutrophic lake

Effects of light and temperature, on the growth of three freshwater green algae isolated from an eutrophic lake and identified as Selenastrum minutum, Coelastrum microporum f. astroidea and Cosmarium subprotumidumwere studied in batch cultures under non-nutrient limited conditions. Experiments were performed to determine the growth rate over a wide range of light intensities (30–456 mol m^–2 s^–1) and temperature (15–35°C), using a 15/9 (light/dark) photoperiod cycle. The maximum growth rates and the optimum light intensities at a temperature of 35°C were 1.73 d^–1 and 420 mol m^–2 s^–1for Selenastrum minutum, 1.64 d^–1 and 400 mol m^–2 s^–1 for Coelastrum microporum and 1.00 d^–1 and 400 mol m^–2 s¹ for Cosmarium subprotumidum. The results were fitted with the mathematical models of Steele (1965), Platt & Jassby (1976) and Peeters & Eilers (1978). Steele's function and equation of Platt & Jassby don't describe correctly the relationship between the growth and light intensity. In the opposite, the equation of Peeters & Eilers provides the best fit for the three species.     

Mathematical characterization of insect cell (Sf-9) death in suspended culture

The effect of baculovirus infection on cell death in suspended cultures was characterized based on work by Wu et al. (1993) Biotech. Bioeng. 41: 104–110 and Wu et al. (1994) Biotechnol. Prog. 10: 55–59. The post infection time can be separated into a constant viability phase characterized by a time delay, td, and a rapid death phase, which is characterized by a specific death rate constant, k. Results indicated that the characteristic time delay decreased with increased multiplicity on infection (MOI). Further, there was only a weak correlation between specific death rate and MOI, for the range of MOI tested. Cell infection and death rates were consistent with a more evenly distributed infection process likely found in suspension cultures.     

Biofilm build-up of Pseudomonas putida in a chemostat at different dilution rates

Summary A test system was set up where the build-up of a biofilm on a defined surface could be studied in a carbon source limited chemostat.The attachment of P. putida ATCC 11172 to glass when growing on L-asparagine was studied at different dilution rates (specific growth rates) from 0.1 to 1.5 h^–1 The number of attached colony forming units (cfu) increased with dilution rate from 1×10⁶ cfu/cm² at 0.1 h^–1 to 4×10⁷ cfu/cm² at 1.0 h^–1 and then the attachment decreased to about 6×10⁶ cfu/cm² at higher dilution rates (1.1–1.5 h^–1). The number of attached cfu was measured after 24 h exposure. The value of the maximum specific growth rate in batch culture was 0.6 h^–1.The total amount of attached cell-mass followed roughly the same pattern as the viable count.The viable count of the cells suspended in the growth medium showed its lowest value at the same dilution rate as resulted in maximum adhesion.It was shown that the effect of growth rate on the biofilm build-up of P. putida is significant, and ought to be borne in mind when continuous culture systems are set up and results evaluated.