Characterization of an oxygen-dependent inducible promoter, the nar promoter of Escherichia coli, to utilize in metabolic engineering

Abstract

The nar promoters, whose transcription is maximally induced under microaerobic conditions in the presence of nitrate ion, were characterized in fed-batch culture to determine whether they can be used for metabolic engineering, by which overall production of valuable chemicals can be increased. For this purpose, we tested whether the expression level of a reporter gene, the lacZ gene from the nar promoter, could be maintained constant throughout the induction period by manipulation of dissolved oxygen (DO) levels at a given nitrate ion concentration. First, E. coli was grown under aerobic conditions (DO 80%) to absorbance at 600 nm (OD600) of 35, then the nar promoter was induced by reduction of DO to different levels, combined with different frequencies and duration of alternating microaerobic and aerobic conditions throughout the entire induction period. For a wild-type nar promoter (pMW61) in a mutant host E. coil with a mutation in the narG gene on the chromosome of the host (RK5265), it was possible to maintain production of beta -galactosidase activity per cell (specific beta -galactosidase activity) at a constant rate at 5000, 10,000, 15,000, and 20,000 Miller units, using different combinations of nitrate ion concentrations (0.1%, 0.5%, and 1%) and DO levels. In addition, it was possible to maintain production of specific beta -galactosidase activity at a constant rate at about 10,000 Miller units in the absence of nitrate ion when a nitrate-independent nar promoter (pMW618) in the narL(-) mutant of the W3110 E. coil strain (W3110narL(-)) was used. Based on these results, we conclude that the nar promoter system provides a convenient expression system for metabolic engineering as well as for maximal production of recombinant proteins under conditions of fed-batch culture. (C) 2001 John Wiley & Sons, Inc.