Antisense Downregulation of ς(32) as a Transient Metabolic Controller in Escherichia coli: Effects on Yield of Active Organophosphorus Hydrolase

Plasmids containing an antisense fragment of the ς(32) gene were constructed and introduced into Escherichia coli cells. Downregulation of the ς(32)-mediated stress response was evaluated under heat shock and ethanol stress and during the production of organophosphorus hydrolase (OPH). Northern blot analyses revealed that ς(32) sense mRNA was virtually undetected in antisense-producing cultures from 5 to 20 min after antisense induction. However, lower-molecular-weight bands were found, presumably due to partial degradation of ς(32) mRNA. While a >10-fold increase in ς(32) protein level was found under ethanol stress in the control cultures, antisense producing cultures resulted in a <3-fold increase, indicating downregulation of ς(32). Correspondingly, antisense synthesis resulted in a decreased level of a ς(32) regulated chaperone (GroEL) for the first 2 h after induction relative to control cultures without ς(32) antisense mRNA. The total yield of OPH in the presence of ς(32) antisense was, on average, 62% of the yield without antisense. However, during ς(32) antisense production, a sixfold-higher specific OPH activity was observed compared to non-antisense-producing cultures.