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Characterization of an oxygen‐dependent inducible promoter, the nar promoter of Escherichia coli , to utilize in metabolic engineering
Author(s) -
Han Se Jong,
Chang Ho Nam,
Lee Jongwon
Publication year - 2001
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/1097-0290(20010305)72:5<573::aid-bit1022>3.0.co;2-i
Subject(s) - mutant , escherichia coli , nitrate , promoter , transcription (linguistics) , chemistry , biochemistry , gene , biology , gene expression , reporter gene , wild type , microbiology and biotechnology , ecology , linguistics , philosophy
The nar promoters, whose transcription is maximally induced under microaerobic conditions in the presence of nitrate ion, were characterized in fed‐batch culture to determine whether they can be used for metabolic engineering, by which overall production of valuable chemicals can be increased. For this purpose, we tested whether the expression level of a reporter gene, the lacZ gene from the nar promoter, could be maintained constant throughout the induction period by manipulation of dissolved oxygen (DO) levels at a given nitrate ion concentration. First, E. coli was grown under aerobic conditions (DO 80%) to absorbance at 600 nm (OD 600 ) of 35, then the nar promoter was induced by reduction of DO to different levels, combined with different frequencies and duration of alternating microaerobic and aerobic conditions throughout the entire induction period. For a wild‐type nar promoter (pMW61) in a mutant host E. coli with a mutation in the narG gene on the chromosome of the host (RK5265), it was possible to maintain production of β‐galactosidase activity per cell (specific β‐galactosidase activity) at a constant rate at 5000, 10,000, 15,000, and 20,000 Miller units, using different combinations of nitrate ion concentrations (0.1%, 0.5%, and 1%) and DO levels. In addition, it was possible to maintain production of specific β‐galactosidase activity at a constant rate at about 10,000 Miller units in the absence of nitrate ion when a nitrate‐independent nar promoter (pMW618) in the narL − mutant of the W3110 E. coli strain (W3110 narL − ) was used. Based on these results, we conclude that the nar promoter system provides a convenient expression system for metabolic engineering as well as for maximal production of recombinant proteins under conditions of fed‐batch culture. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 573–576, 2001.