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

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Abstract
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 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.