In vivo monitoring and alleviation of extracytoplasmic stress to recombinant protein overproduction in the periplasm of Escherichia coli

In Escherichia coli, there are two major pathways, i.e. Cpx and sigma(E), for dealing with the extracytoplasmic stress in the cell envelope. Due to the unique periplasmic processing steps and the tendency to form periplasmic inclusion bodies, penicillin acylase (PAC) offers a model system for studying he induction of extracytoplasmic stress associated with recombinant proteins overproduction in the periplasm of E. coli. In this study, E. coli strains carrying the lacZ reporter gene fusion with the promoters of three stress-responsive genes, i.e. degP, cpxP, and rpoH, were constructed in the JM 109 background for characterization. We demonstrate that pac overexpression induced the extracytoplasmic stress primarily via the Cpx pathway. The upregulated cpxP promoter activity can be a suitable sensor for in vivo monitoring of the extracytoplasmic stress upon pac overexpression. However, such physiological challenge was not observed and all the three promoter activities were reduced when arabinose was used to induce pac overexpression. This result suggests that the physiological impact observed for the IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced cultures could be overcome by the use of arabinose for induction. The extracytoplasmic stress response associated with pac overexpression could be significantly alleviated by the exogenous presence of DegP, but only partially alleviated by its mutant derivative of DegP(S210A.) (C) 2008 Elsevier B.V. All rights reserved.