Development and characterization of an oxygen-dependent inducible promoter system, the modified nar promoter in a mutant Escherichia coli.
A nar promoter system (a modified nar promoter in a mutant host Escherichia coli (pMW618/W3110narL(-))), which is maximally induced under microaerobic conditions, was developed and characterized through batch and fed-batch culture to see whether the modified nar promoter can be used as an oxygen-dependent inducible promoter in the absence of nitrate ion. The modified nar promoter (pMW618) derived by mutations at -10 and -35 regions of the wild-type nar promoter does not require nitrate ion for the full induction, while a mutant host E. coli, W3110narL(-), does not express nitrate-dependent regulatory protein, NARL, from the host chromosome. In this study, it was found from fed-batch culture that the specific beta-galactosidase activity expressed from the lacZ gene fused to the modified nar promoter in the absence of nitrate ion was maximal when E. coli was grown under aerobic conditions (dissolved oxygen (DO) at 80%) to absorbance at 600 nm (OD(600)) of 35, and then the modified nar promoter was induced by lowering DO to 1-2% with alternating microaerobic and aerobic conditions. The maximal specific beta-galactosidase activity became 58,000 Miller at OD(600) of 160 with an induction ratio 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 expression system under conditions of fed-batch culture.
Han
S J
SJ
Department of Chemical Engineering and Bioprocess Engineering Research Center, KAIST, Taejon 305-701, Korea.
Chang
H N
HN
DeMoss
J A
JA
Suh
E J
EJ
Lee
J
J
D001431:Bacteriological Techniques; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D008967:Molecular Biology / Q000379:methods*; D009566:Nitrates / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology*; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011506:Proteins / Q000235:genetics*; D029961:Zebrafish Proteins
D009566:Nitrates; D011506:Proteins; D029961:Zebrafish Proteins; C101996:cpsf4 protein, zebrafish; D010100:Oxygen