Effects of pulse addition of carbon sources on continuous cultivation of Escherichia coli containing a recombinant E. coli gapA gene.
At high glucose concentrations, Escherichia coli produces acetate (Crabtree effect). To look for the influence of glucose and/or acetate in the medium on the expression of a recombinant gene in E. coli, the effect of a pulse addition of glucose, on transcription of a cloned E. coli gapA gene and the resulting glyceraldehyde-3P-dehydrogenase activity (GAPDH), was tested during continuous cultivation of E. coli HB101 transformed with the plasmid pBR::EcogapA. Stable continuous cultures were established in a semi-synthetic medium supplemented with 5 g/L of glucose. After the addition of 7 g of glucose within a few seconds, gapA gene expression was strongly and very rapidly induced. As shown by primer-extension analysis, promoter P1, one of the four transcriptional promoters of the gapA gene, was strongly activated, and GAPDH activity increased. However, after rapid glucose consumption, acetate was produced and acetate concentrations above 2 g/L induced stress conditions. This is shown by a strong activation of promoter P2, that is recognized by the stress specific Esigma32 RNA polymerase. During this period, the total cellular RNA content was strongly diminished. Later, when acetate was partially consumed a high level of total RNA was restored, translation was efficient and a regular increase of the GAPDH-specific activity was observed. The transitions between glucose metabolism, acetate production and the end of acetate consumption, were marked by large increases in RNase and protease activities. For comparison, pulse-addition experiments were also performed with serine and alanine. A transient increase of GAPDH production associated with an increase in biomass was also found for serine that can be utilized as an energy source, whereas the addition of alanine, which is only incorporated into newly synthesized proteins, did not increase GAPDH production. The implication of these data for overproduction of recombinant proteins in E. coli is discussed.
Laboratoire des Sciences du Génie Chimique, CNRS, UPR 6811, ENSAIA, BP 172, F-54505 Vandoeuvre lès Nancy, France.
A
A
Gschaedler
N
N
Robas
J
J
Boudrant
C
C
Branlant
D019342:Acetic Acid / Q000494:pharmacology; D001709:Biotechnology / Q000379:methods; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development*; D005285:Fermentation; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000494:pharmacology
D002352:Carrier Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012694:Serine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005947:Glucose; D019342:Acetic Acid