Development and characterization of an oxygen-dependent inducible promotersystem, the modified nar promoter in a mutant Escherichia coli

A nar promoter system (a modified nar promoter in a mutant host Escherichia coil (pMW618/W3110narL(-))), which is maximally induced under microaerobic conditions, was developed and characterized through batch and fed-batch cu lture to see whether the modified nar promoter can be used as an oxygen-dep endent inducible promoter in the absence of nitrate ion. The modified nar p romoter (pMW618) derived by mutations at -10 and -35 regions of the wild-ty pe nar promoter does not require nitrate ion for the full induction, while a mutant host E. coli, W3110narL(-), does not express nitrate-dependent reg ulatory protein, NARL, from the host chromosome. In this study, it was foun d from fed-batch culture that the specific beta-galactosidase activity expr essed from the lacZ gene fused to the modified nar promoter in the absence of nitrate ion was maximal when E. coil was grown under aerobic conditions (dissolved oxygen (DO) at 80%) to absorbance at 600 nm (OD600) of 35, and t hen the modified nar promoter was induced by lowering DO to 1-2% with alter nating microaerobic and aerobic conditions. The maximal specific beta-galac tosidase activity became 58,000 Miller at OD600 of 160 with an induction ra tio of 20. On the basis of these results, we conclude that the modified nar promoter system (pMW618/W3110narL(-)), requiring only reduction of DO for the full induction, provides a convenient and effective high-level expressi on system under conditions of fed-batch culture. (C) 2000 John Wiley & Sons , Inc.