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.
Institut für Mikrobiologie und experimentelle Therapie, Jena, Federal Republic of Germany.
V
V
Schroeckh
M
M
Hartmann
E
E
Birch-Hirschfeld
D
D
Riesenberg
D001483:Base Sequence; D000242:Cyclic AMP / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics; D005686:Galactokinase / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007370:Interferon Type I / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid
D002373:Cyclic AMP Receptor Protein; D007370:Interferon Type I; D011994:Recombinant Proteins; D000242:Cyclic AMP; D005686:Galactokinase; D005947:Glucose