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

Citation
Sj. Han et al., Characterization of an oxygen-dependent inducible promoter, the nar promoter of Escherichia coli, to utilize in metabolic engineering, BIOTECH BIO, 72(5), 2001, pp. 573-576
Citations number
22
Categorie Soggetti
Biotecnology & Applied Microbiology",Microbiology
Journal title
BIOTECHNOLOGY AND BIOENGINEERING
ISSN journal
00063592 → ACNP
Volume
72
Issue
5
Year of publication
2001
Pages
573 - 576
Database
ISI
SICI code
0006-3592(20010305)72:5<573:COAOIP>2.0.ZU;2-T
Abstract
The nar promoters, whose transcription is maximally induced under microaero bic conditions in the presence of nitrate ion, were characterized in fed-ba tch culture to determine whether they can be used for metabolic engineering , by which overall production of valuable chemicals can be increased. For t his purpose, we tested whether the expression level of a reporter gene, the lacZ gene from the nar promoter, could be maintained constant throughout t he induction period by manipulation of dissolved oxygen (DO) levels at a gi ven nitrate ion concentration. First, E. coli was grown under aerobic condi tions (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 differen t frequencies and duration of alternating microaerobic and aerobic conditio ns throughout the entire induction period. For a wild-type nar promoter (pM W61) in a mutant host E. coil with a mutation in the narG gene on the chrom osome 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 di fferent 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 uni ts in the absence of nitrate ion when a nitrate-independent nar promoter (p MW618) in the narL(-) mutant of the W3110 E. coil strain (W3110narL(-)) was used. Based on these results, we conclude that the nar promoter system pro vides a convenient expression system for metabolic engineering as well as f or maximal production of recombinant proteins under conditions of fed-batch culture. (C) 2001 John Wiley & Sons, Inc.