Cellular Responses to Strong Overexpression of Recombinant Genes in Escherichia Coli

Hong, Lin; Hanschke, Renate; Nicklisch, Silke; Riemschneider, Stephan; Meyer, Sylke; Gupta, Antje; Neubauer, Peter; Nietsche, Thomas; Hecker, Michael; Jarchow, Raymond; Schwahn, Christian

doi:10.1007/978-94-015-9749-4_5

Cited by 10 publications

(10 citation statements)

References 31 publications

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“…The development of other tools for production, such as promoters, is mainly based on the idea that cell mass is produced in the first step and thereafter the cell should be devoted to product formation. However, strong promoters such as the tac and T7 systems can only be employed for a short period of time after induction since the product formation rate often ceases after just 1-2 h. Examples of this can be found in Dedhia et al (1997), Striedner et al (2001), and Lin et al (2001). After this period, production is rapidly reduced to zero and in the worst case the product protein is degraded (Streidner et al, 2001).…”

Section: Introductionmentioning

confidence: 96%

Limiting factors in Escherichia coli fed‐batch production of recombinant proteins

Sandén

Prytz

Tubulekas

et al. 2002

Biotech & Bioengineering

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Fed-batch production of recombinant beta-galactosidase in E. coli was studied with respect to the specific growth rate at induction. The cultivations were designed to induce protein production by IPTG at a glucose feed rate corresponding to high mu = 0.5 h(-1)) or low (mu = 0.1 h(-1)) specific growth rate. Protein production rate was approximately 100% higher at the higher specific growth rate, resulting in the accumulation of beta-galactosidase up to 30% of the total cell protein. Transcription analysis showed that beta-galactosidase-specific messenger RNA was immediately formed after induction (<5 min), but the amount was the same in both cases and was thus not the initial limiting factor. The content of ribosomes, as represented by rRNA, rapidly decreased with specific growth rate from a relative level of 100%, at the high specific growth rate, to 20% at the low specific growth rate. At high specific growth rate, ribosomes were additionally degraded upon induction due to the high production level. Translation therefore seemed to be the initial limiting factor of the protein synthesis capacity. The alarmone guanosine tetraphosphate increased at both high and low feed level inductions, indicating an induction-forced starvation of charged tRNA and/or glucose. The altered physiological status was also detected by the formation of acetic acid. However, the higher production rate resulted in high-level accumulation of acetic acid, which was absent at low feed rate production. Acetic acid production is thus coupled to the high product formation rate and is proposed to be due either to a precursor drain of Krebs cycle intermediates and a time lag before induction of the glyoxalate shunt, or to single amino acid overflow, since the model product is relatively poor in glycin and alanin. In conclusion, it is proposed that production at high specific growth rate becomes precursor-limited, while production at low specific growth rate is carbon- and/or energy-limited.

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Section: Introductionmentioning

confidence: 96%

Limiting factors in Escherichia coli fed‐batch production of recombinant proteins

Sandén

Prytz

Tubulekas

et al. 2002

Biotech & Bioengineering

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138

120

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“…In response to stress ATP or the total adenylate concentration have been followed after cold shock, where both decrease [34], after UV radiation, where the ATP level transiently increased twofold [35], with a following decrease in recA + cells, which the authors relate to the action of substrate level phosphorylation. Also the levels of ATP and other nucleotides have been followed after induction of recombinant proteins by IPTG [36]. …”

Section: Introductionmentioning

confidence: 99%

Transient increase of ATP as a response to temperature up-shift in Escherichia coli

et al. 2005

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SummaryBackground: Escherichia coli induces the heat shock response to a temperature up-shift which is connected to the synthesis of a characteristic set of proteins, including ATP dependent chaperones and proteases. Therefore the balance of the nucleotide pool is important for the adaptation and continuous function of the cell. Whereas it has been observed in eukaryotic cells, that the ATP level immediately decreased after the temperature shift, no data are available for E. coli about the adenosine nucleotide levels during the narrow time range of minutes after a temperature up-shift.

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“…This phenomenon has also been well described, notably in the E. coli host, e.g. the overexpression of recombinant α-glucosidase in E. coli involves changes in the bacterial physiology and inhibition of cellular growth (Lin et al, 2001). The effects of heterologous protein overexpression, disturbing cellular metabolism, are found at various levels, in particular chromosomal replication, transcription, translation, carbon metabolism and respiration (Neubauer and Winter, 2001).…”

mentioning

confidence: 92%

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone

Céline¹,

Chemardin²,

Bigey³

et al. 2004

Yeast

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Ovine leptin was cloned in the methylotrophic yeast Pichia pastoris using a pPIC9K vector. Leptin was produced and secreted into the culture medium using the Saccharomyces cerevisiae α-mating factor prepro signal by five clones. Expression levels of leptin varied from clone to clone, depending on the copy number of the ob gene. Highest expression was observed with the single-copy clone S27 (250 mg/l). The modifications of culture conditions in batch and fed-batch culture increase the yield of protein. The use of higher cell concentration (63 g/l) before induction of oLept associate with a regulation of pH at 3.2, which decreases the effects of proteolysis, increases the expression level of the oLept to 402 mg/l. Moreover, compared with the non-producer clone, we observed a drastic decrease in growth rate and biomass yield in the leptin-producing clones. At the end of the fed-batch phase at pH 3.2 with clone S27, mortality rate reached 17.3%. Results showed that recombinant leptin production induced metabolic stress, and a negative impact on biomass yield and growth rate. We characterized the recombinant leptin produced by clone S27. It exhibited a molecular mass of 16 kDa, an N-terminal amino acid sequence identical to that of ovine leptin but with an additional tyrosine introduced by the cloning site. Moreover, it was found to be biologically active in vitro. The available production of a large quantity of oLept will strengthen the functional study for theorical and practical purposes.

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Cellular Responses to Strong Overexpression of Recombinant Genes in Escherichia Coli

Cited by 10 publications

References 31 publications

Limiting factors in Escherichia coli fed‐batch production of recombinant proteins

Limiting factors in Escherichia coli fed‐batch production of recombinant proteins

Transient increase of ATP as a response to temperature up-shift in Escherichia coli

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone

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