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 dur ing continuous cultivation of E. coli HB101 transformed with the plasmid pB R::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 gl ucose within a few seconds, gapA gene expression was strongly and very rapi dly induced. As shown by primer-extension analysis, promoter P1, one of the four transcriptional promoters of the gapA gene, was strongly activated, a nd GAPDH activity increased. However, after rapid glucose consumption, acet ate was produced and acetate concentrations above 2 g/L induced stress cond itions. This is shown by a strong activation of promoter P2, that is recogn ized by the stress specific E sigma(32) RNA polymerase. During this period, the total cellular RNA content was strongly diminished. Later, when acetat e was partially consumed a high level of total RNA was restored, translatio n was efficient and a regular increase of the GAPDH-specific activity was o bserved. The transitions between glucose metabolism, acetate production and the end of acetate consumption, were marked by large increases in RNase an d protease activities. For comparison, pulse-addition experiments were also performed with serine and alanine. A transient increase of GAPDH productio n 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. (C) 1999 John Wiley & Sons, Inc.