Fed-batch cultivation of an oxygen-dependent inducible promoter system, the nar promoter in Escherichia coli with an inactivated nar operon.
The nar promoter of Escherichia coli is maximally induced under anaerobic or microaerobic conditions in the presence of nitrate. We previously demonstrated in batch experiments that the intact nar promoter of E. coli cloned into a pBR322-based plasmid serves as a high-level expression system in a nar mutant of E. coli (Lee et al., 1996b). In this study, we extend characterization of the nar promoter expression system to the fed-batch culture mode, which is widely used in industrial-scale fermentation. From these experiments, it was found that the specific beta-galactosidase activity expressed from the lacZ gene fused to the nar promoter was maximal when host cells were grown under aerobic conditions [dissolved oxygen, (DO) = 80%] to absorbance at 600 nm (OD600) = 35 before induction of the nar promoter by lowering DO to 1-2% with alternating microaerobic and aerobic conditions. Approximately 15 h after induction, the OD600 of the culture reached 135 and the specific beta-galactosidase activity increased to 40,000 Miller units, equivalent to approximately 35% of the total cellular proteins. The specific beta-galactosidase activity before induction was approximately 1,000 Miller units, giving an induction ratio of approximately 40. Based on these results, we conclude that the nar promoter provides a convenient and effective high level expression system under conditions of fed-batch culture.
Han
S J
SJ
Department of Chemical Engineering and Bioprocess Engineering Research Center, KAIST, Taejon 305-701, Korea.
Chang
H N
HN
Lee
J
J
D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division; D004926:Escherichia coli / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
D001616:beta-Galactosidase; D010100:Oxygen