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
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.