Flow cytometric analysis of bacterial physiology during induction of foreign protein synthesis in recombinant Escherichia coli cells.
The production of foreign proteins at high yields represents a severe metabolic stress for Escherichia coli cells. In many cases, induction of protein synthesis results in rapid exhaustion of the cellular energy and metabolic precursors and thus in cell death. Therefore, sustained production of foreign proteins requires some fine tuning of the specific production rate to meet the capabilities of the cell. This has stimulated us to analyze by flow cytometry the physiological behaviour of recombinant E. coli cells producing human superoxide dismutase (SOD). Two strains that produce SOD under the control of either a combined T7/lac promoter or the phi10 promoter were compared by using the following parameters: (a) total DNA content as an indicator of cell division, (b) total RNA content as a measure for protein synthesis activity, (c) total protein content representing cell size, and (d) intracellular SOD content as a measure for productivity. Results show that those cells that continue to increase their biomass after induction of foreign protein synthesis also have the highest specific production rate. Cells, however, do not divide to a measureable degree but rather increase their size. The results confirm the importance of fine-tuning expression systems to prolong the lifetime of cells after induction. This will result in an increased yield.
Borth
N
N
Institute of Applied Microbiology, University of Agriculture, Vienna, Austria. borth@mail.boku.ac.at
Mitterbauer
R
R
Mattanovich
D
D
Kramer
W
W
Bayer
K
K
Katinger
H
H
D001426:Bacterial Proteins / Q000096:biosynthesis; D018533:Biomass; D004269:DNA, Bacterial / Q000096:biosynthesis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D012329:RNA, Bacterial / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D013482:Superoxide Dismutase / Q000096:biosynthesis* / Q000235:genetics
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; D013482:Superoxide Dismutase