Antisense Downregulation of ς32 as a Transient Metabolic Controller in Escherichia coli: Effects on Yield of Active Organophosphorus Hydrolase

Plasmids containing an antisense fragment of the ς³² gene were constructed and introduced into Escherichia coli cells. Downregulation of the ς³²-mediated stress response was evaluated under heat shock and ethanol stress and during the production of organophosphorus hydrolase (OPH). Northern blot analyses revealed that ς³² 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 ς³² mRNA. While a >10-fold increase in ς³² protein level was found under ethanol stress in the control cultures, antisense producing cultures resulted in a <3-fold increase, indicating downregulation of ς³². Correspondingly, antisense synthesis resulted in a decreased level of a ς³² regulated chaperone (GroEL) for the first 2 h after induction relative to control cultures without ς³² antisense mRNA. The total yield of OPH in the presence of ς³² antisense was, on average, 62% of the yield without antisense. However, during ς³² antisense production, a sixfold-higher specific OPH activity was observed compared to non-antisense-producing cultures.