Secretion-dependent proteolysis of heterologous protein by recombinant Escherichia coli is connected to an increased activity of the energy-generating dissimilatory pathway

The synthesis of a proteolytically unstable protein, originally designed fo r periplasmic export in recombinant Escherichia coil BL21(DE3), a strain na turally deficient for the ATP-dependent protease Lon (or La) and the outer membrane protease OmpT, is associated with a severe growth inhibition. This inhibition is not observed in BL21(DE3) synthesizing a closely related but proteolytically stable protein that is sequestered into inclusion bodies. It is shown that the growth inhibition is mainly caused by a slower cell di vision rate and a reduced growth yield and not by a general loss of cell di vision competence. Cells proceed with their normal growth characteristics w hen exposed again to conditions that do not sustain the expression of the h eterologous gene. The performance of cells synthesizing either the stable o r the degraded protein was also studied in high cell density cultures by em ploying a new method to calculate the actual specific growth rate, the biom ass yield coefficient, and the dissimilated fraction of the carbon substrat e in real-time. It is shown that the growth inhibition of cells synthesizin g the proteolytically degraded protein is connected to an increased dissimi lation of the carbon substrate resulting in a concomitant reduction of the growth rate and the biomass yield coefficient with respect to the carbon so urce. It is postulated that the increased dissimilation of the carbon subst rate by lon-deficient BI21(DE3) cells synthesizing the proteolytically unst able protein may result from a higher energy demand required for the in viv o degradation of this protein by ATP-dependent proteases different from the protease Lon. (C) 1999 John Wiley & Sons, Inc.