Fed-batch cultivation of an oxygen-dependent inducible promoter system, the nar promoter in Escherichia coli with an inactivated nar operon
โ Scribed by Se Jong Han; Ho Nam Chang; Jongwon Lee
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 206 KB
- Volume
- 59
- Category
- Article
- ISSN
- 0006-3592
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โฆ Synopsis
The nar promoter of Escherichia coli is maximally induced under anaerobic or microaerobic conditions in the presence of nitrate. We previously demonstrated in batch experiments that the intact nar promoter of E. coli cloned into a pBR322-based plasmid serves as a high-level expression system in a nar mutant of E. coli (Lee et al., 1996b). In this study, we extend characterization of the nar promoter expression system to the fedbatch culture mode, which is widely used in industrialscale fermentation. From these experiments, it was found that the specific โค-galactosidase activity expressed from the lacZ gene fused to the nar promoter was maximal when host cells were grown under aerobic conditions [dissolved oxygen, (DO) = 80%] to absorbance at 600 nm (OD 600 ) = 35 before induction of the nar promoter by lowering DO to 1-2% with alternating microaerobic and aerobic conditions. Approximately 15 h after induction, the OD 600 of the culture reached 135 and the specific โค-galactosidase activity increased to 40,000 Miller units, equivalent to approximately 35% of the total cellular proteins. The specific โค-galactosidase activity before induction was approximately 1,000 Miller units, giving an induction ratio of approximately 40. Based on these results, we conclude that the nar promoter provides a convenient and effective high level expression system under conditions of fed-batch culture.
๐ SIMILAR VOLUMES
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-dep