| 2. |
- Lin, H. Y., et al.
(författare)
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Change of extracellular cAMP concentration is a sensitive reporter for bacterial fitness in high-cell-density cultures of Escherichia coli
- 2004
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 87:5, s. 602-613
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Tidskriftsartikel (refereegranskat)abstract
- Guanosine-3'5'-tetraphosphate (ppGpp) and as, two regulators of the starvation response of Escherichia coli, have received increasing attention for monitoring cell physiological changes in production processes, although both are difficult to quantify. The kinetics of cAMP formation and degradation were not yet investigated in such processes, although the complex regulation of cAMP by synthesis, release, and degradation in connection with straightforward methods for analysis renders it a highly informative target. Therefore, we followed the cAMP concentration in various nonrecombinant and in four different recombinant glucose-limited fed-batch processes in different production scales. The intracellular cAMP concentration increases strongly at the end of the batch phase. Most cAMP is released to the cultivation medium. The rates of accumulation and degradation of extracellular cAMP are growth-rate-dependent and show a distinct maximum at a growth rate of about 0.35 h(-1). At very low growth rates, below 0.05 h(-1), extracellular cAMP is not produced but rather degraded, independent of whether this low growth rate is caused by glucose limitation or by the high metabolic load of recombinant protein production. In contrast to intracellular cAMP, which is highly unstable, analysis of extracellular cAMP is simpler and the kinetics of accumulation and degradation reflect well the physiological situation, including unlimited growth, limitation, and severe starvation of a production host.
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| 3. |
- Lin, H. Y., et al.
(författare)
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Determination of the maximum specific uptake capacities for glucose and oxygen in glucose-limited fed-batch cultivations of Escherichia coli
- 2001
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Ingår i: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 73:5, s. 347-357
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Tidskriftsartikel (refereegranskat)abstract
- A simple pulse-based method for the determination of the maximum uptake capacities for glucose a nd oxygen in glucose limited cultivations of E. coli is presented. The method does not depend on the time-consuming analysis of glucose or acetate, and therefore can be used to control the feed rate in glucose limited cultivations, such as fed-batch processes. The application of this method in fed-batch processes of E. coli showed that the uptake capacity for neither glucose nor oxygen is a constant parameter, as often is assumed in fed-batch models. The glucose uptake capacity decreased significantly when the specific growth rate decreased below 0.15 h(-1) and fell to about 0.6 mmol g(-1) h(-1) (mmol per g cell dry weight and hour) at the end of fed-batch fermentations, where mu was approximately 0.02 h(-1). The oxygen uptake capacity started to decrease somewhat earlier when mu declined below 0.25 h(-1) and was 5 mmol g(-1) h(-1) at the end of the fermentations. The behavior of both uptake systems is integrated in a dynamic model which allows a better fitting of experimental values for glucose in fed-batch processes in comparison to generally used unstructured kinetic models.
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