Escherichia coli growth and plasmid copy numbers in continuous cultivations

Reinikainen, P.; Virkajärvi, Ilkka

doi:10.1007/bf01031568

Cited by 30 publications

(17 citation statements)

References 7 publications

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“…Using a higher growth rate of 0.28 h ¡1 , we reduced the DNA yields achieved prior to the heat treatment by 28% and 20% for SCS1-L and MC4100, respectively, as compared to the low growth rate. It is widely accepted that copy numbers of ColE1-type plasmids are inversely proportional to the speciWc growth rate [21,25,34,35,38,39], and the results of this study are consistent with previous observations. The conclusions are more complicated, however, when the impact of heat treatment is also considered.…”

Section: Discussionsupporting

confidence: 94%

“…Although some studies indicate an optimal speciWc growth rate for maximal DNA yield [35,39], low speciWc growth rates are generally associated with favoring plasmid DNA synthesis on a per cell basis [2,12,22,25,43]. Since a nutrient-limited fed-batch process routinely achieves cell densities of 40 g/L, volumetric plasmid yield is maximized by a process operated at a low feeding (i.e., growth) rate.…”

Section: Introductionmentioning

confidence: 98%

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DNA plasmid production in different host strains of Escherichia coli

Singer¹,

Eiteman

Altman

2009

J Ind Microbiol Biotechnol

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We compared plasmid DNA production in 13 strains of Escherichia coli in shake flasks using media containing glucose or glycerol. DNA yield from either carbon source showed small correlation with maximum growth rate. Three strains, SCS1-L, BL21 and MC4100, were selected for a controlled exponential fed-batch process at a growth rate of 0.14 h(-1) to an optical density of about 70, followed by a four-hour heat treatment. Prior to heat treatment, SCS1-L generated 15.4 mg DNA/g, BL21 generated 11.0 mg DNA/g and MC4100 generated 7.9 mg DNA/g, while after heat treatment the strains attained DNA yields, respectively, of 18.0, 15.0 and 6.8 mg/g. The strains also varied in their percentage of supercoiled DNA after heat treatment, with SCS1-L averaging 66% supercoiled, BL21 17% and MC4100 40%. We further investigated the two strains that yielded the highest percentage of supercoiled DNA (SCS1-L and MC4100) at a higher growth rate of 0.28 h(-1). At this condition, a slightly lower DNA yield was generated faster, and the percentage of supercoiled DNA increased. Heat treatment improved DNA yield, and surprisingly did so to a greater extent at the higher growth rate. As a consequence of these factors, higher growth rates might be advantageous for DNA production.

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Section: Discussionsupporting

confidence: 94%

Section: Introductionmentioning

confidence: 98%

DNA plasmid production in different host strains of Escherichia coli

Singer¹,

Eiteman

Altman

2009

J Ind Microbiol Biotechnol

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“…in opposing findings, either with a positive correlation between a high copy number plasmid and high growth rates (Reinikainen and Virkajarvi, 1989) or a negative correlation (Kim and Ryu, 1991;Ryan and Parulekar, 1991).…”

Section: Correlation Of Copy Number and Growth Ratementioning

confidence: 87%

Host strain influences on supercoiled plasmid DNA production in Escherichia coli: Implications for efficient design of large‐scale processes

Yau

Keshavarz-Moore

Ward

2008

Biotech & Bioengineering

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We set out to investigate if E. coli genotype plays a significant role in host strain selection for optimal processing of plasmid DNA based on both quality and quantity of supercoiling. Firstly 17 E. coli commercial and non-commercial strains were selected and their available genetic backgrounds were researched in the open literature. Growth characteristics of all the strains were considered and made impartial by using a common medium and growth condition platform. By keeping the growth conditions constant for each strain/plasmid combination, we are only looking at one variable which is the host strain. The second step was to attempt to correlate the findings with common genotype characteristics (e.g. mutations such as endA or recA). We found that one can screen the number of strains which are likely to give good productivity early on, before any further optimisation and verification is performed, both for small and large plasmids. Also, it is worth noting that complex plasmid interactions with each strain prevent the use of genotype alone in making an intelligent choice for supercoiling optimisation. This leads to a third optimisation step selecting a few of the potentially high performing strains based on high DNA yield and supercoiling, with a view to identify the factors which need improvement in strain design and bioreactor optimisation. We found that high specific growth rates of some strains did not affect the level of DNA supercoiling but did influence the total plasmid yield, potentially an important aspect in the design of fermentation strategy. Interestingly, a few host/plasmid combinations result in what appears to be runaway plasmid replication.

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“…When D > 1/t B or F > V/t B , the P 1 metric is monotonically increasing. This condition for monotonic increase is typically satisfied for the initial batch operation times that are required for fermentation systems that produce DNA plasmids [8][9][10][11][12][13][14][15]. In this case, the optimal operation using this metric is continuous operation for as long as possible.…”

Section: Cumulative Mass/time Productivity With Constant Product Concmentioning

confidence: 99%

“…Productivity (dilution rate times the amount of b-lactamase per milliliter of culture) showed a maximum of 3.4 units/ml at a dilution rate of 0.44 h -1 , demonstrating the existence of two potential optimal dilution rates for maximizing the plasmid concentration and protein expression. The effect of dilution rate on microbial growth and copy number was also examined by Reinikainen [9] using E. coli K-12 HB101 strain, harboring either the plasmid pBR322 (4.36 kb), or a recombinant plasmid pKTH1220 (14 kb), or no plasmid. The LB medium containing ampicillin at a concentration of 100 mg/L was used for both inoculum and cultivations.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Optimization Metrics for Continuous Fermentation of Plasmid DNA

Kelly¹,

Balguri²,

Asoor³

et al. 2009

Chem Eng & Technol

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An evaluation of cumulative productivity metrics for optimizing the continuous fermentation of plasmid DNA (pDNA) is carried out in this work. DNA plasmids provide a good illustrative example for comparison because of the plasmid instability issues associated with their production. However, the analysis presented in this work can also be applied to other intracellular bioproduct continuous fermentation systems. The productivity metrics considered are cumulative product mass per time, profit, and cost objective functions. These metrics are used to determine the optimal continuous fermentation run time at a given dilution rate, during the dynamic period associated with switching from batch to continuous fermentation. The results of this study indicate that different optimum continuous fermentation run times are predicted based upon the choice of the continuous fermentation operating conditions and optimization metric. In particular, the dilution rate must be larger than the inverse of a characteristic time, that is a function of the initial batch operating and plasmid stability times before continuous operation becomes optimal. Provided that this condition is met, different optimal continuous operation run times are obtained depending on the cumulative productivity metric, or operating objective, that is employed.

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Escherichia coli growth and plasmid copy numbers in continuous cultivations

Cited by 30 publications

References 7 publications

DNA plasmid production in different host strains of Escherichia coli

DNA plasmid production in different host strains of Escherichia coli

Host strain influences on supercoiled plasmid DNA production in Escherichia coli: Implications for efficient design of large‐scale processes

Evaluation of Optimization Metrics for Continuous Fermentation of Plasmid DNA

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