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 energ y and metabolic precursors and thus in cell death, Therefore, sustaine d production of foreign proteins requires some fine tuning of the spec ific production rate to meet the capabilities of the cell. This has st imulated us to analyze by flow cytometry the physiological behaviour o f 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 phi 10 promoter were compared by using the foll owing parameters: (a) total DNA content as an indicator of cell divisi on, (b) total RNA content as a measure for protein synthesis activity, (c) total protein content representing cell size, and (d) intracellul ar SOD content as a measure for productivity. Results show that those cells that continue to increase their biomass after induction of forei gn protein synthesis also have the highest specific production rate, C ells, however; do not divide to a measureable degree but rather increa se their size, The results confirm the importance of fine-tuning expre ssion systems to prolong the lifetime of cells after induction. This w ill result in an increased yield. (C) 1998 Wiley-Liss, Inc.