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D Riesenberg

Publications and source records attributed to D Riesenberg.

At least 19 recordsLinked to original sources
PubMed1992

Improvement of recombinant gene expression in Escherichia coli for glucose-controlled continuous and fed-batch cultures.

Escherichia coli TG1 (pHRW500) permanently expressed the human interferon alpha 1 gene (ifn alpha 1) directed by the tryptophan promoter (trpP.O) during continuous and fed-batch cultivation with a limited supply of glucose. The expression of ifn alpha 1 could be improved after insertion of the catabolite activator region (cap) upstream to trpP.O during cultivation of the modified E. coli TG1(pHRW500cap) in glucose-controlled continuous and fed-batch cultures. The cap-mediated stimulatory effect on the expression of cap-trpP.O-ifn alpha 1 increased with decreasing dilution rate. These results are in line with the increase in the level of cAMP with declining dilution rate and the well-known positive effects of cAMP-catabolite gene activator protein (CAP) at the transcriptional level. In addition, expression of the galactokinase gene (trpP.O-galK) in E. coli TG1(pDR720) could be improved in the same way with cap-trpP.O-galK in E. coli TG1(pDR720cap). Determinations of plasmid copy numbers, cellular amounts of galactokinase-mRNA, activity of galactokinase (AGalK) and the concentration of galactokinase at various dilution rates (D) strengthen the conclusion that the increase in AGalK with decreasing D was indeed due to the cap-mediated enhancement of transcription of the galK gene. We suggest that expression of other recombinant genes directed by various promoters that allow permanent transcription during growth with limited glucose supply in chemostat and fed-batch fermentors can be improved by appropriate insertion of the cap region.

Base Sequence↗
PubMed1991

High cell density fermentation of recombinant Escherichia coli with computer-controlled optimal growth rate.

In recent years recombinant DNA technology has enabled us to produce various proteins of therapeutic importance with microorganisms. As an appropriate host organism, E. coli plays a dominant role. Yields of E. coli dry cell mass in shaker flask culture range from 1-2 g/L, whereas in fermentors up to 10 g dry cells/L can be achieved. ZIMET and GBF have developed a high cell density fermentation process that produces E. coli (on a glucose/mineral salt medium) up to more than 100 g dry cells/L in a special fed-batch mode. This cultivation strategy prevents oxygen limitation and hence the accumulation of acetate and other metabolic byproducts. The specific growth rate can be adjusted so that product formation reaches its optimum value. An example of the production of alpha1-interferon is presented. The high cell density fermentations were realized in 30- and 450-L Chemap fermentors (ZIMET) and in a three-stage bioreactor scale-up system (72, 300, and 1,500 L) developed in cooperation with GBF and B. Braun Melsungen AG. Multiloop controllers were used to control the process variables.

Biotechnology↗
PubMed1991

High cell density cultivation of Escherichia coli at controlled specific growth rate.

A high cell density cultivation (HCDC) for growth of Escherichia coli in an especially designed glucose/mineral salt medium is proposed. The HCDC essentially starts as a batch process which is followed by a two-phase fed-batch cultivation. After unlimited growth at mu max = 0.45 h-1 in the batch part, growth was controlled at a reduced specific growth rate (mu = 0.11 h-1 less than mu max) over a period of 3 doubling times in which the biomass concentration increased from 12 to 95 g 1(-1) (phase 1 of fed-batch cultivation). Control of growth (mu) was realized by a PO2 control loop (by variation of glucose feeding) and a mu control loop (by variation of agitation speed N) while the actual mu was calculated from the off-gas composition. If the agitation rate cannot be increased anymore the mu controller is switched off (end of phase 1). In the following phase 2, mu declines, however, the still acting pO2 (glucose) controller guarantees sufficient O2 supply till the end of the cultivation with a biomass concentration of 110 g 1(-1) (dry mass). The proposed HCDC suppresses generation of inhibitory by-products and the high yield coefficients indicate the economy of the process.

Bacteriological Techniques↗
PubMed1991

High-cell-density cultivation of Escherichia coli.

High-cell-density cultivations of Escherichia coli in glucose-mineral-salt media produce more than 100 g dry cells litre-1 in special fed-batch modes with feeding of glucose and ammonia only. The specific growth rate can be adjusted to allow optimum recombinant protein generation.

Biotechnology↗
PubMed1990

High cell density fermentation of recombinant Escherichia coli expressing human interferon alpha 1.

A defined medium was developed which, by means of a specific fed-batch mode, allows growth of the recombinant Escherichia coli strain TG1 (pBB210) up to a cell density of 60 g dry weight/l. Apart from glucose and aqueous ammonia fed as carbon and nitrogen sources, it was necessary to supply other nutrients or O2-enriched air. Aqueous ammonia also served for pH control. The pO2 level was kept at 20% saturation via closed-loop controls operating the two output variables of stirrer speed and glucose feeding rate. This fed-batch method prevented significant accumulation of acetate and other metabolic by-products. The recombinant E. coli expressed interferon alpha 1 more efficiently at a lower specific growth rate (muPr approximately 0.15 h-1) than at the maximum specific growth rate (mu max = 0.45 h-1). Therefore, fermentation in the batch phase at mu max was only allowed to continue up to a medium cell density. In the succeeding fed-batch phase, the specific growth rate was reduced to muPr by increasing the stirrer speed according to an empirically developed time scale.

Ammonia↗
PubMed1987

Consensus conferences.

Explore the source record for details and available documents.

Consensus Statements as Topic↗
PubMed1985

A radioimmunoassay for (p)ppGpp and its application to Streptomyces hygroscopicus.

Aradioimmunoassay for (p)ppGpp was set up and applied to Streptomyces hygroscopicus. The (p)ppGpp specific antibodies obtained from rabbits immunized with ppGpp-human serum albumin could not distinguish between guanosine-5'-triphosphate-3'-diphosphate (pppGpp) and guanosine-5'-diphosphate-3'-diphosphate (ppGpp) whereas the cross reacitivity against structurally related nucleotides was negligible. Therefore, the antibodies were used for determination of (p)ppGpp, i.e. the sum of pppGpp and ppGpp. The pretreatment of biological samples for quantification of (p)ppGpp included formic acid extraction of cell pool followed by step gradient elution on DEAE-Sephadex A25 (Cl-) column for separation of (p)ppGpp from nucleotides with three or less phosphate groups. The very sensitive radioimmunoassay allowed determination of very low amounts: 1--50 pmoles (p)ppGpp per assay tube. The application of the RIA to S. hygroscopicus revealed a basal level in exponentially growing mycelium of about 2 pmoles (p)ppGpp per mg dry weight. The equilibrated pool size after induction of stringent response with serine hydroxamate was found to be about 30 pmoles (p)ppGpp per mg dry weight.

Chromatography, DEAE-Cellulose↗
PubMed1985

Synergistic effect of cobalt on the induction by A-factor of the formation of aerial mycelium and anthracyclines by a blocked mutant of Streptomyces griseus.

Cobalt ions synergistically supported the inducing effect of A-factor 1a on the formation of aerial mycelium and anthracyclines by the blocked S. griseus mutant 86. This suggested an involvement of effector 1a in the transport or metabolism of this trace element. In accordance with the proposed role of 1a as an autoregulatory secondary metabolite, the parent strain S. griseus JA 3933 secreted 1a into the medium after a decrease of the specific growth rate and before the onset of antibiotic production.

4-Butyrolactone↗