Escherichia coli oxygen-dependent nar promoter systme in the fed-batch cultures for the production of recombinant β-Galactosidase

Abstract

The nar promoter of Escherichia coli, which is maximally induced under anaerobic conditions in the presence of nitrate, was characterized to investigate the feasibility of utilizing the nar promoter system for industrial-scale fermentation to produce recombinant proteins. To find the most suitable system, several different combinations of the nar promoters (the wild type nar promoter (pMW61) and the modified nar promoter (pMW618)) and E. coli strain (nar operon mutant (RK5265) and narL mutant (RK5278, $W3110narL^-$) were studied. For both pMW61 and pMW618, the nar promoters were cloned onto pBR322, and the lacZ gene expressing β-galactosidase instead of the structural genes of the nar operon was cloned for reporter gene downstream of the nar promoter. The modified nar promoter (pMW618) was prepared by changing several bases on the -10 and -35 region of the nar promoter, making the modified nar promoter insensitive to nitrate. The nar operation mutant (RK5265) cannot produce nitrate reductase by insertional mutation of transposon into narG gene, one of the structural genes of the nar operon. The narL mutants cannot produce narL, one of the activator proteins binding to the nar promoter once activated by nitrate. Here combinations of pMW61/RK5265 and pMW618/narL mutants will be described in more details as they have more desirable characteristics. From the studies using the pMW61/RK5265 system, it was found that the best growth medium for the fed-batch culture was modified R medium, and it was necessary to keep the glucose concentration as low as possible during fed-batch operation to maximize induction of the nar promoter. The continuous low DO (Dissolved Oxygen) level used to induce the nar promoter enhanced formation of acetic acid, resulting in the inhibition of cell growth. To reduce the production of acetic acid during the induction period, alternating microaerobic and aerobic conditions achieved by exponential feeding with pH-stat mode of cultivation was e...