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 anaero bic or microaerobic conditions in the presence of nitrate. We previous ly demonstrated in batch experiments that the intact nar promoter of E . coli cloned into a pBR322-based plasmid serves as a high-level expre ssion system in a nar mutant of E. coli (Lee et al., 1996b). In this s tudy, we extend characterization of the nar promoter expression system to the fed-batch culture mode, which is widely used in industrial-sca le fermentation. From these experiments, it was found that the specifi c beta-galactosidase activity expressed from the lacZ gene fused to th e nar promoter was maximal when host cells were grown under aerobic co nditions [dissolved oxygen, (DO) = 80%] to absorbance at 600 nm (OD600 ) = 35 before induction of the nar promoter by lowering DO to 1-2% wit h 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, equival ent to approximately 35% of the total cellular proteins. The specific beta-galactosidase activity before induction was approximately 1,000 M iller units, giving an induction ratio of approximately 40. Based on t hese results, we conclude that the nar promoter provides a convenient and effective high level expression system under conditions of fed-bat ch culture. (C) 1998 John Wiley & Sons, Inc.