Premium
Continuous, high level production and excretion of a plasmid‐encoded protein by Escherichia coli in a two‐stage chemostat
Author(s) -
Fu Jeffrey,
Wilson David B.,
Shuler Michael L.
Publication year - 1993
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/bit.260411004
Subject(s) - chemostat , dilution , lac operon , escherichia coli , excretion , enterobacteriaceae , overproduction , substrate (aquarium) , biology , plasmid , strain (injury) , microbiology and biotechnology , biochemistry , chemistry , enzyme , bacteria , chromatography , genetics , gene , ecology , physics , anatomy , thermodynamics
The stable continuous overproduction of a plasmidencoded protein, β‐lactamase, for at least 50 days by Escherichia coli K‐12, RB791(pKN), with release into the culture medium has been demonstrated in two‐stage chemostats. The second‐stage culture was continuously induced with 0.1 m M IPTG. Continuous expression of β‐lactamase could not be sustained with this strain in a single‐stage chemostat because of cell death and selection for lac −1 cells. β‐Lactamase production in the second stage was sensitive to the second‐stage dilution rate and the distribution of the limiting substrate (i.e., glucose) between the first and second stages. The fraction of viable, excreting cells and the average copy number in the induced culture was measurably higher under those conditions of dilution rate and substrate distribution which yielded high β‐lactamase levels. The best operating conditions found at 20°C were a first‐stage dilution rate of 0.12 h −1 , a second‐stage dilution rate of 0.03 h −1 , and equal glucose feed supplied to each stage. Enzymatically active β‐lactamase was produced at a level of 25% of total cellular protein with 90% excretion yielding 300 mg β‐lactamase/L that was 50% pure at an OD 600 < 6. © 1993 Wiley & Sons, Inc.